Lucy Shapiro

All Publications

• Identification and Demonstration of roGFP2 as an Environmental Sensor for Cryogenic Correlative Light and Electron Microscopy. Journal of structural biology Perez, D., Dahlberg, P. D., Wang, J., Sartor, A. M., Borden, J. S., Shapiro, L., Moerner, W. E. 2022: 107881

  Abstract

  Cryogenic correlative light and electron microscopy (cryo-CLEM) seeks to leverage orthogonal information present in two powerful imaging modalities. While recent advances in cryogenic electron microscopy (cryo-EM) allow for the visualization and identification of structures within cells at the nanometer scale, information regarding the cellular environment, such as pH, membrane potential, ionic strength etc. that influence the observed structures remains absent. Fluorescence microscopy can potentially be used to reveal this information when specific labels, known as fluorescent biosensors, are used, but there has been minimal use of such biosensors in cryo-CLEM to date. Here we demonstrate the applicability of one such biosensor, the fluorescent protein roGFP2, for cryo-CLEM experiments. At room temperature, the ratio of roGFP2 emission brightness when excited at 425 nm or 488 nm is known to report on the local redox potential. When samples containing roGFP2 are rapidly cooled to 77K in a manner compatible with cryo-EM, the ratio of excitation peaks remains a faithful indicator of the redox potential at the time of freezing. Using purified protein in different oxidizing/reducing environments, we generate a calibration curve which can be used to analyze in situ measurements. As a proof-of-principle demonstration, we investigate the oxidation/reduction state within vitrified Caulobacter crescentus cells. The polar organizing protein Z (PopZ) localizes to the polar regions of C. crescentus where it is known to form a distinct microdomain. By expressing an inducible roGFP2-PopZ fusion we can visualize individual microdomains in the context of their redox environment.

  View details for DOI 10.1016/j.jsb.2022.107881

  View details for PubMedID 35811036

• ATP-responsive biomolecular condensates tune bacterial kinase signaling. Science advances Saurabh, S., Chong, T. N., Bayas, C., Dahlberg, P. D., Cartwright, H. N., Moerner, W. E., Shapiro, L. 2022; 8 (7): eabm6570

  Abstract

  Biomolecular condensates formed via liquid-liquid phase separation enable spatial and temporal organization of enzyme activity. Phase separation in many eukaryotic condensates has been shown to be responsive to intracellular adenosine triphosphate (ATP) levels, although the consequences of these mechanisms for enzymes sequestered within the condensates are unknown. Here, we show that ATP depletion promotes phase separation in bacterial condensates composed of intrinsically disordered proteins. Enhanced phase separation promotes the sequestration and activity of a client kinase enabling robust signaling and maintenance of viability under the stress posed by nutrient scarcity. We propose that a diverse repertoire of condensates can serve as control knobs to tune enzyme sequestration and reactivity in response to the metabolic state of bacterial cells.

  View details for DOI 10.1126/sciadv.abm6570

  View details for PubMedID 35171683

• Delivering the message: How a novel technology enabled the rapid development of effective vaccines. Cell Shapiro, L., Losick, R. 2021

  Abstract

  This year's LaskerDebakey Clinical Research Award honors Katalin Kariko and Drew Weissman for the development of a therapeutic technology based on nucleoside-modification of messenger RNA, enabling the rapid development of the highly effective COVID-19 vaccines.

  View details for DOI 10.1016/j.cell.2021.08.019

  View details for PubMedID 34562362

• A localized adaptor protein performs distinct functions at the Caulobacter cell poles PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, J., Moerner, W. E., Shapiro, L. 2021; 118 (13)

  View details for Web of Science ID 000637394200069

• A localized adaptor protein performs distinct functions at the Caulobacter cell poles. Proceedings of the National Academy of Sciences of the United States of America Wang, J., Moerner, W. E., Shapiro, L. 2021; 118 (13)

  Abstract

  Asymmetric cell division generates two daughter cells with distinct characteristics and fates. Positioning different regulatory and signaling proteins at the opposing ends of the predivisional cell produces molecularly distinct daughter cells. Here, we report a strategy deployed by the asymmetrically dividing bacterium Caulobacter crescentus where a regulatory protein is programmed to perform distinct functions at the opposing cell poles. We find that the CtrA proteolysis adaptor protein PopA assumes distinct oligomeric states at the two cell poles through asymmetrically distributed c-di-GMP: dimeric at the stalked pole and monomeric at the swarmer pole. Different polar organizing proteins at each cell pole recruit PopA where it interacts with and mediates the function of two molecular machines: the ClpXP degradation machinery at the stalked pole and the flagellar basal body at the swarmer pole. We discovered a binding partner of PopA at the swarmer cell pole that together with PopA regulates the length of the flagella filament. Our work demonstrates how a second messenger provides spatiotemporal cues to change the physical behavior of an effector protein, thereby facilitating asymmetry.

  View details for DOI 10.1073/pnas.2024705118

  View details for PubMedID 33753507

• A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence. mBio Zhou, X., Eckart, M. R., Shapiro, L. 2021; 12 (1)

  Abstract

  Bacterial cells utilize toxin-antitoxin systems to inhibit self-reproduction, while maintaining viability, when faced with environmental challenges. The activation of the toxin is often coupled to the induction of cellular response pathways, such as the stringent response, in response to multiple stress conditions. Under these conditions, the cell enters a quiescent state referred to as dormancy or persistence. How toxin activation triggers persistence and induces a systemic stress response in the alphaproteobacteria remains unclear. Here, we report that in Caulobacter, a hipA2-encoded bacterial toxin contributes to bacterial persistence by manipulating intracellular amino acid balance. HipA2 is a serine/threonine kinase that deactivates tryptophanyl-tRNA synthetase by phosphorylation, leading to stalled protein synthesis and the accumulation of free tryptophan. An increased level of tryptophan allosterically activates the adenylyltransferase activity of GlnE that, in turn, deactivates glutamine synthetase GlnA by adenylylation. The inactivation of GlnA promotes the deprivation of glutamine in the cell, which triggers a stringent response. By screening 69 stress conditions, we find that HipBA2 responds to multiple stress signals through the proteolysis of HipB2 antitoxin by the Lon protease and the release of active HipA2 kinase, revealing a molecular mechanism that allows disparate stress conditions to be sensed and funneled into a single response pathway.IMPORTANCE To overcome various environmental challenges, bacterial cells can enter a physiologically quiescent state, known as dormancy or persistence, which balances growth and viability. In this study, we report a new mechanism by which a toxin-antitoxin system responds to harsh environmental conditions or nutrient deprivation by orchestrating a dormant state while preserving viability. The hipA2-encoded kinase functions as a toxin in Caulobacter, inducing bacterial persistence by disturbing the intracellular tryptophan-glutamine balance. A nitrogen regulatory circuit can be regulated by the intracellular level of tryptophan, which mimics the allosteric role of glutamine in this feedback loop. The HipBA2 module senses different types of stress conditions by increasing the intracellular level of tryptophan, which in turn breaks the tryptophan-glutamine balance and induces glutamine deprivation. Our results reveal a molecular mechanism that allows disparate environmental challenges to converge on a common pathway that results in a dormant state.

  View details for DOI 10.1128/mBio.03020-20

  View details for PubMedID 33622732

• A Bacterial Toxin Perturbs Intracellular Amino Acid Balance To Induce Persistence MBIO Zhou, X., Eckart, M. R., Shapiro, L. 2021; 12 (1)

  View details for DOI 10.1128/mBio.03020-20.

  View details for Web of Science ID 000668756400004

• The Nucleoid-Associated Protein GapR Uses Conserved Structural Elements To Oligomerize and Bind DNA. mBio Lourenco, R. F., Saurabh, S., Herrmann, J., Wakatsuki, S., Shapiro, L. 2020; 11 (3)

  Abstract

  Nucleoid-associated proteins (NAPs) are DNA binding proteins critical for the organization and function of the bacterial chromosome. A newly discovered NAP in Caulobacter crescentus, GapR, is thought to facilitate the movement of the replication and transcription machines along the chromosome by stimulating type II topoisomerases to remove positive supercoiling. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recently published DNA-bound crystal structure of GapR, we identified the structural elements involved in oligomerization and DNA binding. Moreover, we show that GapR is maintained as a tetramer upon its dissociation from DNA and that tetrameric GapR is capable of binding DNA molecules in vitro Analysis of protein chimeras revealed that two helices of GapR are functionally conserved in H-NS, demonstrating that two evolutionarily distant NAPs with distinct mechanisms of action utilize conserved structural elements to oligomerize and bind DNA.IMPORTANCE Bacteria organize their genetic material in a structure called the nucleoid, which needs to be compact to fit inside the cell and, at the same time, dynamic to allow high rates of replication and transcription. Nucleoid-associated proteins (NAPs) play a pivotal role in this process, so their detailed characterization is crucial for our understanding of DNA organization into bacterial cells. Even though NAPs affect DNA-related processes differently, all of them have to oligomerize and bind DNA for their function. The significance of this study is the identification of structural elements involved in the oligomerization and DNA binding of a newly discovered NAP in C. crescentus and the demonstration that structural elements are conserved in evolutionarily distant and functionally distinct NAPs.

  View details for DOI 10.1128/mBio.00448-20

  View details for PubMedID 32518183

• Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in Caulobacter. Proceedings of the National Academy of Sciences of the United States of America Dahlberg, P. D., Saurabh, S., Sartor, A. M., Wang, J., Mitchell, P. G., Chiu, W., Shapiro, L., Moerner, W. E. 2020

  Abstract

  Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of 30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.

  View details for DOI 10.1073/pnas.2001849117

  View details for PubMedID 32513734

• Cryogenic Superresolution Fluorescence Correlated with Cryogenic Electron Tomography: Combining Specific Labeling and High Resolution Dahlberg, P. D., Saurabh, S., Wang, J., Sartor, A. M., Chiu, W., Shapiro, L., Moerner, W. E. CELL PRESS. 2020: 20A–21A

  View details for Web of Science ID 000513023200098

• Continuous, Topologically Guided Protein Crystallization Drives Self-Assembly of a Bacterial Surface Layer Comerci, C. J., Herrmann, J., Yoon, J., Jabbarpour, F., Zhou, X., Nomellini, J. F., Smit, J., Shapiro, L., Wakatsuki, S., Moerner, W. E. CELL PRESS. 2020: 201A–202A

  View details for Web of Science ID 000513023201258

• Robust Modulation of a Bacterial Kinase by Protein Phase Separation Saurabh, S., Chong, T., Bayas, C., Dahlberg, P. D., Moerner, W. E., Shapiro, L. CELL PRESS. 2020: 203A

  View details for Web of Science ID 000513023201267

• Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus. Nature microbiology Lasker, K., von Diezmann, L., Zhou, X., Ahrens, D. G., Mann, T. H., Moerner, W. E., Shapiro, L. 2020

  Abstract

  Selective recruitment and concentration of signalling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization1-3. The dynamic flow of molecules into and out of these compartments occurs on faster timescales than for membrane-enclosed organelles, presenting a possible mechanism to control spatial patterning within cells. Here, we combine single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modelling and a spatially resolved promoter activation assay to study signal exchange in and out of the 200nm cytoplasmic pole-organizing protein popZ (PopZ) microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus4-8. Two phospho-signalling proteins, the transmembrane histidine kinase CckA and the cytoplasmic phosphotransferase ChpT, provide the only phosphate source for the cell fate-determining transcription factor CtrA9-18. We find that all three proteins exhibit restricted rates of entry into and escape from the microdomain as well as enhanced phospho-signalling within, leading to a submicron gradient of activated CtrA-P19 that is stable and sublinear. Entry into the microdomain is selective for cytosolic proteins and requires a binding pathway to PopZ. Our work demonstrates how nanoscale protein assemblies can modulate signal propagation with fine spatial resolution, and that in Caulobacter, this modulation serves to reinforce asymmetry and differential cell fate of the two daughter cells.

  View details for DOI 10.1038/s41564-019-0647-7

  View details for PubMedID 31959967

• Cryogenic single-molecule active control microscopy with a photoactivatable fluorescent protein Sartor, A. M., Dahlberg, P. D., Wang, J., Saurabh, S., Shapiro, L., Moerner, W. E., Gregor, Koberling, F., Erdmann, R. SPIE-INT SOC OPTICAL ENGINEERING. 2020

  View details for DOI 10.1117/12.2546333

  View details for Web of Science ID 000546225400006

• A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly. Proceedings of the National Academy of Sciences of the United States of America Herrmann, J., Li, P., Jabbarpour, F., Chan, A. C., Rajkovic, I., Matsui, T., Shapiro, L., Smit, J., Weiss, T. M., Murphy, M. E., Wakatsuki, S. 2019

  Abstract

  Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

  View details for DOI 10.1073/pnas.1909798116

  View details for PubMedID 31848245

• Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation. Proceedings of the National Academy of Sciences of the United States of America Zhou, X., Wang, J., Herrmann, J., Moerner, W. E., Shapiro, L. 2019

  Abstract

  The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

  View details for DOI 10.1073/pnas.1906119116

  View details for PubMedID 31315982

• Protein Self-Assembly Drives Surface Layer Biogenesis and Maintenance in C. crescentus Herrmann, J., Comerci, C., Yoon, J., Jabbarpour, F., Shapiro, L., Wakatsuki, S., Moerner, W. E. CELL PRESS. 2019: 159A

  View details for DOI 10.1016/j.bpj.2018.11.881

  View details for Web of Science ID 000460779800789

• Biomolecular Condensates at Bacterial Cell Poles Function to Drive Spatially Restricted Signal Propagation Shapiro, L. CELL PRESS. 2019: 5A

  View details for DOI 10.1016/j.bpj.2018.11.056

  View details for Web of Science ID 000460779800028

• Multi-Step 2D Protein Crystallization via Structural Changes within an Ordered Lattice Herrmann, J., Comerci, C. J., Jabbarpour, F., Shapiro, L., Moerner, W. E., Wakatsuki, S. CELL PRESS. 2019: 194A

  View details for DOI 10.1016/j.bpj.2018.11.1077

  View details for Web of Science ID 000460779800965

• Dissection of Protein Function Within a Bacterial Biomolecular Condensate by In Vitro Reconstitution Saurabh, S., Shapiro, L. CELL PRESS. 2019: 458A–459A

  View details for DOI 10.1016/j.bpj.2018.11.2475

  View details for Web of Science ID 000460779802305

• A Bacterial Biomolecular Condensate Sequesters a Signaling Pathway that Drives Spatial Regulation of Gene Expression and Asymmetric Cell Division Lasker, K., von Diezmann, A., Moerner, W. E., Shapiro, L. CELL PRESS. 2019: 453A

  View details for DOI 10.1016/j.bpj.2018.11.2446

  View details for Web of Science ID 000460779802279

• Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly. Nature communications Comerci, C. J., Herrmann, J. n., Yoon, J. n., Jabbarpour, F. n., Zhou, X. n., Nomellini, J. F., Smit, J. n., Shapiro, L. n., Wakatsuki, S. n., Moerner, W. E. 2019; 10 (1): 2731

  Abstract

  Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C. crescentus, we show that 2D protein self-assembly is sufficient to build and maintain the S-layer in living cells by efficient protein crystal nucleation and growth. We propose a model supported by single-molecule tracking whereby randomly secreted SLP monomers diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated at the edges of growing 2D S-layer crystals. Surface topology creates crystal defects and boundaries, thereby guiding S-layer assembly. Unsupervised assembly poses challenges for therapeutics targeting S-layers. However, protein crystallization as an evolutionary driver rationalizes S-layer diversity and raises the potential for biologically inspired self-assembling macromolecular nanomaterials.

  View details for DOI 10.1038/s41467-019-10650-x

  View details for PubMedID 31227690

• Identification of PAmKate as a Red Photoactivatable Fluorescent Protein for Cryogenic Super-Resolution Imaging. Journal of the American Chemical Society Dahlberg, P. D., Sartor, A. M., Wang, J., Saurabh, S., Shapiro, L., Moerner, W. E. 2018; 140 (39): 12310–13

  Abstract

  Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.

  View details for PubMedID 30222332

• Integration of cell cycle signals by multi-PAS domain kinases. Proceedings of the National Academy of Sciences of the United States of America Mann, T. H., Shapiro, L. 2018

  Abstract

  Spatial control of intracellular signaling relies on signaling proteins sensing their subcellular environment. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. As a model for how subcellular spatial information can control signaling activity, we have reconstituted the cell pole-specific control of the master regulator kinase/phosphatase CckA from the asymmetrically dividing bacterium Caulobacter crescentus CckA is active as a kinase only when it accumulates within a microdomain at the new cell pole, where it colocalizes with the pseudokinase DivL. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. Here, we show that CckA uses its PAS domains to integrate information from DivL and its own oligomerization state to control the balance of its kinase and phosphatase activities. We reconstituted the DivL-CckA complex on liposomes in vitro and found that DivL directly controls the CckA kinase/phosphatase switch, and that stimulation of either CckA catalytic activity depends on the second of its two PAS domains. We further show that CckA oligomerizes through a multidomain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homooligomerization. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling.

  View details for PubMedID 29987042

• Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bayas, C. A., Wang, J., Lee, M. K., Schrader, J. M., Shapiro, L., Moerner, W. E. 2018; 115 (16): E3712–E3721

  Abstract

  We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

  View details for PubMedID 29610352

• A Polar Matrix Microdomain Constrains Diffusion and Regulates Intracellular Signaling von Diezmann, A., Lasker, K., Mann, T. H., Ahrens, D. G., Shapiro, L., Moerner, W. E. CELL PRESS. 2018: 548A

  View details for Web of Science ID 000430563200493

• Probing Asymmetric Behavior of a Cell Cycle Regupatory Protein in Live Caulobacter using Single-Molecule Imaging Wang, J., Shapiro, L., Moerner, W. E. CELL PRESS. 2018: 350A

  View details for Web of Science ID 000430450000249

• A Red Fluorescent Protein for Cryogenic Single-Molecule Superresolution Imaging Sorter, A. M., Dahlberg, P. D., Wang, J., Shapiro, L., Moerner, W. E. CELL PRESS. 2018: 529A–530A

  View details for Web of Science ID 000430563200402

• Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer Herrmann, J., Smit, J., Shapiro, L., Wakatsuki, S. CELL PRESS. 2018: 404A

  View details for Web of Science ID 000430450000508

• Two-Color Sted Microscopy to Visualize S-Layer Biogenesis in Caulobacter Crescentus Comerci, C. J., Herrmann, J., Shapiro, L., Wakatsuki, S., Moerner, W. E. CELL PRESS. 2018: 613A

  View details for Web of Science ID 000430563300065

• Spatial Organization and Dynamics of RNA Processing in Caulobacter Crescentus Bayas, C., Wang, J., Lee, M. K., Schrader, J. M., Shapiro, L., Moerner, W. E. CELL PRESS. 2018: 251A

  View details for Web of Science ID 000430439600509

• Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer BIOPHYSICAL JOURNAL Herrmann, J., Jabbarpour, F., Bargar, P. G., Nomellini, J. F., Li, P., Lane, T. J., Weiss, T. M., Smit, J., Shapiro, L., Wakatsuki, S. 2017; 112 (9): 1841-1851

  Abstract

  Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state. This finding invalidates the common assumption that S-layers serve only as static wall-like structures. In vitro, the Caulobacter S-layer protein, RsaA, enters the aggregate state at physiological temperatures and low divalent calcium ion concentrations. At higher concentrations, calcium ions stabilize monomeric RsaA, which can then transition to the two-dimensional crystalline state. Caulobacter requires micromolar concentrations of calcium for normal growth and development. Without an S-layer, Caulobacter is even more sensitive to changes in environmental calcium concentration. Therefore, this structurally dynamic S-layer responds to environmental conditions as an ion sensor and protects Caulobacter from calcium deficiency stress, a unique mechanism of bacterial adaptation. These findings provide a biochemical and physiological basis for RsaA's calcium-binding behavior, which extends far beyond calcium's commonly accepted role in aiding S-layer biogenesis or oligomerization and demonstrates a connection to cellular fitness.

  View details for DOI 10.1016/j.bpj.2017.04.003

  View details for Web of Science ID 000401301600013

  View details for PubMedID 28494955

• Ultra-photostable, genetically directed fluoromodule enables STED nanoscopy and long time scale single protein tracks in live bacteria Saurabh, S., Perez, A., Comerci, C., Shapiro, L., Moerner, W. AMER CHEMICAL SOC. 2017

  View details for Web of Science ID 000430568500763

• A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity. mBio Perez, A. M., Mann, T. H., Lasker, K., Ahrens, D. G., Eckart, M. R., Shapiro, L. 2017; 8 (1)

  Abstract

  Signaling hubs at bacterial cell poles establish cell polarity in the absence of membrane-bound compartments. In the asymmetrically dividing bacterium Caulobacter crescentus, cell polarity stems from the cell cycle-regulated localization and turnover of signaling protein complexes in these hubs, and yet the mechanisms that establish the identity of the two cell poles have not been established. Here, we recapitulate the tripartite assembly of a cell fate signaling complex that forms during the G1-S transition. Using in vivo and in vitro analyses of dynamic polar protein complex formation, we show that a polymeric cell polarity protein, SpmX, serves as a direct bridge between the PopZ polymeric network and the cell fate-directing DivJ histidine kinase. We demonstrate the direct binding between these three proteins and show that a polar microdomain spontaneously assembles when the three proteins are coexpressed heterologously in an Escherichia coli test system. The relative copy numbers of these proteins are essential for complex formation, as overexpression of SpmX in Caulobacter reorganizes the polarity of the cell, generating ectopic cell poles containing PopZ and DivJ. Hierarchical formation of higher-order SpmX oligomers nucleates new PopZ microdomain assemblies at the incipient lateral cell poles, driving localized outgrowth. By comparison to self-assembling protein networks and polar cell growth mechanisms in other bacterial species, we suggest that the cooligomeric PopZ-SpmX protein complex in Caulobacter illustrates a paradigm for coupling cell cycle progression to the controlled geometry of cell pole establishment.IMPORTANCE Lacking internal membrane-bound compartments, bacteria achieve subcellular organization by establishing self-assembling protein-based microdomains. The asymmetrically dividing bacterium Caulobacter crescentus uses one such microdomain to link cell cycle progression to morphogenesis, but the mechanism for the generation of this microdomain has remained unclear. Here, we demonstrate that the ordered assembly of this microdomain occurs via the polymeric network protein PopZ directly recruiting the polarity factor SpmX, which then recruits the histidine kinase DivJ to the developing cell pole. Further, we find that overexpression of the bridge protein SpmX in Caulobacter disrupts this ordered assembly, generating ectopic cell poles containing both PopZ and DivJ. Together, PopZ and SpmX assemble into a cooligomeric network that forms the basis for a polar microdomain that coordinates bacterial cell polarity.

  View details for DOI 10.1128/mBio.02238-16

  View details for PubMedID 28246363

  View details for PubMedCentralID PMC5347347

• Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule. Current protocols in cell biology Saurabh, S. n., Perez, A. M., Comerci, C. J., Shapiro, L. n., Moerner, W. E. 2017; 75: 4.32.1–4.32.22

  Abstract

  Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super-resolution microscopy using stimulated emission depletion. © 2017 by John Wiley & Sons, Inc.

  View details for PubMedID 28627757

• Dynamic translation regulation in Caulobacter cell cycle control. Proceedings of the National Academy of Sciences of the United States of America Schrader, J. M., Li, G., Childers, W. S., Perez, A. M., Weissman, J. S., Shapiro, L., McAdams, H. H. 2016; 113 (44): E6859-E6867

  Abstract

  Progression of the Caulobacter cell cycle requires temporal and spatial control of gene expression, culminating in an asymmetric cell division yielding distinct daughter cells. To explore the contribution of translational control, RNA-seq and ribosome profiling were used to assay global transcription and translation levels of individual genes at six times over the cell cycle. Translational efficiency (TE) was used as a metric for the relative rate of protein production from each mRNA. TE profiles with similar cell cycle patterns were found across multiple clusters of genes, including those in operons or in subsets of operons. Collections of genes associated with central cell cycle functional modules (e.g., biosynthesis of stalk, flagellum, or chemotaxis machinery) have consistent but different TE temporal patterns, independent of their operon organization. Differential translation of operon-encoded genes facilitates precise cell cycle-timing for the dynamic assembly of multiprotein complexes, such as the flagellum and the stalk and the correct positioning of regulatory proteins to specific cell poles. The cell cycle-regulatory pathways that produce specific temporal TE patterns are separate from-but highly coordinated with-the transcriptional cell cycle circuitry, suggesting that the scheduling of translational regulation is organized by the same cyclical regulatory circuit that directs the transcriptional control of the Caulobacter cell cycle.

  View details for PubMedID 27791168

• Cell cycle progression in Caulobacter requires a nucleoid-associated protein with high AT sequence recognition PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ricci, D. P., Melfi, M. D., Lasker, K., Dill, D. L., McAdams, H. H., Shapiro, L. 2016; 113 (40): E5952-E5961

  Abstract

  Faithful cell cycle progression in the dimorphic bacterium Caulobacter crescentus requires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required for Caulobacter growth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed in Caulobacter The Escherichia coli nucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously in Caulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed in E. coli, suggesting that GapR and H-NS have distinct functions. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression.

  View details for DOI 10.1073/pnas.1612579113

  View details for Web of Science ID 000384528900022

  View details for PubMedID 27647925

  View details for PubMedCentralID PMC5056096

• An intracellular compass spatially coordinates cell cycle modules in Caulobacter crescentus. Current opinion in microbiology Lasker, K., Mann, T. H., Shapiro, L. 2016; 33: 131-139

  Abstract

  Cellular functions in Bacteria, such as chromosome segregation and cytokinesis, result from cascades of molecular events operating largely as self-contained modules. Regulated timing of these cellular modules stems from global genetic circuits that allow precise temporal activation with respect to cell cycle progression and cell differentiation. Critically, many of these functions occur at defined locations within the cell, and therefore regulators of each module must communicate to remain coordinated in space. In this perspective, we highlight recent discoveries in Caulobacter crescentus asymmetric cell division to illuminate diverse mechanisms by which a cellular compass, composed of scaffolding and signaling proteins, directs cell cycle modules to their exact cellular addresses.

  View details for DOI 10.1016/j.mib.2016.06.007

  View details for PubMedID 27517351

  View details for PubMedCentralID PMC5069156

• Super-resolution Imaging of Live Bacteria Cells Using a Genetically Directed, Highly Photostable Fluoromodule. Journal of the American Chemical Society Saurabh, S., Perez, A. M., Comerci, C. J., Shapiro, L., Moerner, W. E. 2016; 138 (33): 10398-10401

  Abstract

  The rapid development in fluorescence microscopy and imaging techniques has greatly benefited our understanding of the mechanisms governing cellular processes at the molecular level. In particular, super-resolution microscopy methods overcome the diffraction limit to observe nanoscale cellular structures with unprecedented detail, and single-molecule tracking provides precise dynamic information about the motions of labeled proteins and oligonucleotides. Enhanced photostability of fluorescent labels (i.e., maximum emitted photons before photobleaching) is a critical requirement for achieving the ultimate spatio-temporal resolution with either method. While super-resolution imaging has greatly benefited from highly photostable fluorophores, a shortage of photostable fluorescent labels for bacteria has limited its use in these small but relevant organisms. In this study, we report the use of a highly photostable fluoromodule, dL5, to genetically label proteins in the Gram-negative bacterium Caulobacter crescentus, enabling long-time-scale protein tracking and super-resolution microscopy. dL5 imaging relies on the activation of the fluorogen Malachite Green (MG) and can be used to label proteins sparsely, enabling single-protein detection in live bacteria without initial bleaching steps. dL5-MG complexes emit 2-fold more photons before photobleaching compared to organic dyes such as Cy5 and Alexa 647 in vitro, and 5-fold more photons compared to eYFP in vivo. We imaged fusions of dL5 to three different proteins in live Caulobacter cells using stimulated emission depletion microscopy, yielding a 4-fold resolution enhancement compared to diffraction-limited imaging. Importantly, dL5 fusions to an intermediate filament protein CreS are significantly less perturbative compared to traditional fluorescent protein fusions. To the best of our knowledge, this is the first demonstration of the use of fluorogen activating proteins for super-resolution imaging in live bacterial cells.

  View details for DOI 10.1021/jacs.6b05943

  View details for PubMedID 27479076

  View details for PubMedCentralID PMC4996739

• A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues NATURE COMMUNICATIONS Mann, T. H., Childers, W. S., Blair, J. A., Eckart, M. R., Shapiro, L. 2016; 7

  Abstract

  All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation.

  View details for DOI 10.1038/ncomms11454

  View details for Web of Science ID 000374896400001

  View details for PubMedCentralID PMC4853435

• Three-Dimensional Super-Resolution Imaging of the RNA Degradation Machinery in Caulobacter Crescentus Bayas, C. A., Schrader, J. M., Lee, M. K., Shapiro, L., Moerner, W. E. CELL PRESS. 2016: 163A–164A

  View details for DOI 10.1016/j.bpj.2015.11.913

  View details for Web of Science ID 000375093800305

• CauloBrowser: A systems biology resource for Caulobacter crescentus. Nucleic acids research Lasker, K., Schrader, J. M., Men, Y., Marshik, T., Dill, D. L., McAdams, H. H., Shapiro, L. 2016; 44 (D1): D640-5

  Abstract

  Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of high-throughput spatiotemporal databases including data from gene expression profiling experiments (microarrays, RNA-seq, ChIP-seq, ribosome profiling, LC-ms proteomics), gene essentiality studies (Tn-seq), genome wide protein localization studies, and global chromosome methylation analyses (SMRT sequencing). A major challenge involves the integration of these diverse data sets into one comprehensive community resource. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource.

  View details for DOI 10.1093/nar/gkv1050

  View details for PubMedID 26476443

  View details for PubMedCentralID PMC4702786

• CauloBrowser: A systems biology resource for Caulobacter crescentus. Nucleic acids research Lasker, K., Schrader, J. M., Men, Y., Marshik, T., Dill, D. L., McAdams, H. H., Shapiro, L. 2016; 44 (D1): D640-5

  Abstract

  Caulobacter crescentus is a premier model organism for studying the molecular basis of cellular asymmetry. The Caulobacter community has generated a wealth of high-throughput spatiotemporal databases including data from gene expression profiling experiments (microarrays, RNA-seq, ChIP-seq, ribosome profiling, LC-ms proteomics), gene essentiality studies (Tn-seq), genome wide protein localization studies, and global chromosome methylation analyses (SMRT sequencing). A major challenge involves the integration of these diverse data sets into one comprehensive community resource. To address this need, we have generated CauloBrowser (www.caulobrowser.org), an online resource for Caulobacter studies. This site provides a user-friendly interface for quickly searching genes of interest and downloading genome-wide results. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. In addition, the site provides a genome viewer that enables customizable visualization of all published high-throughput genomic data. The depth and diversity of data sets collected by the Caulobacter community makes CauloBrowser a unique and valuable systems biology resource.

  View details for DOI 10.1093/nar/gkv1050

  View details for PubMedID 26476443

  View details for PubMedCentralID PMC4702786

• A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues. Nature communications Mann, T. H., Seth Childers, W., Blair, J. A., Eckart, M. R., Shapiro, L. 2016; 7: 11454-?

  Abstract

  All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation.

  View details for DOI 10.1038/ncomms11454

  View details for PubMedID 27117914

• Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Schrader, J. M., Shapiro, L. 2015

  Abstract

  The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

  View details for DOI 10.3791/52633

  View details for Web of Science ID 000361534800042

  View details for PubMedID 25938623

  View details for PubMedCentralID PMC4541484

• The global regulatory architecture of transcription during the Caulobacter cell cycle. PLoS genetics Zhou, B., Schrader, J. M., Kalogeraki, V. S., Abeliuk, E., Dinh, C. B., Pham, J. Q., Cui, Z. Z., Dill, D. L., McAdams, H. H., Shapiro, L. 2015; 11 (1)

  Abstract

  Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

  View details for DOI 10.1371/journal.pgen.1004831

  View details for PubMedID 25569173

  View details for PubMedCentralID PMC4287350

• A pseudokinase couples signaling pathways to enable asymmetric cell division in a bacterium MICROBIAL CELL Childers, W., Shapiro, L. 2015; 2 (1): 29–32

  Abstract

  Bacteria face complex decisions when initiating developmental events such as sporulation, nodulation, virulence, and asymmetric cell division. These developmental decisions require global changes in genomic readout, and bacteria typically employ intricate (yet poorly understood) signaling networks that enable changes in cell function. The bacterium Caulobacter crescentus divides asymmetrically to yield two functionally distinct cells: a motile, chemotactic swarmer cell, and a sessile stalked cell with replication and division capabilities. Work from several Caulobacter labs has revealed that differentiation requires concerted regulation by several two-component system (TCS) signaling pathways that are differentially positioned at the poles of the predivisional cell (Figure 1). The strict unidirectional flow from histidine kinase (HK) to the response regulator (RR), observed in most studied TCS, is difficult to reconcile with the notion that information can be transmitted between two or more TCS signaling pathways. In this study, we uncovered a mechanism by which daughter cell fate, which is specified by the DivJ-DivK-PleC system and effectively encoded in the phosphorylation state of the single-domain RR DivK, is communicated to the CckA-ChpT-CtrA signaling pathway that regulates more than 100 genes for polar differentiation, replication initiation and cell division. Using structural biology and biochemical findings we proposed a mechanistic basis for TCS pathway coupling in which the DivL pseudokinase is repurposed as a sensor rather than participant in phosphotransduction.

  View details for PubMedID 28357261

• The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle. PLoS genetics Zhou, B., Schrader, J. M., Kalogeraki, V. S., Abeliuk, E., Dinh, C. B., Pham, J. Q., Cui, Z. Z., Dill, D. L., McAdams, H. H., Shapiro, L. 2015; 11 (1)

  View details for DOI 10.1371/journal.pgen.1004831

  View details for PubMedID 25569173

• Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mera, P. E., Kalogeraki, V. S., Shapiro, L. 2014; 111 (45): 16100-16105

  Abstract

  During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.

  View details for DOI 10.1073/pnas.1418989111

  View details for Web of Science ID 000344526800061

  View details for PubMedCentralID PMC4234595

• Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation. Proceedings of the National Academy of Sciences of the United States of America Mera, P. E., Kalogeraki, V. S., Shapiro, L. 2014; 111 (45): 16100-5

  Abstract

  During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.

  View details for DOI 10.1073/pnas.1418989111

  View details for PubMedID 25349407

  View details for PubMedCentralID PMC4234595

• Cell Fate Regulation Governed by a Repurposed Bacterial Histidine Kinase PLOS BIOLOGY Childers, W. S., Xu, Q., Mann, T. H., Mathews, I. I., Blair, J. A., Deacon, A. M., Shapiro, L. 2014; 12 (10)

  Abstract

  One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  View details for DOI 10.1371/journal.pbio.1001979

  View details for Web of Science ID 000344461700017

  View details for PubMedCentralID PMC4211667

• Cell fate regulation governed by a repurposed bacterial histidine kinase. PLoS biology Childers, W. S., Xu, Q., Mann, T. H., Mathews, I. I., Blair, J. A., Deacon, A. M., Shapiro, L. 2014; 12 (10)

  Abstract

  One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  View details for DOI 10.1371/journal.pbio.1001979

  View details for PubMedID 25349992

• ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division MOLECULAR MICROBIOLOGY Williams, B., Bhat, N., Chien, P., Shapiro, L. 2014; 93 (5): 853-866

  Abstract

  Proteolytic control of Caulobacter cell cycle proteins is primarily executed by ClpXP, a dynamically localized protease implicated in turnover of several factors critical for faithful cell cycle progression. Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAP in vivo and in vitro. A peptide containing the C-terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAP in vitro but is primarily degraded by ClpAP in vivo. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non-replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. Taken together, these results suggest that the activity of both ClpXP and ClpAP on divisome substrates is differentially regulated in daughter cells.

  View details for DOI 10.1111/mmi.12698

  View details for Web of Science ID 000341639600002

  View details for PubMedID 24989075

• The coding and noncoding architecture of the Caulobacter crescentus genome. PLoS genetics Schrader, J. M., Zhou, B., Li, G., Lasker, K., Childers, W. S., Williams, B., Long, T., Crosson, S., McAdams, H. H., Weissman, J. S., Shapiro, L. 2014; 10 (7)

  Abstract

  Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. We provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5'-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5'-RACE. Additionally, using ribosome profiling and LC-MS, we mapped translation start sites and coding regions with near complete coverage. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the coding DNA sequence (CDS). These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Overall 19% of the transcribed and translated genomic elements were newly identified or significantly improved by this approach, providing a valuable genomic resource to elucidate the complete C. crescentus genetic circuitry that controls asymmetric cell division.

  View details for DOI 10.1371/journal.pgen.1004463

  View details for PubMedID 25078267

  View details for PubMedCentralID PMC4117421

• The Coding and Noncoding Architecture of the Caulobacter crescentus Genome. PLoS genetics Schrader, J. M., Zhou, B., Li, G., Lasker, K., Childers, W. S., Williams, B., Long, T., Crosson, S., McAdams, H. H., Weissman, J. S., Shapiro, L. 2014; 10 (7)

  View details for DOI 10.1371/journal.pgen.1004463

  View details for PubMedID 25078267

• Bacterial scaffold directs pole-specific centromere segregation. Proceedings of the National Academy of Sciences of the United States of America Ptacin, J. L., Gahlmann, A., Bowman, G. R., Perez, A. M., von Diezmann, A. R., Eckart, M. R., Moerner, W. E., Shapiro, L. 2014; 111 (19): E2046-55

  Abstract

  Bacteria use partitioning systems based on the ParA ATPase to actively mobilize and spatially organize molecular cargoes throughout the cytoplasm. The bacterium Caulobacter crescentus uses a ParA-based partitioning system to segregate newly replicated chromosomal centromeres to opposite cell poles. Here we demonstrate that the Caulobacter PopZ scaffold creates an organizing center at the cell pole that actively regulates polar centromere transport by the ParA partition system. As segregation proceeds, the ParB-bound centromere complex is moved by progressively disassembling ParA from a nucleoid-bound structure. Using superresolution microscopy, we show that released ParA is recruited directly to binding sites within a 3D ultrastructure composed of PopZ at the cell pole, whereas the ParB-centromere complex remains at the periphery of the PopZ structure. PopZ recruitment of ParA stimulates ParA to assemble on the nucleoid near the PopZ-proximal cell pole. We identify mutations in PopZ that allow scaffold assembly but specifically abrogate interactions with ParA and demonstrate that PopZ/ParA interactions are required for proper chromosome segregation in vivo. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. PopZ therefore functions as a polar hub complex at the cell pole to directly regulate the directionality and destination of transfer of the mitotic segregation machine.

  View details for DOI 10.1073/pnas.1405188111

  View details for PubMedID 24778223

• The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach. Nucleic acids research Gonzalez, D., Kozdon, J. B., McAdams, H. H., Shapiro, L., Collier, J. 2014; 42 (6): 3720-3735

  Abstract

  DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

  View details for DOI 10.1093/nar/gkt1352

  View details for PubMedID 24398711

  View details for PubMedCentralID PMC3973325

• Systems architecture of a bacterial cell cycle Shapiro, L. FEDERATION AMER SOC EXP BIOL. 2014

  View details for Web of Science ID 000346646705186

• Ribosome Profiling of the Caulobacter Cell-Cycle 58th Annual Meeting of the Biophysical-Society Schrader, J. M., Li, G., Zhou, B., Weissman, J. S., Shapiro, L. CELL PRESS. 2014: 375A–376A

  View details for Web of Science ID 000337000402130

• Quantifying the Spatial Organization of Bacterial Ribosomes using Three-Dimensional Super-Resolution Microscopy Lee, M., Schrader, J., Li, G., Weissman, J., McAdams, H., Shapiro, L., Moerner, W. E. CELL PRESS. 2014: 492A

  View details for DOI 10.1016/j.bpj.2013.11.2753

  View details for Web of Science ID 000337000402726

• DNA Segregation and Partitioning in Caulobacter Crescentus: Super-Resolving Protein Colocalization at the Cell Pole Gahlmann, A., Ptacin, J. L., von Diezmann, A. S., Shapiro, L., Moerner, W. E. CELL PRESS. 2014: 59A–60A

  View details for DOI 10.1016/j.bpj.2013.11.408

  View details for Web of Science ID 000337000400306

• Quantitative Registration and Distribution Analysis of Multicolor 3D Super-Resolution Images of Proteins Reveals Nanoscale Spatial Organization von Diezmann, A. S., Gahlmann, A., Ptacin, J. L., Shapiro, L., Moerner, W. E. CELL PRESS. 2014: 203A

  View details for DOI 10.1016/j.bpj.2013.11.1192

  View details for Web of Science ID 000337000401138

• A Novel Function of the Bacterial Replication Initiator Protein DnaA Mera, P. E., Kalogeraki, V., Shapiro, L. CELL PRESS. 2014: 271A

  View details for Web of Science ID 000337000401495

• Unique Signaling Logic within a Bacterial Cell Cycle Circuit Childers, W., Xu, Q., Mathews, I. I., Mann, T. H., Blair, J. A., Deacon, A. M., Shapiro, L. CELL PRESS. 2014: 309A

  View details for DOI 10.1016/j.bpj.2013.11.1789

  View details for Web of Science ID 000337000401688

• Using Optically Reversible Spatial Mutations to Dissect the Asymmetric Developmental Program of a Bacterium Lasker, K., Abraham, A., Childers, W., Shapiro, L. CELL PRESS. 2014: 594A

  View details for DOI 10.1016/j.bpj.2013.11.3287

  View details for Web of Science ID 000337000403342

• Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proceedings of the National Academy of Sciences of the United States of America Kozdon, J. B., Melfi, M. D., Luong, K., Clark, T. A., Boitano, M., Wang, S., Zhou, B., Gonzalez, D., Collier, J., Turner, S. W., Korlach, J., Shapiro, L., McAdams, H. H. 2013; 110 (48): E4658-67

  View details for DOI 10.1073/pnas.1319315110

  View details for PubMedID 24218615

• Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle. Proceedings of the National Academy of Sciences of the United States of America Kozdon, J. B., Melfi, M. D., Luong, K., Clark, T. A., Boitano, M., Wang, S., Zhou, B., Gonzalez, D., Collier, J., Turner, S. W., Korlach, J., Shapiro, L., McAdams, H. H. 2013; 110 (48): E4658-67

  Abstract

  The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N(6)-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.

  View details for DOI 10.1073/pnas.1319315110

  View details for PubMedID 24218615

• Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold. Molecular microbiology Bowman, G. R., Perez, A. M., Ptacin, J. L., Ighodaro, E., Folta-Stogniew, E., Comolli, L. R., Shapiro, L. 2013; 90 (4): 776-795

  Abstract

  In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub-cellular localization and recruitment activity. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C-terminal domain also blocked discrete steps in the assembly of higher-order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub-cellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.

  View details for DOI 10.1111/mmi.12398

  View details for PubMedID 24102805

  View details for PubMedCentralID PMC3859194

• Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein. Structure Blair, J. A., Xu, Q., Childers, W. S., Mathews, I. I., Kern, J. W., Eckart, M., Deacon, A. M., Shapiro, L. 2013; 21 (9): 1590-1601

  Abstract

  Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus, ChpT is an essential mediator within the cell-cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (CtrA or CpdR) that controls cell-cycle progression. To understand how ChpT interacts with multiple signaling partners, we solved the crystal structure of ChpT at 2.3 Å resolution. ChpT adopts a pseudo-HK architecture but does not bind ATP. We identified two point mutation classes affecting phosphotransfer and cell morphology: one that globally impairs ChpT phosphotransfer, and a second that mediates partner selection. Importantly, a small set of conserved ChpT residues promotes signaling crosstalk and contributes to the branched signaling that activates the master regulator CtrA while inactivating the CtrA degradation signal, CpdR.

  View details for DOI 10.1016/j.str.2013.06.024

  View details for PubMedID 23932593

  View details for PubMedCentralID PMC3787845

• Retrospective. Francois Jacob (1920-2013). Science Shapiro, L., Losick, R. 2013; 340 (6135): 939-?

  View details for DOI 10.1126/science.1239975

  View details for PubMedID 23704565

• Francois Jacob (1920-2013) SCIENCE Shapiro, L., Losick, R. 2013; 340 (6135): 939-939

  View details for DOI 10.1126/science.1239975

  View details for Web of Science ID 000319344100035

• Quantitative Multicolor Subdiffraction Imaging of Bacterial Protein Ultrastructures in Three Dimensions NANO LETTERS Gahlmann, A., Ptacin, J. L., Grover, G., Quirin, S., von Diezmann, A. R., Lee, M. K., Backlund, M. P., Shapiro, L., Piestun, R., Moerner, W. E. 2013; 13 (3): 987-993

  Abstract

  We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D.

  View details for DOI 10.1021/nl304071h

  View details for Web of Science ID 000316243800020

  View details for PubMedID 23414562

  View details for PubMedCentralID PMC3599789

• Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hong, S., Toro, E., Mortensen, K. I., de la Rosa, M. A., Doniach, S., Shapiro, L., Spakowitz, A. J., McAdams, H. H. 2013; 110 (5): 1674-1679

  Abstract

  We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 μm. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

  View details for DOI 10.1073/pnas.1220824110

  View details for Web of Science ID 000314558100027

  View details for PubMedID 23319648

  View details for PubMedCentralID PMC3562846

• Chromosome architecture is a key element of bacterial cellular organization CELLULAR MICROBIOLOGY Ptacin, J. L., Shapiro, L. 2013; 15 (1): 45-52

  Abstract

  The bacterial chromosome encodes information at multiple levels. Beyond direct protein coding, genomes encode regulatory information required to orchestrate the proper timing and levels of gene expression and protein synthesis, and contain binding sites and regulatory sequences to co-ordinate the activities of proteins involved in chromosome repair and maintenance. In addition, it is becoming increasingly clear that yet another level of information is encoded by the bacterial chromosome - the three-dimensional packaging of the chromosomal DNA molecule itself and its positioning relative to the cell. This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. Recent studies have begun to identify and characterize novel systems that utilize the three-dimensional spatial information encoded by chromosomal architecture to co-ordinate and direct fundamental cellular processes within the cytoplasm, providing large-scale order within the complex clutter of the cytoplasmic compartment.

  View details for DOI 10.1111/cmi.12049

  View details for Web of Science ID 000314586600005

  View details for PubMedID 23078580

  View details for PubMedCentralID PMC3660146

• Deciphering the Transcriptional Landscape of Caulobacter crescentus at Base Pair Resolution 11th International Conference on Computational Methods in Systems Biology (CMSB) Zhou, B., Schrader, J., Christen, B., McAdams, H., Shapiro, L. SPRINGER-VERLAG BERLIN. 2013: 247–247

  View details for Web of Science ID 000342772500028

• Life in a Three-dimensional Grid JOURNAL OF BIOLOGICAL CHEMISTRY Shapiro, L. 2012; 287 (45)

  Abstract

  There have been two sharp demarcations in my life in science: the transition from fine arts to chemistry, which happened early in my career, and the move from New York to Stanford University, which initiated an ongoing collaboration with the physicist Harley McAdams. Both had a profound effect on the kinds of questions I posed and the means I used to arrive at answers. The outcome of these experiences, together with the extraordinary scientists I came to know along the way, was and is an abiding passion to fully understand a simple cell in all its complexity and beauty.

  View details for DOI 10.1074/jbc.X112.422337

  View details for Web of Science ID 000310642200061

  View details for PubMedID 23007401

  View details for PubMedCentralID PMC3488097

• Osmolality-Dependent Relocation of Penicillin-Binding Protein PBP2 to the Division Site in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Hocking, J., Priyadarshini, R., Takacs, C. N., Costa, T., Dye, N. A., Shapiro, L., Vollmer, W., Jacobs-Wagner, C. 2012; 194 (12): 3116-3127

  Abstract

  The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.

  View details for DOI 10.1128/JB.00260-12

  View details for Web of Science ID 000304978400010

  View details for PubMedID 22505677

  View details for PubMedCentralID PMC3370875

• Three-Dimensional Super-Resolution Imaging of the Midplane Protein FtsZ in Live Caulobacter crescentus Cells Using Astigmatism CHEMPHYSCHEM Biteen, J. S., Goley, E. D., Shapiro, L., Moerner, W. E. 2012; 13 (4): 1007-1012

  Abstract

  Single-molecule super-resolution imaging provides a non-invasive method for nanometer-scale imaging and is ideally suited to investigations of quasi-static structures within live cells. Here, we extend the ability to image subcellular features within bacteria cells to three dimensions based on the introduction of a cylindrical lens in the imaging pathway. We investigate the midplane protein FtsZ in Caulobacter crescentus with super-resolution imaging based on fluorescent-protein photoswitching and the natural polymerization/depolymerization dynamics of FtsZ associated with the Z-ring. We quantify these dynamics and determine the FtsZ depolymerization time to be <100 ms. We image the Z-ring in live and fixed C. crescentus cells at different stages of the cell cycle and find that the FtsZ superstructure is dynamic with the cell cycle, forming an open shape during the stalked stage and a dense focus during the pre-divisional stage.

  View details for DOI 10.1002/cphc.201100686

  View details for Web of Science ID 000301537300020

  View details for PubMedID 22262316

• Structure of the pilus assembly protein TadZ from Eubacterium rectale: implications for polar localization MOLECULAR MICROBIOLOGY Xu, Q., Christen, B., Chiu, H., Jaroszewski, L., Klock, H. E., Knuth, M. W., Miller, M. D., Elsliger, M., Deacon, A. M., Godzik, A., Lesley, S. A., Figurski, D. H., Shapiro, L., Wilson, I. A. 2012; 83 (4): 712-727

  Abstract

  The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from Eubacterium rectale (ErTadZ), in complex with ATP and Mg(2+) , was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down 'active site'. Homology modelling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during an early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.

  View details for DOI 10.1111/j.1365-2958.2011.07954.x

  View details for Web of Science ID 000299779200005

  View details for PubMedID 22211578

  View details for PubMedCentralID PMC3272108

• An SMC ATPase mutant disrupts chromosome segregation in Caulobacter MOLECULAR MICROBIOLOGY Schwartz, M. A., Shapiro, L. 2011; 82 (6): 1359-1374

  Abstract

  Accurate replication and segregation of the bacterial genome are essential for cell cycle progression. We have identified a single amino acid substitution in the Caulobacter structural maintenance of chromosomes (SMC) protein that disrupts chromosome segregation and cell division. The E1076Q point mutation in the SMC ATPase domain caused a dominant-negative phenotype in which DNA replication was able to proceed, but duplicated parS centromeres, normally found at opposite cell poles, remained at one pole. The cellular positions of other chromosomal loci were in the wild-type order relative to the parS centromere, but chromosomes remained unsegregated and appeared to be stacked upon one another. Purified SMC-E1076Q was deficient in ATP hydrolysis and exhibited abnormally stable binding to DNA. We propose that SMC spuriously links the duplicated chromosome immediately after passage of the replication fork. In wild-type cells, ATP hydrolysis opens the SMC dimer, freeing one chromosome to segregate to the opposite pole. The loss of ATP hydrolysis causes the SMC-E1076Q dimer to remain bound to both chromosomes, inhibiting segregation.

  View details for DOI 10.1111/j.1365-2958.2011.07836.x

  View details for Web of Science ID 000298087300007

  View details for PubMedID 21923769

  View details for PubMedCentralID PMC3273039

• Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lew, M. D., Lee, S. F., Ptacin, J. L., Lee, M. K., Twieg, R. J., Shapiro, L., Moerner, W. E. 2011; 108 (46): E1102-E1110

  Abstract

  Recently, single-molecule imaging and photocontrol have enabled superresolution optical microscopy of cellular structures beyond Abbe's diffraction limit, extending the frontier of noninvasive imaging of structures within living cells. However, live-cell superresolution imaging has been challenged by the need to image three-dimensional (3D) structures relative to their biological context, such as the cellular membrane. We have developed a technique, termed superresolution by power-dependent active intermittency and points accumulation for imaging in nanoscale topography (SPRAIPAINT) that combines imaging of intracellular enhanced YFP (eYFP) fusions (SPRAI) with stochastic localization of the cell surface (PAINT) to image two different fluorophores sequentially with only one laser. Simple light-induced blinking of eYFP and collisional flux onto the cell surface by Nile red are used to achieve single-molecule localizations, without any antibody labeling, cell membrane permeabilization, or thiol-oxygen scavenger systems required. Here we demonstrate live-cell 3D superresolution imaging of Crescentin-eYFP, a cytoskeletal fluorescent protein fusion, colocalized with the surface of the bacterium Caulobacter crescentus using a double-helix point spread function microscope. Three-dimensional colocalization of intracellular protein structures and the cell surface with superresolution optical microscopy opens the door for the analysis of protein interactions in living cells with excellent precision (20-40 nm in 3D) over a large field of view (12 12 μm).

  View details for DOI 10.1073/pnas.1114444108

  View details for PubMedID 22031697

• The Three-Dimensional Architecture of a Bacterial Genome and Its Alteration by Genetic Perturbation MOLECULAR CELL Umbarger, M. A., Toro, E., Wright, M. A., Porreca, G. J., Bau, D., Hong, S., Fero, M. J., Zhu, L. J., Marti-Renom, M. A., McAdams, H. H., Shapiro, L., Dekker, J., Church, G. M. 2011; 44 (2): 252-264

  Abstract

  We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.

  View details for DOI 10.1016/j.molcel.2011.09.010

  View details for Web of Science ID 000296212100011

  View details for PubMedID 22017872

• Live-cell single-molecule and super-resolution imaging in bacteria Coupland, B., Haas, B. L., Hoye, E., Koropatkin, N., Matson, J., DiRita, V., Martens, E., Shapiro, L., Moerner, W. E., Biteen, J. S. AMER CHEMICAL SOC. 2011

  View details for Web of Science ID 000299378306327

• The essential genome of a bacterium MOLECULAR SYSTEMS BIOLOGY Christen, B., Abeliuk, E., Collier, J. M., Kalogeraki, V. S., Passarelli, B., Coller, J. A., Fero, M. J., McAdams, H. H., Shapiro, L. 2011; 7

  Abstract

  Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.

  View details for DOI 10.1038/msb.2011.58

  View details for Web of Science ID 000294537800007

  View details for PubMedID 21878915

  View details for PubMedCentralID PMC3202797

• Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter MOLECULAR MICROBIOLOGY Dye, N. A., Pincus, Z., Fisher, I. C., Shapiro, L., Theriot, J. A. 2011; 81 (2): 368-394

  Abstract

  The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape.

  View details for DOI 10.1111/j.1365-2958.2011.07698.x

  View details for Web of Science ID 000292567200009

  View details for PubMedID 21564339

  View details for PubMedCentralID PMC3137890

• Assembly of the Caulobacter cell division machine MOLECULAR MICROBIOLOGY Goley, E. D., Yeh, Y., Hong, S., Fero, M. J., Abeliuk, E., McAdams, H. H., Shapiro, L. 2011; 80 (6): 1680-1698

  Abstract

  Cytokinesis in Gram-negative bacteria is mediated by a multiprotein machine (the divisome) that invaginates and remodels the inner membrane, peptidoglycan and outer membrane. Understanding the order of divisome assembly would inform models of the interactions among its components and their respective functions. We leveraged the ability to isolate synchronous populations of Caulobacter crescentus cells to investigate assembly of the divisome and place the arrival of each component into functional context. Additionally, we investigated the genetic dependence of localization among divisome proteins and the cell cycle regulation of their transcript and protein levels to gain insight into the control mechanisms underlying their assembly. Our results revealed a picture of divisome assembly with unprecedented temporal resolution. Specifically, we observed (i) initial establishment of the division site, (ii) recruitment of early FtsZ-binding proteins, (iii) arrival of proteins involved in peptidoglycan remodelling, (iv) arrival of FtsA, (v) assembly of core divisome components, (vi) initiation of envelope invagination, (vii) recruitment of polar markers and cytoplasmic compartmentalization and (viii) cell separation. Our analysis revealed differences in divisome assembly among Caulobacter and other bacteria that establish a framework for identifying aspects of bacterial cytokinesis that are widely conserved from those that are more variable.

  View details for DOI 10.1111/j.1365-2958.2011.07677.x

  View details for Web of Science ID 000292106100020

  View details for PubMedID 21542856

• The Architecture and Conservation Pattern of Whole-Cell Control Circuitry JOURNAL OF MOLECULAR BIOLOGY McAdams, H. H., Shapiro, L. 2011; 409 (1): 28-35

  Abstract

  The control circuitry that directs and paces Caulobacter cell cycle progression involves the entire cell operating as an integrated system. This control circuitry monitors the environment and the internal state of the cell, including the cell topology, as it orchestrates orderly activation of cell cycle subsystems and Caulobacter's asymmetric cell division. The proteins of the Caulobacter cell cycle control system and its internal organization are co-conserved across many alphaproteobacteria species, but there are great differences in the regulatory apparatus' functionality and peripheral connectivity to other cellular subsystems from species to species. This pattern is similar to that observed for the "kernels" of the regulatory networks that regulate development of metazoan body plans. The Caulobacter cell cycle control system has been exquisitely optimized as a total system for robust operation in the face of internal stochastic noise and environmental uncertainty. When sufficient details accumulate, as for Caulobacter cell cycle regulation, the system design has been found to be eminently rational and indeed consistent with good design practices for human-designed asynchronous control systems.

  View details for DOI 10.1016/j.jmb.2011.02.041

  View details for Web of Science ID 000290779500004

  View details for PubMedID 21371478

  View details for PubMedCentralID PMC3108490

• Regulatory Response to Carbon Starvation in Caulobacter crescentus PLOS ONE Britos, L., Abeliuk, E., Taverner, T., Lipton, M., McAdams, H., Shapiro, L. 2011; 6 (4)

  Abstract

  Bacteria adapt to shifts from rapid to slow growth, and have developed strategies for long-term survival during prolonged starvation and stress conditions. We report the regulatory response of C. crescentus to carbon starvation, based on combined high-throughput proteome and transcriptome analyses. Our results identify cell cycle changes in gene expression in response to carbon starvation that involve the prominent role of the FixK FNR/CAP family transcription factor and the CtrA cell cycle regulator. Notably, the SigT ECF sigma factor mediates the carbon starvation-induced degradation of CtrA, while activating a core set of general starvation-stress genes that respond to carbon starvation, osmotic stress, and exposure to heavy metals. Comparison of the response of swarmer cells and stalked cells to carbon starvation revealed four groups of genes that exhibit different expression profiles. Also, cell pole morphogenesis and initiation of chromosome replication normally occurring at the swarmer-to-stalked cell transition are uncoupled in carbon-starved cells.

  View details for DOI 10.1371/journal.pone.0018179

  View details for Web of Science ID 000289354100006

  View details for PubMedID 21494595

  View details for PubMedCentralID PMC3073932

• Super-Resolution Imaging of the Nucleoid-Associated Protein HU in Caulobacter crescentus BIOPHYSICAL JOURNAL Lee, S. F., Thompson, M. A., Schwartz, M. A., Shapiro, L., Moerner, W. E. 2011; 100 (7): L31-L33

  Abstract

  Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.

  View details for DOI 10.1016/j.bpj.2011.02.022

  View details for Web of Science ID 000289494200001

  View details for PubMedID 21463569

  View details for PubMedCentralID PMC3072666

• Bacterial polarity CURRENT OPINION IN CELL BIOLOGY Bowman, G. R., Lyuksyutova, A. I., Shapiro, L. 2011; 23 (1): 71-77

  Abstract

  Many recent studies have revealed exquisite subcellular localization of proteins, DNA, and other molecules within bacterial cells, giving credence to the concept of prokaryotic anatomy. Common sites for localized components are the poles of rod-shaped cells, which are dynamically modified in composition and function in order to control cellular physiology. An impressively diverse array of mechanisms underlies bacterial polarity, including oscillatory systems, phospho-signaling pathways, the sensing of membrane curvature, and the integration of cell cycle regulators with polar maturation.

  View details for DOI 10.1016/j.ceb.2010.10.013

  View details for Web of Science ID 000288349000010

  View details for PubMedID 21095111

• Assembly of a bacterial cell division machine. Annual Meeting of the American-Society-for-Cell-Biology (ASCB) Goley, E. D., Yeh, Y., McAdams, H., Shapiro, L. AMER SOC CELL BIOLOGY. 2011

  View details for Web of Science ID 000305505503547

• The role of a bacterial SMC in chromosome segregation Annual Meeting of the American-Society-for-Cell-Biology (ASCB) Schwartz, M., Shapiro, L. AMER SOC CELL BIOLOGY. 2011

  View details for Web of Science ID 000305505504226

• Exploring protein superstructures and dynamics in live bacterial cells using single-molecule and superresolution imaging. Methods in molecular biology (Clifton, N.J.) Biteen, J. S., Shapiro, L., Moerner, W. E. 2011; 783: 139-158

  Abstract

  Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the optical diffraction limit, and enables superresolution reconstruction of structures beyond the diffraction limit. This work summarizes how single-molecule and superresolution imaging can be applied to the study of protein dynamics and superstructures in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells.

  View details for DOI 10.1007/978-1-61779-282-3_8

  View details for PubMedID 21909887

• Superresolution Imaging of Targeted Proteins in Fixed and Living Cells Using Photoactivatable Organic Fluorophores JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lee, H. D., Lord, S. J., Iwanaga, S., Zhan, K., Xie, H., Williams, J. C., Wang, H., Bowman, G. R., Goley, E. D., Shapiro, L., Twieg, R. J., Rao, J., Moerner, W. E. 2010; 132 (43): 15099-15101

  Abstract

  Superresolution imaging techniques based on sequential imaging of sparse subsets of single molecules require fluorophores whose emission can be photoactivated or photoswitched. Because typical organic fluorophores can emit significantly more photons than average fluorescent proteins, organic fluorophores have a potential advantage in super-resolution imaging schemes, but targeting to specific cellular proteins must be provided. We report the design and application of HaloTag-based target-specific azido DCDHFs, a class of photoactivatable push-pull fluorogens which produce bright fluorescent labels suitable for single-molecule superresolution imaging in live bacterial and fixed mammalian cells.

  View details for DOI 10.1021/ja1044192

  View details for PubMedID 20936809

• An essential transcription factor, SciP, enhances robustness of Caulobacter cell cycle regulation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tan, M. H., Kozdon, J. B., Shen, X., Shapiro, L., McAdams, H. H. 2010; 107 (44): 18985-18990

  Abstract

  A cyclical control circuit composed of four master regulators drives the Caulobacter cell cycle. We report that SciP, a helix-turn-helix transcription factor, is an essential component of this circuit. SciP is cell cycle-controlled and co-conserved with the global cell cycle regulator CtrA in the α-proteobacteria. SciP is expressed late in the cell cycle and accumulates preferentially in the daughter swarmer cell. At least 58 genes, including many flagellar and chemotaxis genes, are regulated by a type 1 incoherent feedforward motif in which CtrA activates sciP, followed by SciP repression of ctrA and CtrA target genes. We demonstrate that SciP binds to DNA at a motif distinct from the CtrA binding motif that is present in the promoters of genes co-regulated by SciP and CtrA. SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. The type 1 incoherent feedforward circuit motif enhances the pulse-like expression of the downstream genes, and the negative feedback to ctrA expression reduces peak CtrA accumulation. The presence of SciP in the control network enhances the robustness of the cell cycle to varying growth rates.

  View details for DOI 10.1073/pnas.1014395107

  View details for Web of Science ID 000283749000046

  View details for PubMedID 20956288

  View details for PubMedCentralID PMC2973855

• Initiating bacterial mitosis Understanding the mechanism of ParA-mediated chromosome segregation CELL CYCLE Ptacin, J. L., Shapiro, L. 2010; 9 (20): 4033-4034

  View details for DOI 10.4161/cc.9.20.13521

  View details for Web of Science ID 000283058300001

  View details for PubMedID 20948303

• The Caulobacter Tol-Pal Complex Is Essential for Outer Membrane Integrity and the Positioning of a Polar Localization Factor JOURNAL OF BACTERIOLOGY Yeh, Y., Comolli, L. R., Downing, K. H., Shapiro, L., McAdams, H. H. 2010; 192 (19): 4847-4858

  Abstract

  Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscopy images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor.

  View details for DOI 10.1128/JB.00607-10

  View details for Web of Science ID 000281866900006

  View details for PubMedID 20693330

  View details for PubMedCentralID PMC2944545

• Imaging-Based Identification of a Critical Regulator of FtsZ Protofilament Curvature in Caulobacter MOLECULAR CELL Goley, E. D., Dye, N. A., Werner, J. N., Gitai, Z., Shapiro, L. 2010; 39 (6): 975-987

  Abstract

  FtsZ is an essential bacterial GTPase that polymerizes at midcell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. However, many of the mechanistic details underlying these functions are unknown. We sought to identify FtsZ-binding proteins that influence FtsZ function in Caulobacter crescentus. Here, we present a microscopy-based screen through which we discovered two FtsZ-binding proteins, FzlA and FzlC. FzlA is conserved in α-proteobacteria and was found to be functionally critical for cell division in Caulobacter. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher-order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. Induction of FtsZ curvature by FzlA carries implications for regulating FtsZ function by modulating its superstructure.

  View details for DOI 10.1016/j.molcel.2010.08.027

  View details for Web of Science ID 000282377200016

  View details for PubMedID 20864042

  View details for PubMedCentralID PMC2945607

• CrfA, a small noncoding RNA regulator of adaptation to carbon starvation in Caulobacter crescentus. Journal of bacteriology Landt, S. G., Lesley, J. A., Britos, L., Shapiro, L. 2010; 192 (18): 4763-4775

  Abstract

  Small noncoding regulatory RNAs (sRNAs) play a key role in the posttranscriptional regulation of many bacterial genes. The genome of Caulobacter crescentus encodes at least 31 sRNAs, and 27 of these sRNAs are of unknown function. An overexpression screen for sRNA-induced growth inhibition along with sequence conservation in a related Caulobacter species led to the identification of a novel sRNA, CrfA, that is specifically induced upon carbon starvation. Twenty-seven genes were found to be strongly activated by CrfA accumulation. One-third of these target genes encode putative TonB-dependent receptors, suggesting CrfA plays a role in the surface modification of C. crescentus, facilitating the uptake of nutrients during periods of carbon starvation. The mechanism of CrfA-mediated gene activation was investigated for one of the genes predicted to encode a TonB-dependent receptor, CC3461. CrfA functions to stabilize the CC3461 transcript. Complementarity between a region of CrfA and the terminal region of the CC3461 5'-untranslated region (5'-UTR) and also the behavior of a deletion of this region and a site-specific base substitution and a 3-base deletion in the CrfA complementary sequence suggest that CrfA binds to a stem-loop structure upstream of the CC3461 Shine-Dalgarno sequence and stabilizes the transcript.

  View details for DOI 10.1128/JB.00343-10

  View details for PubMedID 20601471

  View details for PubMedCentralID PMC2937403

• A spindle-like apparatus guides bacterial chromosome segregation NATURE CELL BIOLOGY Ptacin, J. L., Lee, S. F., Garner, E. C., Toro, E., Eckart, M., Comolli, L. R., Moerner, W., Shapiro, L. 2010; 12 (8): 791-U46

  Abstract

  Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

  View details for DOI 10.1038/ncb2083

  View details for Web of Science ID 000280561600011

  View details for PubMedID 20657594

  View details for PubMedCentralID PMC3205914

• Polar Remodeling and Histidine Kinase Activation, Which Is Essential for Caulobacter Cell Cycle Progression, Are Dependent on DNA Replication Initiation JOURNAL OF BACTERIOLOGY Iniesta, A. A., Hillson, N. J., Shapiro, L. 2010; 192 (15): 3893-3902

  Abstract

  Caulobacter crescentus initiates a single round of DNA replication during each cell cycle. Following the initiation of DNA replication, the essential CckA histidine kinase is activated by phosphorylation, which (via the ChpT phosphotransferase) enables the phosphorylation and activation of the CtrA global regulator. CtrA approximately P then blocks the reinitiation of replication while regulating the transcription of a large number of cell cycle-controlled genes. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Because CckA approximately P promotes the activation of CtrA, we addressed the question of what controls the temporal activation of CckA. We found that the initiation of DNA replication is a prerequisite for remodeling the new cell pole, which includes the localization of the DivL protein kinase to that pole and, consequently, the localization, autophosphorylation, and activation of CckA at that pole. Thus, CckA activation is dependent on polar remodeling and a DNA replication initiation checkpoint that is tightly integrated with the polar phospho-signaling cascade governing cell cycle progression.

  View details for DOI 10.1128/JB.00468-10

  View details for Web of Science ID 000279782000006

  View details for PubMedID 20525830

  View details for PubMedCentralID PMC2916389

• DipM links peptidoglycan remodelling to outer membrane organization in Caulobacter MOLECULAR MICROBIOLOGY Goley, E. D., Comolli, L. R., Fero, K. E., Downing, K. H., Shapiro, L. 2010; 77 (1): 56-73

  Abstract

  Cell division in Gram-negative organisms requires coordinated invagination of the multilayered cell envelope such that each daughter receives an intact inner membrane, peptidoglycan (PG) layer and outer membrane (OM). Here, we identify DipM, a putative LytM endopeptidase in Caulobacter crescentus, and show that it plays a critical role in maintaining cell envelope architecture during growth and division. DipM localized to the division site in an FtsZ-dependent manner via its PG-binding LysM domains. Although not essential for viability, DeltadipM cells exhibited gross morphological defects, including cell widening and filamentation, indicating a role in cell shape maintenance and division that we show requires its LytM domain. Strikingly, cells lacking DipM also showed OM blebbing at the division site, at cell poles and along the cell body. Cryo electron tomography of sacculi isolated from cells depleted of DipM revealed marked thickening of the PG as compared to wild type, which we hypothesize leads to loss of trans-envelope contacts between components of the Tol-Pal complex. We conclude that DipM is required for normal envelope invagination during division and to maintain a sacculus of constant thickness that allows for maintenance of OM connections throughout the cell envelope.

  View details for DOI 10.1111/j.1365-2958.2010.07222.x

  View details for Web of Science ID 000279168200007

  View details for PubMedID 20497504

  View details for PubMedCentralID PMC2915588

• Cell pole-specific activation of a critical bacterial cell cycle kinase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iniesta, A. A., Hillson, N. J., Shapiro, L. 2010; 107 (15): 7012-7017

  Abstract

  Caulobacter crescentus integrates phospho-signaling pathways and transcription factor regulatory cascades to drive the cell cycle. Despite the essential role of the CckA histidine kinase in the control of cell cycle events, the factors that signal its activation at a specific time in the cell cycle have remained elusive. A conditional genetic screen for CckA mislocalization mutants, using automated fluorescence microscopy and an image processing platform, revealed that the essential DivL protein kinase promotes CckA localization, autophosphorylation, and activity at the new cell pole. The transient accumulation of DivL at the new cell pole, but not its kinase activity, is required for the localization and activation of CckA. Because DivL and CckA accumulate at the same cell pole after the initiation of DNA replication and were found to interact in vivo, we propose that DivL recruits CckA to the pole, thereby promoting its autophosphorylation and activity.

  View details for DOI 10.1073/pnas.1001767107

  View details for Web of Science ID 000276642100081

  View details for PubMedID 20351295

  View details for PubMedCentralID PMC2872457

• Caulobacter PopZ forms a polar subdomain dictating sequential changes in pole composition and function MOLECULAR MICROBIOLOGY Bowman, G. R., Comolli, L. R., Gaietta, G. M., Fero, M., Hong, S., Jones, Y., Lee, J. H., Downing, K. H., Ellisman, M. H., McAdams, H. H., Shapiro, L. 2010; 76 (1): 173-189

  Abstract

  The bacterium Caulobacter crescentus has morphologically and functionally distinct cell poles that undergo sequential changes during the cell cycle. We show that the PopZ oligomeric network forms polar ribosome exclusion zones that change function during cell cycle progression. The parS/ParB chromosomal centromere is tethered to PopZ at one pole prior to the initiation of DNA replication. During polar maturation, the PopZ-centromere tether is broken, and the PopZ zone at that pole then switches function to act as a recruitment factor for the ordered addition of multiple proteins that promote the transformation of the flagellated pole into a stalked pole. Stalked pole assembly, in turn, triggers the initiation of chromosome replication, which signals the formation of a new PopZ zone at the opposite cell pole, where it functions to anchor the newly duplicated centromere that has traversed the long axis of the cell. We propose that pole-specific control of PopZ function co-ordinates polar development and cell cycle progression by enabling independent assembly and tethering activities at the two cell poles.

  View details for DOI 10.1111/j.1365-2958.2010.07088.x

  View details for Web of Science ID 000276036000013

  View details for PubMedID 20149103

  View details for PubMedCentralID PMC2935252

• High-throughput identification of protein localization dependency networks PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Christen, B., Fero, M. J., Hillson, N. J., Bowman, G., Hong, S., Shapiro, L., McAdams, H. H. 2010; 107 (10): 4681-4686

  Abstract

  Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

  View details for DOI 10.1073/pnas.1000846107

  View details for Web of Science ID 000275368400036

  View details for PubMedID 20176934

  View details for PubMedCentralID PMC2842071

• Bacterial Chromosome Organization and Segregation COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Toro, E., Shapiro, L. 2010; 2 (2)

  Abstract

  Bacterial chromosomes are generally approximately 1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. Recent advances in cell-imaging technology with subdiffraction resolution have revealed that the bacterial nucleoid is reliably oriented and highly organized within the cell. Such organization is transmitted from one generation to the next by progressive segregation of daughter chromosomes and anchoring of DNA to the cell envelope. Active segregation by a mitotic machinery appears to be common; however, the mode of chromosome segregation varies significantly from species to species.

  View details for DOI 10.1101/cshperspect.a000349

  View details for Web of Science ID 000279881400003

  View details for PubMedID 20182613

  View details for PubMedCentralID PMC2828278

• System-level design of bacterial cell cycle control 146th Nobel Symposium on Systems Biology McAdams, H. H., Shapiro, L. ELSEVIER SCIENCE BV. 2009: 3984–91

  Abstract

  Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of an entire bacterial cell cycle system functioning as a state machine. Oscillating levels of a few temporally-controlled master regulator proteins in a cyclical circuit drive cell cycle progression. To a striking degree, the cell cycle regulation is a whole cell phenomenon. Phospho-signaling proteins and proteases dynamically deployed to specific locations on the cell wall are vital. An essential phospho-signaling system integral to the cell cycle circuitry is central to accomplishing asymmetric cell division.

  View details for DOI 10.1016/j.febslet.2009.09.030

  View details for Web of Science ID 000273208000016

  View details for PubMedID 19766635

  View details for PubMedCentralID PMC2795017

• Why and How Bacteria Localize Proteins SCIENCE Shapiro, L., McAdams, H. H., Losick, R. 2009; 326 (5957): 1225-1228

  Abstract

  Despite their small size, bacteria have a remarkably intricate internal organization. Bacteria deploy proteins and protein complexes to particular locations and do so in a dynamic manner in lockstep with the organized deployment of their chromosome. The dynamic subcellular localization of protein complexes is an integral feature of regulatory processes of bacterial cells.

  View details for DOI 10.1126/science.1175685

  View details for Web of Science ID 000272117900037

  View details for PubMedID 19965466

• Feedback Control of DnaA-Mediated Replication Initiation by Replisome-Associated HdaA Protein in Caulobacter JOURNAL OF BACTERIOLOGY Collier, J., Shapiro, L. 2009; 191 (18): 5706-5716

  Abstract

  Chromosome replication in Caulobacter crescentus is tightly regulated to ensure that initiation occurs at the right time and only once during the cell cycle. The timing of replication initiation is controlled by both CtrA and DnaA. CtrA binds to and silences the origin. Upon the clearance of CtrA from the cell, the DnaA protein accumulates and allows loading of the replisome at the origin. Here, we identify an additional layer of replication initiation control that is mediated by the HdaA protein. In Escherichia coli, the Hda protein inactivates DnaA after replication initiation. We show that the Caulobacter HdaA homologue is necessary to restrict the initiation of DNA replication to only once per cell cycle and that it dynamically colocalizes with the replisome throughout the cell cycle. Moreover, the transcription of hdaA is directly activated by DnaA, providing a robust feedback regulatory mechanism that adjusts the levels of HdaA to inactivate DnaA.

  View details for DOI 10.1128/JB.00525-09

  View details for Web of Science ID 000269372600017

  View details for PubMedID 19633089

  View details for PubMedCentralID PMC2737981

• Superresolution imaging of protein superstructures in live Caulobacter crescentus cells with EYFP Biteen, J. S., Thompson, M. A., Tselentis, N. K., Shapiro, L., Moerner, W. E. AMER CHEMICAL SOC. 2009

  View details for Web of Science ID 000207861909276

• Imaging beyond the diffraction limit in cells using single-molecule active control Moerner, W. E., Biteen, J., Conley, N. R., Lee, H., Lord, S. J., Thompson, M. A., Shapiro, L., Liu, N., Samuel, R., Twieg, R. J. AMER CHEMICAL SOC. 2009: 555–555

  View details for Web of Science ID 000207857800509

• Dynamic chromosome organization and protein localization coordinate the regulatory circuitry that drives the bacterial cell cycle. Cold Spring Harbor symposia on quantitative biology Goley, E. D., Toro, E., McAdams, H. H., Shapiro, L. 2009; 74: 55-64

  Abstract

  The bacterial cell has less internal structure and genetic complexity than cells of eukaryotic organisms, yet it is a highly organized system that uses both temporal and spatial cues to drive its cell cycle. Key insights into bacterial regulatory programs that orchestrate cell cycle progression have come from studies of Caulobacter crescentus, a bacterium that divides asymmetrically. Three global regulatory proteins cycle out of phase with one another and drive cell cycle progression by directly controlling the expression of 200 cell-cycle-regulated genes. Exploration of this system provided insights into the evolution of regulatory circuits and the plasticity of circuit structure. The temporal expression of the modular subsystems that implement the cell cycle and asymmetric cell division is also coordinated by differential DNA methylation, regulated proteolysis, and phosphorylation signaling cascades. This control system structure has parallels to eukaryotic cell cycle control architecture. Remarkably, the transcriptional circuitry is dependent on three-dimensional dynamic deployment of key regulatory and signaling proteins. In addition, dynamically localized DNA-binding proteins ensure that DNA segregation is coupled to the timing and cellular position of the cytokinetic ring. Comparison to other organisms reveals conservation of cell cycle regulatory logic, even if regulatory proteins, themselves, are not conserved.

  View details for DOI 10.1101/sqb.2009.74.005

  View details for PubMedID 19687139

• Superresolution Imaging in Live Caulobacter Crescentus Cells Using Photoswitchable Enhanced Yellow Fluorescent Protein Conference on Single Molecule Spectroscopy and Imaging II Biteen, J. S., Thompson, M. A., Tselentis, N. K., Shapiro, L., Moerner, W. E. SPIE-INT SOC OPTICAL ENGINEERING. 2009

  View details for DOI 10.1117/12.809080

  View details for Web of Science ID 000285712600011

• Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP NATURE METHODS Biteen, J. S., Thompson, M. A., Tselentis, N. K., Bowman, G. R., Shapiro, L., Moerner, W. E. 2008; 5 (11): 947-949

  Abstract

  The commonly used, monomeric EYFP enabled imaging of intracellular protein structures beyond the optical resolution limit ('super-resolution' imaging) in living cells. By combining photoinduced activation of single EYFP fusions and time-lapse imaging, we obtained sub-40 nm resolution images of the filamentous superstructure of the bacterial actin protein MreB in live Caulobacter crescentus cells. These studies demonstrated that EYFP is a useful emitter for in vivo super-resolution imaging.

  View details for DOI 10.1038/nmeth.1258

  View details for Web of Science ID 000260532500013

  View details for PubMedID 18794860

  View details for PubMedCentralID PMC2655310

• A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iniesta, A. A., Shapiro, L. 2008; 105 (43): 16602-16607

  Abstract

  Dynamic protein localization is an integral component of the regulatory circuit that drives the Caulobacter cell cycle. The ClpXP protease is localized to the Caulobacter cell pole, where it catalyzes the degradation of the CtrA master regulator at specific times in the cell cycle. Clearance of active CtrA at the G1/S transition allows the initiation of DNA replication and cell-cycle progression. The polar localization of ClpXP is dependent on the polar positioning of the CpdR single-domain response regulator. Only the unphosphorylated form of CpdR localizes and activates ClpXP. We demonstrate that another single domain response regulator, DivK, promotes the polar accumulation of unphosphorylated CpdR and that CpdR is subsequently degraded at the cell pole by the localized ClpXP protease. Thus, CpdR function is regulated by a feedback loop that incorporates its differential phosphorylation, the transient polar localization and activity of the ClpXP protease, and the clearance of the CpdR by polar ClpXP that, in turn, releases ClpXP from the pole relieving the degradation of CtrA. CtrA approximately P then accumulates and activates the transcription of cpdR, completing the regulatory loop, establishing an integrated network that controls a robust cell-cycle transition.

  View details for DOI 10.1073/pnas.0808807105

  View details for Web of Science ID 000260913500037

  View details for PubMedID 18946044

  View details for PubMedCentralID PMC2575466

• Caulobacter requires a dedicated mechanism to initiate chromosome segregation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Toro, E., Hong, S., McAdams, H. H., Shapiro, L. 2008; 105 (40): 15435-15440

  Abstract

  Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show that Caulobacter crescentus makes use of and requires a dedicated mechanism to initiate chromosome segregation. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement.

  View details for DOI 10.1073/pnas.0807448105

  View details for Web of Science ID 000260360500041

  View details for PubMedID 18824683

  View details for PubMedCentralID PMC2563096

• SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus JOURNAL OF BACTERIOLOGY Lesley, J. A., Shapiro, L. 2008; 190 (20): 6867-6880

  Abstract

  Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.

  View details for DOI 10.1128/JB.00700-08

  View details for Web of Science ID 000259631900035

  View details for PubMedID 18723629

  View details for PubMedCentralID PMC2566184

• A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole CELL Bowman, G. R., Comolli, L. R., Zhu, J., Eckart, M., Koenig, M., Downing, K. H., Moerner, W. E., Earnest, T., Shapiro, L. 2008; 134 (6): 945-955

  Abstract

  Bacterial replication origins move towards opposite ends of the cell during DNA segregation. We have identified a proline-rich polar protein, PopZ, required to anchor the separated Caulobacter crescentus chromosome origins at the cell poles, a function that is essential for maintaining chromosome organization and normal cell division. PopZ interacts directly with the ParB protein bound to specific DNA sequences near the replication origin. As the origin/ParB complex is being replicated and moved across the cell, PopZ accumulates at the cell pole and tethers the origin in place upon arrival. The polar accumulation of PopZ occurs by a diffusion/capture mechanism that requires the MreB cytoskeleton. High molecular weight oligomers of PopZ assemble in vitro into a filamentous network with trimer junctions, suggesting that the PopZ network and ParB-bound DNA interact in an adhesive complex, fixing the chromosome origin at the cell pole.

  View details for DOI 10.1016/j.cell.2008.07.015

  View details for Web of Science ID 000259318100015

  View details for PubMedID 18805088

  View details for PubMedCentralID PMC2745220

• Architecture and inherent robustness of a bacterial cell-cycle control system PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shen, X., Collier, J., Dill, D., Shapiro, L., Horowitz, M., McAdams, H. H. 2008; 105 (32): 11340-11345

  Abstract

  A closed-loop control system drives progression of the coupled stalked and swarmer cell cycles of the bacterium Caulobacter crescentus in a near-mechanical step-like fashion. The cell-cycle control has a cyclical genetic circuit composed of four regulatory proteins with tight coupling to processive chromosome replication and cell division subsystems. We report a hybrid simulation of the coupled cell-cycle control system, including asymmetric cell division and responses to external starvation signals, that replicates mRNA and protein concentration patterns and is consistent with observed mutant phenotypes. An asynchronous sequential digital circuit model equivalent to the validated simulation model was created. Formal model-checking analysis of the digital circuit showed that the cell-cycle control is robust to intrinsic stochastic variations in reaction rates and nutrient supply, and that it reliably stops and restarts to accommodate nutrient starvation. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. The hybrid cell-cycle simulation implementation is inherently extensible and provides a promising approach for development of whole-cell behavioral models that can replicate the observed functionality of the cell and its responses to changing environmental conditions.

  View details for DOI 10.1073/pnas.0805258105

  View details for Web of Science ID 000258560700056

  View details for PubMedID 18685108

  View details for PubMedCentralID PMC2516238

• Small non-coding RNAs in Caulobacter crescentus MOLECULAR MICROBIOLOGY Landt, S. G., Abeliuk, E., McGrath, P. T., Lesley, J. A., McAdams, H. H., Shapiro, L. 2008; 68 (3): 600-614

  Abstract

  Small non-coding RNAs (sRNAs) are active in many bacterial cell functions, including regulation of the cell's response to environmental challenges. We describe the identification of 27 novel Caulobacter crescentus sRNAs by analysis of RNA expression levels assayed using a tiled Caulobacter microarray and a protocol optimized for detection of sRNAs. The principal analysis method involved identification of sets of adjacent probes with unusually high correlation between the individual intergenic probes within the set, suggesting presence of a sRNA. Among the validated sRNAs, two are candidate transposase gene antisense RNAs. The expression of 10 of the sRNAs is regulated by either entry into stationary phase, carbon starvation, or rich versus minimal media. The expression of four of the novel sRNAs changes as the cell cycle progresses. One of these shares a promoter motif with several genes expressed at the swarmer-to-stalked cell transition; while another appears to be controlled by the CtrA global transcriptional regulator. The probe correlation analysis approach reported here is of general use for large-scale sRNA identification for any sequenced microbial genome.

  View details for DOI 10.1111/j.1365-2958.2008.06172.x

  View details for Web of Science ID 000254641600007

  View details for PubMedID 18373523

• Coordination of chromosome replication and cell cycle progression in Caulobacter Shapiro, L. FEDERATION AMER SOC EXP BIOL. 2008

  View details for Web of Science ID 000208467800418

• Coordination of DNA replication and cell division in Caulobacter crescentus 31st Annual Meeting of the German-Society-for-Cell-Biology Tan, M. H., McAdams, H., Shapiro, L. ELSEVIER GMBH, URBAN & FISCHER VERLAG. 2008: 23–23

  View details for Web of Science ID 000255316100052

• Getting organized - how bacterial cells move proteins and DNA NATURE REVIEWS MICROBIOLOGY Thanbichler, M., Shapiro, L. 2008; 6 (1): 28-40

  Abstract

  In recent years, the subcellular organization of prokaryotic cells has become a focal point of interest in microbiology. Bacteria have evolved several different mechanisms to target protein complexes, membrane vesicles and DNA to specific positions within the cell. This versatility allows bacteria to establish the complex temporal and spatial regulatory networks that couple morphological and physiological differentiation with cell-cycle progression. In addition to stationary localization factors, dynamic cytoskeletal structures also have a fundamental role in many of these processes. In this Review, we summarize the current knowledge on localization mechanisms in bacteria, with an emphasis on the role of polymeric protein assemblies in the directed movement and positioning of macromolecular complexes.

  View details for DOI 10.1038/nrmicro1495

  View details for Web of Science ID 000252065900012

  View details for PubMedID 18059290

• Multiplexed Quantitative Proteomics Using Mass Spectrometry PROTEIN MASS SPECTROMETRY Ross, P. L., Chen, X., Toro, E., Britos, L., Shapiro, L., Pappin, D., Whitelegge, J. P. 2008; 52: 449–66

  View details for DOI 10.1016/S0166-526X(08)00218-3

  View details for Web of Science ID 000310710400021

• Caulobacter crescentus as a whole-cell uranium biosensor APPLIED AND ENVIRONMENTAL MICROBIOLOGY Hillson, N. J., Hu, P., Andersen, G. L., Shapiro, L. 2007; 73 (23): 7615-7621

  Abstract

  We engineered a strain of the bacterium Caulobacter crescentus to fluoresce in the presence of micromolar levels of uranium at ambient temperatures when it is exposed to a hand-held UV lamp. Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. We designated one of these genes urcA (for uranium response in caulobacter). We constructed a reporter that utilizes the urcA promoter to produce a UV-excitable green fluorescent protein in the presence of the uranyl cation, a soluble form of uranium. This reporter is specific for uranium and has little cross specificity for nitrate (<400 microM), lead (<150 microM), cadmium (<48 microM), or chromium (<41.6 microM). The uranium reporter construct was effective for discriminating contaminated groundwater samples (4.2 microM uranium) from uncontaminated groundwater samples (<0.1 microM uranium) collected at the Oak Ridge Field Research Center. In contrast to other uranium detection methodologies, the Caulobacter reporter strain can provide on-demand usability in the field; it requires minimal sample processing and no equipment other than a hand-held UV lamp, and it may be sprayed directly on soil, groundwater, or industrial surfaces.

  View details for DOI 10.1128/AEM.01566-07

  View details for Web of Science ID 000251474400017

  View details for PubMedID 17905881

  View details for PubMedCentralID PMC2168040

• A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus NUCLEIC ACIDS RESEARCH Thanbichler, M., Iniesta, A. A., Shapiro, L. 2007; 35 (20)

  Abstract

  Caulobacter crescentus is widely used as a powerful model system for the study of prokaryotic cell biology and development. Analysis of this organism is complicated by a limited selection of tools for genetic manipulation and inducible gene expression. This study reports the identification and functional characterization of a vanillate-regulated promoter (P(van)) which meets all requirements for application as a multi-purpose expression system in Caulobacter, thus complementing the established xylose-inducible system (P(xyl)). Furthermore, we introduce a newly constructed set of integrating and replicating shuttle vectors that considerably facilitate cell biological and physiological studies in Caulobacter. Based on different narrow and broad-host range replicons, they offer a wide choice of promoters, resistance genes, and fusion partners for the construction of fluorescently or affinity-tagged proteins. Since many of these constructs are also suitable for use in other bacteria, this work provides a comprehensive collection of tools that will enrich many areas of microbiological research.

  View details for DOI 10.1093/nar/gkm818

  View details for Web of Science ID 000251336000004

  View details for PubMedID 17959646

  View details for PubMedCentralID PMC2175322

• A DNA methylation ratchet governs progression through a bacterial cell cycle PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Collier, J., McAdams, H. H., Shapiro, L. 2007; 104 (43): 17111-17116

  Abstract

  The Caulobacter cell cycle is driven by a cascade of transient regulators, starting with the expression of DnaA in G(1) and ending with the expression of the essential CcrM DNA methyltransferase at the completion of DNA replication. The timing of DnaA accumulation was found to be regulated by the methylation state of the dnaA promoter, which in turn depends on the chromosomal position of dnaA near the origin of replication and restriction of CcrM synthesis to the end of the cell cycle. The dnaA gene is preferentially transcribed from a fully methylated promoter. DnaA initiates DNA replication and activates the transcription of the next cell-cycle regulator, GcrA. With the passage of the replication fork, the dnaA promoter becomes hemimethylated, and DnaA accumulation drops. GcrA then activates the transcription of the next cell-cycle regulator, CtrA, once the replication fork passes through the ctrA P1 promoter, generating two hemimethylated copies of ctrA. The ctrA gene is preferentially transcribed from a hemimethylated promoter. CtrA then activates the transcription of ccrM, to bring the newly replicated chromosome to the fully methylated state, promoting dnaA transcription and the start of a new cell cycle. We show that the cell-cycle timing of CcrM is critical for Caulobacter fitness. The sequential changes in the chromosomal methylation state serve to couple the progression of DNA replication to cell-cycle events regulated by the master transcriptional regulatory cascade, thus providing a ratchet mechanism for robust cell-cycle control.

  View details for Web of Science ID 000250487600069

  View details for PubMedID 17942674

• Cell cycle regulation in Caulobacter: location, location, location JOURNAL OF CELL SCIENCE Goley, E. D., Iniesta, A. A., Shapiro, L. 2007; 120 (20): 3501-3507

  Abstract

  Cellular reproduction in all organisms requires temporal and spatial coordination of crucial events, notably DNA replication, chromosome segregation and cytokinesis. Recent studies on the dimorphic bacterium Caulobacter crescentus (Caulobacter) highlight mechanisms by which positional information is integrated with temporal modes of cell cycle regulation. Caulobacter cell division is inherently asymmetric, yielding progeny with different fates: stalked cells and swarmer cells. Cell type determinants in stalked progeny promote entry into S phase, whereas swarmer progeny remain in G1 phase. Moreover, initiation of DNA replication is allowed only once per cell cycle. This finite window of opportunity is imposed by coordinating spatially constrained proteolysis of CtrA, an inhibitor of DNA replication initiation, with forward progression of the cell cycle. Positional cues are equally important in coordinating movement of the chromosome with cell division site selection in Caulobacter. The chromosome is specifically and dynamically localized over the course of the cell cycle. As the duplicated chromosomes are partitioned, factors that restrict assembly of the cell division protein FtsZ associate with a chromosomal locus near the origin, ensuring that the division site is located towards the middle of the cell.

  View details for DOI 10.1242/jcs.005967

  View details for Web of Science ID 000250043300002

  View details for PubMedID 17928306

• Spatial complexity and control of a bacterial cell cycle CURRENT OPINION IN BIOTECHNOLOGY Collier, J., Shapiro, L. 2007; 18 (4): 333-340

  Abstract

  A major breakthrough in understanding the bacterial cell cycle is the discovery that bacteria exhibit a high degree of intracellular organization. Chromosomal loci and many protein complexes are positioned at particular subcellular sites. In this review, we examine recently discovered control mechanisms that make use of dynamically localized protein complexes to orchestrate the Caulobacter crescentus cell cycle. Protein localization, notably of signal transduction proteins, chromosome partition proteins, and proteases, serves to coordinate cell division with chromosome replication and cell differentiation. The developmental fate of daughter cells is decided before completion of cytokinesis, via the early establishment of cell polarity by the distribution of activated signaling proteins, bacterial cytoskeleton, and landmark proteins.

  View details for DOI 10.1016/j.copbio.2007.07.007

  View details for Web of Science ID 000249980400008

  View details for PubMedID 17709236

  View details for PubMedCentralID PMC2716793

• An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site SCIENCE Rock, F. L., Mao, W., Yaremchuk, A., Tukalo, M., Crepin, T., Zhou, H., Zhang, Y., Hernandez, V., Akama, T., Baker, S. J., Plattner, J. J., Shapiro, L., Martinis, S. A., Benkovic, S. J., Cusack, S., Alley, M. R. 2007; 316 (5832): 1759-1761

  Abstract

  Aminoacyl-transfer RNA (tRNA) synthetases, which catalyze the attachment of the correct amino acid to its corresponding tRNA during translation of the genetic code, are proven antimicrobial drug targets. We show that the broad-spectrum antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), in development for the treatment of onychomycosis, inhibits yeast cytoplasmic leucyl-tRNA synthetase by formation of a stable tRNA(Leu)-AN2690 adduct in the editing site of the enzyme. Adduct formation is mediated through the boron atom of AN2690 and the 2'- and 3'-oxygen atoms of tRNA's3'-terminal adenosine. The trapping of enzyme-bound tRNA(Leu) in the editing site prevents catalytic turnover, thus inhibiting synthesis of leucyl-tRNA(Leu) and consequentially blocking protein synthesis. This result establishes the editing site as a bona fide target for aminoacyl-tRNA synthetase inhibitors.

  View details for DOI 10.1126/science.1142189

  View details for Web of Science ID 000247400500051

  View details for PubMedID 17588934

• High-throughput identification of transcription start sites, conserved promoter motifs and predicted regulons NATURE BIOTECHNOLOGY McGrath, P. T., Lee, H., Zhang, L., Iniesta, A. A., Hottes, A. K., Tan, M. H., Hillson, N. J., Hu, P., Shapiro, L., McAdams, H. H. 2007; 25 (5): 584-592

  Abstract

  Using 62 probe-level datasets obtained with a custom-designed Caulobacter crescentus microarray chip, we identify transcriptional start sites of 769 genes, 53 of which are transcribed from multiple start sites. Transcriptional start sites are identified by analyzing probe signal cross-correlation matrices created from probe pairs tiled every 5 bp upstream of the genes. Signals from probes binding the same message are correlated. The contribution of each promoter for genes transcribed from multiple promoters is identified. Knowing the transcription start site enables targeted searching for regulatory-protein binding motifs in the promoter regions of genes with similar expression patterns. We identified 27 motifs, 17 of which share no similarity to the characterized motifs of other C. crescentus transcriptional regulators. Using these motifs, we predict coregulated genes. We verified novel promoter motifs that regulate stress-response genes, including those responding to uranium challenge, a stress-response sigma factor and a stress-response noncoding RNA.

  View details for DOI 10.1038/nbt1294

  View details for Web of Science ID 000246369400027

  View details for PubMedID 17401361

• The push and pull of the bacterial cytoskeleton TRENDS IN CELL BIOLOGY Dye, N. A., Shapiro, L. 2007; 17 (5): 239-245

  Abstract

  A crucial function for eukaryotic cytoskeletal filaments is to organize the intracellular space: facilitate communication across the cell and enable the active transport of cellular components. It was assumed for many years that the small size of the bacterial cell eliminates the need for a cytoskeleton, because simple diffusion of proteins is rapid over micron-scale distances. However, in the last decade, cytoskeletal proteins have indeed been found to exist in bacteria where they have an important role in organizing the bacterial cell. Here, we review the progress that has been made towards understanding the mechanisms by which bacterial cytoskeletal proteins influence cellular organization. These discoveries have advanced our understanding of bacterial physiology and provided insight into the evolution of the eukaryotic cytoskeleton.

  View details for DOI 10.1016/j.tcb.2007.03.005

  View details for Web of Science ID 000246939100005

  View details for PubMedID 17434308

• Systems biology of Caulobacter ANNUAL REVIEW OF GENETICS Laub, M. T., Shapiro, L., McAdams, H. H. 2007; 41: 429-441

  Abstract

  The dynamic range of a bacterial species' natural environment is reflected in the complexity of its systems that control cell cycle progression and its range of adaptive responses. We discuss the genetic network and integrated three-dimensional sensor/response systems that regulate the cell cycle and asymmetric cell division in the bacterium Caulobacter crescentus. The cell cycle control circuitry is tied closely to chromosome replication and morphogenesis by multiple feedback pathways from the modular functions that implement the cell cycle. The sophistication of the genetic regulatory circuits and the elegant integration of temporally controlled transcription and protein synthesis with spatially dynamic phosphosignaling and proteolysis pathways, and epigenetic regulatory mechanisms, form a remarkably robust living system.

  View details for DOI 10.1146/annurev.genet.41.110306.130346

  View details for Web of Science ID 000252359500018

  View details for PubMedID 18076330

• Chromosome organization and segregation in bacteria JOURNAL OF STRUCTURAL BIOLOGY Thanbichler, M., Shapiro, L. 2006; 156 (2): 292-303

  Abstract

  In the recent years, considerable advances have been made towards understanding the structure and function of the bacterial chromosome. A number of different factors appear to cooperate in condensing DNA into a highly dynamic assembly of supercoiled loops. Despite this variability in the lower levels of chromatin structure, the global arrangement of chromosomal DNA within the cell is surprisingly conserved, with loci being arrayed along the cellular long axis in line with their order on the genomic map. This conserved pattern is propagated during the course of DNA segregation. First, after entry into S-phase, the newly synthesized origin regions are segregated in an active and directed process, involving the bacterial actin homolog MreB. Subsequent DNA segments then follow by different mechanisms. They are separated immediately after release from the replisome and move rapidly to their conserved positions in the incipient daughter cell compartments. Partitioning of the bacterial chromosome thus takes place while DNA replication is in progress.

  View details for DOI 10.1016/j.jsb.2006.05.007

  View details for Web of Science ID 000242008100007

  View details for PubMedID 16860572

• PHYS 489-Direct observation of MreB treadmilling in Caulobacter by single-molecule fluorescence microscopy Kim, S., Gitai, Z., Kinkhabwala, A., Shapiro, L., Moerner, W. E. AMER CHEMICAL SOC. 2006

  View details for Web of Science ID 000207781609166

• Single molecules of the bacterial actin MreB undergo directed treadmilling motion in Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kim, S. Y., Gitai, Z., Kinkhabwala, A., Shapiro, L., Moerner, W. E. 2006; 103 (29): 10929-10934

  Abstract

  The actin cytoskeleton represents a key regulator of multiple essential cellular functions in both eukaryotes and prokaryotes. In eukaryotes, these functions depend on the orchestrated dynamics of actin filament assembly and disassembly. However, the dynamics of the bacterial actin homolog MreB have yet to be examined in vivo. In this study, we observed the motion of single fluorescent MreB-yellow fluorescent protein fusions in living Caulobacter cells in a background of unlabeled MreB. With time-lapse imaging, polymerized MreB [filamentous MreB (fMreB)] and unpolymerized MreB [globular MreB (gMreB)] monomers could be distinguished: gMreB showed fast motion that was characteristic of Brownian diffusion, whereas the labeled molecules in fMreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer provides an indication that, like actin, MreB monomers treadmill through MreB filaments by preferential polymerization at one filament end and depolymerization at the other filament end. From these data, we extract several characteristics of single MreB filaments, including that they are, on average, much shorter than the cell length and that the direction of their polarized assembly seems to be independent of the overall cellular polarity. Thus, MreB, like actin, exhibits treadmilling behavior in vivo, and the long MreB structures that have been visualized in multiple bacterial species seem to represent bundles of short filaments that lack a uniform global polarity.

  View details for DOI 10.1073/pnas.0604503103

  View details for Web of Science ID 000239327200021

  View details for PubMedID 16829583

  View details for PubMedCentralID PMC1544151

• A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Iniesta, A. A., McGrath, P. T., Reisenauer, A., McAdams, H. H., Shapiro, L. 2006; 103 (29): 10935-10940

  Abstract

  Temporally and spatially controlled master regulators drive the Caulobacter cell cycle by regulating the expression of >200 genes. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. We show here that a previously unidentified single domain-response regulator, CpdR, when in the unphosphorylated state, binds to ClpXP and, thereby, causes its localization to the cell pole. We further show that ClpXP localization is required for CtrA proteolysis. When CpdR is phosphorylated, ClpXP is delocalized, and CtrA is not degraded. Both CtrA and CpdR are phosphorylated via the same CckA histidine kinase phospho-signaling pathway, providing a reinforcing mechanism that simultaneously activates CtrA and prevents its degradation by delocalizing the CpdR/ClpXP complex. In swarmer cells, CpdR is in the phosphorylated state, thus preventing ClpXP localization and CtrA degradation. As swarmer cells differentiate into stalked cells (G1/S transition), unphosphorylated CpdR accumulates and is localized to the stalked cell pole, where it enables ClpXP localization and CtrA proteolysis, allowing the initiation of DNA replication. Dynamic protease localization mediated by a phospho-signaling pathway is a novel mechanism to integrate spatial and temporal control of bacterial cell cycle progression.

  View details for DOI 10.1073/pnas.0604554103

  View details for Web of Science ID 000239327200022

  View details for PubMedID 16829582

  View details for PubMedCentralID PMC1544152

• MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter CELL Thanbichler, M., Shapiro, L. 2006; 126 (1): 147-162

  Abstract

  Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. After replication of the polarly located origin region, one copy moves rapidly to the opposite end of the cell in an MreB-dependent manner. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated.

  View details for DOI 10.1016/j.cell.2006.05.038

  View details for Web of Science ID 000239224800023

  View details for PubMedID 16839883

• A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator CELL McGrath, P. T., Iniesta, A. A., Ryan, K. R., Shapiro, L., McAdams, H. H. 2006; 124 (3): 535-547

  Abstract

  Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.

  View details for DOI 10.1016/j.cell.2005.12.033

  View details for PubMedID 16469700

• The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter JOURNAL OF BACTERIOLOGY Wang, S. C., West, L., Shapiro, L. 2006; 188 (4): 1497-1508

  Abstract

  Bacterial chromosome partitioning and cell division are tightly connected cellular processes. We show here that the Caulobacter crescentus FtsK protein localizes to the division plane, where it mediates multiple functions involved in chromosome segregation and cytokinesis. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. The FtsK C terminus is essential in Caulobacter and is involved in maintaining accurate chromosome partitioning. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. These results suggest that the interdependence between chromosome partitioning and cell division in Caulobacter is mediated, in part, by the FtsK protein.

  View details for DOI 10.1128/JB.188.4.1497-1508.2006

  View details for PubMedID 16452433

  View details for PubMedCentralID PMC1367234

• DnaA couples DNA replication and the expression of two cell cycle master regulators EMBO JOURNAL Collier, J., Murray, S. R., Shapiro, L. 2006; 25 (2): 346-356

  Abstract

  Cell cycle progression in Caulobacter is driven by the master transcriptional regulators CtrA and GcrA. The cellular levels of CtrA and GcrA are temporally and spatially out-of-phase during the cell cycle, with CtrA repressing gcrA transcription and GcrA activating ctrA transcription. Here, we show that DnaA, a protein required for the initiation of DNA replication, also functions as a transcriptional activator of gcrA, which in turn activates multiple genes, notably those involved in chromosome replication and segregation. The cellular concentration of DnaA is cell cycle-controlled, peaking at the time of replication initiation and gcrA induction. Regulated proteolysis of GcrA contributes to the cell cycle variations in GcrA abundance. We propose that DnaA couples DNA replication initiation with the expression of the two oscillating regulators GcrA and CtrA and that the DnaA/GcrA/CtrA regulatory cascade drives the forward progression of the Caulobacter cell cycle.

  View details for DOI 10.1038/sj.emboj.7600927

  View details for Web of Science ID 000234952500008

  View details for PubMedID 16395331

  View details for PubMedCentralID PMC1383511

• Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease EMBO JOURNAL Chen, J. C., Hottes, A. K., McAdams, H. H., McGrath, P. T., Viollier, P. H., Shapiro, L. 2006; 25 (2): 377-386

  Abstract

  We demonstrate that successive cleavage events involving regulated intramembrane proteolysis (Rip) occur as a function of time during the Caulobacter cell cycle. The proteolytic substrate PodJ(L) is a polar factor that recruits proteins required for polar organelle biogenesis to the correct cell pole at a defined time in the cell cycle. We have identified a periplasmic protease (PerP) that initiates the proteolytic sequence by truncating PodJ(L) to a form with altered activity (PodJ(S)). Expression of perP is regulated by a signal transduction system that activates cell type-specific transcription programs and conversion of PodJ(L) to PodJ(S) in response to the completion of cytokinesis. PodJ(S), sequestered to the progeny swarmer cell, is subsequently released from the polar membrane by the membrane metalloprotease MmpA for degradation during the swarmer-to-stalked cell transition. This sequence of proteolytic events contributes to the asymmetric localization of PodJ isoforms to the appropriate cell pole. Thus, temporal activation of the PerP protease and spatial restriction of the polar PodJ(L) substrate cooperatively control the cell cycle-dependent onset of Rip.

  View details for DOI 10.1038/sj.emboj.7600935

  View details for Web of Science ID 000234952500011

  View details for PubMedID 16395329

  View details for PubMedCentralID PMC1383518

• Two independent spiral structures control cell shape in Caulobacter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dye, N. A., Pincus, Z., Theriot, J. A., Shapiro, L., Gitai, Z. 2005; 102 (51): 18608-18613

  Abstract

  The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.

  View details for DOI 10.1073/pnas.0507708102

  View details for Web of Science ID 000234174300065

  View details for PubMedID 16344481

  View details for PubMedCentralID PMC1317941

• DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus MOLECULAR MICROBIOLOGY Hottes, A. K., Shapiro, L., McAdams, H. H. 2005; 58 (5): 1340-1353

  Abstract

  The level of DnaA, a key bacterial DNA replication initiation factor, increases during the Caulobacter swarmer-to-stalked transition just before the G1/S transition. We show that DnaA coordinates DNA replication initiation with cell cycle progression by acting as a global transcription factor. Using DnaA depletion and induction in synchronized cell populations, we have analysed global transcription patterns to identify the differential regulation of normally co-expressed genes. The DnaA regulon includes genes encoding several replisome components, the GcrA global cell cycle regulator, the PodJ polar localization protein, the FtsZ cell division protein, and nucleotide biosynthesis enzymes. In cells depleted of DnaA, the G1/S transition is temporally separated from the swarmer-to-stalked cell differentiation, which is normally coincident. In the absence of DnaA, the CtrA master regulator is cleared by proteolysis during the swarmer-to-stalked cell transition as usual, but DNA replication initiation is blocked. In this case, expression of gcrA, which is directly repressed by CtrA, does not increase in conjunction with the disappearance of CtrA until DnaA is subsequently induced, showing that gcrA expression requires DnaA. DnaA boxes are present upstream of many genes whose expression requires DnaA, and His6-DnaA binds to the promoters of gcrA, ftsZ and podJ in vitro. This redundant control of gcrA transcription by DnaA (activation) and CtrA (repression) forms a robust switch controlling the decision to proceed through the cell cycle or to remain in the G1 stage.

  View details for DOI 10.1111/j.1365-2958.2005.04912.x

  View details for Web of Science ID 000233170700012

  View details for PubMedID 16313620

• Identification of borinic esters as inhibitors of bacterial cell growth and bacterial methyltransferases, CcrM and MenH JOURNAL OF MEDICINAL CHEMISTRY Benkovic, S. J., Baker, S. J., Alley, M. R., Woo, Y. H., Zhang, Y. K., Akama, T., Mao, W. M., Baboval, J., Rajagopalan, P. T., Wall, M., Kahng, L. S., Tavassoli, A., Shapiro, L. 2005; 48 (23): 7468-7476

  Abstract

  As bacteria continue to develop resistance toward current antibiotics, we find ourselves in a continual battle to identify new antibacterial agents and targets. We report herein a class of boron-containing compounds termed borinic esters that have broad spectrum antibacterial activity with minimum inhibitory concentrations (MIC) in the low microgram/mL range. These compounds were identified by screening for inhibitors against Caulobacter crescentus CcrM, an essential DNA methyltransferase from gram negative alpha-proteobacteria. In addition, we demonstrate that borinic esters inhibit menaquinone methyltransferase in gram positive bacteria using a new biochemical assay for MenH from Bacillus subtilis. Our data demonstrate the potential for further development of borinic esters as antibacterial agents as well as leads to explore more specific inhibitors against two essential bacterial enzymes.

  View details for DOI 10.1021/jm050676a

  View details for Web of Science ID 000233399500043

  View details for PubMedID 16279806

• The bacterial nucleoid: A highly organized and dynamic structure JOURNAL OF CELLULAR BIOCHEMISTRY Thanbichler, M., Wang, S. C., Shapiro, L. 2005; 96 (3): 506-521

  Abstract

  Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.

  View details for DOI 10.1002/jcb.20519

  View details for Web of Science ID 000232262800007

  View details for PubMedID 15988757

• Conserved modular design of an oxygen sensory/signaling network with species-specific output PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Crosson, S., McGrath, P. T., Stephens, C., McAdams, H. H., Shapiro, L. 2005; 102 (22): 8018-8023

  Abstract

  Principles of modular design are evident in signaling networks that detect and integrate a given signal and, depending on the organism in which the network module is present, transduce this signal to affect different metabolic or developmental pathways. Here we report a global transcriptional analysis of an oxygen sensory/signaling network in Caulobacter crescentus consisting of the sensor histidine kinase FixL, its cognate response regulator FixJ, the transcriptional regulator FixK, and the kinase inhibitor FixT. It is known that in rhizobial bacteria these proteins form a network that regulates transcription of genes required for symbiotic nitrogen fixation, anaerobic and microaerobic respiration, and hydrogen metabolism under hypoxic conditions. We have identified a positive feedback loop in this network and present evidence that the negative feedback regulator, FixT, acts to inhibit FixL by mimicking a response regulator. Overall, the core circuit topology of the Fix network is conserved between the rhizobia and C. crescentus, a free-living aerobe that cannot fix nitrogen, respire anaerobically, or metabolize hydrogen. In C. crescentus, the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. Thus, the Fix network is a conserved sensory/signaling module whose transcriptional output has been adapted to the unique physiologies of C. crescentus and the nitrogen-fixing rhizobia.

  View details for DOI 10.1073/pnas.0503022102

  View details for Web of Science ID 000229531000043

  View details for PubMedID 15911751

  View details for PubMedCentralID PMC1142393

• The choreographed dynamics of bacterial chromosomes TRENDS IN MICROBIOLOGY Gitai, Z., Thanbichler, M., Shapiro, L. 2005; 13 (5): 221-228

  Abstract

  Despite decades of study, the exquisite temporal and spatial organization of bacterial chromosomes has only recently been appreciated. The direct visualization of specific chromosomal loci has revealed that bacteria condense, move and position their chromosomes in a reproducible fashion. The realization that bacterial chromosomes are actively translocated through the cell suggests the existence of specific mechanisms that direct this process. Here, we review bacterial chromosome dynamics and our understanding of the mechanisms that direct and coordinate them.

  View details for DOI 10.1016/j.tim.2005.03.006

  View details for Web of Science ID 000229467100008

  View details for PubMedID 15866039

• The structure and function of the bacterial chromosome CURRENT OPINION IN GENETICS & DEVELOPMENT Thanbichler, M., Viollier, P. H., Shapiro, L. 2005; 15 (2): 153-162

  Abstract

  Advances in microscopic and cell biological techniques have considerably improved our understanding of bacterial chromosome organization and dynamics. The nucleoid was formerly perceived to be an amorphous entity divided into ill-defined domains of supercoiling that are randomly deposited in the cell. Recent work, however, has demonstrated a remarkable degree of spatial organization. A highly ordered chromosome structure, established while DNA replication and partitioning are in progress, is maintained and propagated during growth. Duplication of the chromosome and partitioning of the newly generated daughter strands are interwoven processes driven by the dynamic interplay between the synthesis, segregation and condensation of DNA. These events are intimately coupled with the bacterial cell cycle and exhibit a previously unanticipated complexity reminiscent of eukaryotic systems.

  View details for Web of Science ID 000228496100006

  View details for PubMedID 15797198

• MreB actin-mediated segregation of a specific region of a bacterial chromosome CELL Gital, Z., Dye, N. A., Reisenauer, A., Wachi, M., Shapiro, L. 2005; 120 (3): 329-341

  Abstract

  Faithful chromosome segregation is an essential component of cell division in all organisms. The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. To investigate the potential role of the actin-like MreB protein in bacterial chromosome segregation, we first demonstrate that MreB is the direct target of the small molecule A22. We then demonstrate that A22 completely blocks the movement of newly replicated loci near the origin of replication but has no qualitative or quantitative effect on the segregation of other loci if added after origin segregation. MreB selectively interacts, directly or indirectly, with origin-proximal regions of the chromosome, arguing that the origin-proximal region segregates via an MreB-dependent mechanism not used by the rest of the chromosome.

  View details for Web of Science ID 000227028900009

  View details for PubMedID 15707892

• A membrane metalloprotease participates in the sequential degradation of a Caulobacter polarity determinant MOLECULAR MICROBIOLOGY Chen, J. C., Viollier, P. H., Shapiro, L. 2005; 55 (4): 1085-1103

  Abstract

  Caulobacter crescentus assembles many of its cellular machines at distinct times and locations during the cell cycle. PodJ provides the spatial cues for the biogenesis of several polar organelles, including the pili, adhesive holdfast and chemotactic apparatus, by recruiting structural and regulatory proteins, such as CpaE and PleC, to a specific cell pole. PodJ is a protein with a single transmembrane domain that exists in two forms, full-length (PodJL) and truncated (PodJS), each appearing during a specific time period of the cell cycle to control different aspects of polar organelle development. PodJL is synthesized in the early predivisional cell and is later proteolytically converted to PodJS. During the swarmer-to-stalked transition, PodJS must be degraded to preserve asymmetry in the next cell cycle. We found that MmpA facilitates the degradation of PodJS. MmpA belongs to the site-2 protease (S2P) family of membrane-embedded zinc metalloproteases, which includes SpoIVFB and YluC of Bacillus subtilis and YaeL of Escherichia coli. MmpA appears to cleave within or near the transmembrane segment of PodJS, releasing it into the cytoplasm for complete proteolysis. While PodJS has a specific temporal and spatial address, MmpA is present throughout the cell cycle; furthermore, periplasmic fusion to mRFP1 suggested that MmpA is uniformly distributed around the cell. We also determined that mmpA and yaeL can complement each other in C. crescentus and E. coli, indicating functional conservation. Thus, the sequential degradation of PodJ appears to involve regulated intramembrane proteolysis (Rip) by MmpA.

  View details for DOI 10.1111/j.1365-2958.2004.04443.x

  View details for Web of Science ID 000226707700011

  View details for PubMedID 15686556

• Spatial complexity of mechanisms controlling a bacterial cell cycle CURRENT OPINION IN MICROBIOLOGY Viollier, P. H., Shapiro, L. 2004; 7 (6): 572-578

  Abstract

  Cell cycle progression in Caulobacter is governed by a multilayered regulatory network linking chromosome replication with polar morphogenesis and cell division. Temporal and spatial regulation have emerged as the central themes, with the abundance, activity and subcellular location of key structural and regulatory proteins changing over the course of the cell cycle. An additional layer of complexity was recently uncovered, showing that each segment of the chromosome is located at a specific cellular position both during and after the completion of DNA replication, raising the possibility that this positioning contributes to temporal and spatial control of gene expression.

  View details for DOI 10.1016/j.mib.2004.10.005

  View details for Web of Science ID 000225782400003

  View details for PubMedID 15556028

• An actin-like gene can determine bacterial cell polarity 44th Annual Meeting of the American-Society-for-Cell-Biology Gitai, Z., Dye, N., Shapiro, L. AMER SOC CELL BIOLOGY. 2004: 10A–10A

  View details for Web of Science ID 000224648800052

• A genetic oscillator and the regulation of cell cycle progression in Caulobacter crescentus CELL CYCLE Crosson, S., McAdams, H., Shapiro, L. 2004; 3 (10): 1252-1254

  Abstract

  Analyses of cell polarity, division, and differentiation in prokaryotes have identified several regulatory proteins that exhibit dramatic changes in expression and spatial localization over the course of a cell cycle. The dynamic behavior of these proteins is often intrinsically linked to their function as polarity determinants.(1-3) In the alpha-proteobacterium, Caulobacter crescentus, the CtrA global transcriptional regulator exhibits a spatially and temporally dynamic expression pattern across the cell cycle. CtrA plays key roles in asymmetric cell division and in the timing of chromosome replication.(3,4) An additional global regulator, GcrA, has recently been discovered that both regulates and is regulated by CtrA.(5) Together, these regulatory proteins create a genetic circuit in which the cellular concentrations of CtrA and GcrA oscillate spatially and temporally to control daughter cell differentiation and cell cycle progression.

  View details for Web of Science ID 000225590800012

  View details for PubMedID 15467452

• Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Viollier, P. H., Thanbichler, M., McGrath, P. T., West, L., Meewan, M., McAdams, H. H., Shapiro, L. 2004; 101 (25): 9257-9262

  Abstract

  The chromosomal origin and terminus of replication are precisely localized in bacterial cells. We examined the cellular position of 112 individual loci that are dispersed over the circular Caulobacter crescentus chromosome and found that in living cells each locus has a specific subcellular address and that these loci are arrayed in linear order along the long axis of the cell. Time-lapse microscopy of the location of the chromosomal origin and 10 selected loci in the origin-proximal half of the chromosome showed that during DNA replication, as the replisome sequentially copies each locus, the newly replicated DNA segments are moved in chronological order to their final subcellular destination in the nascent half of the predivisional cell. Thus, the remarkable organization of the chromosome is being established while DNA replication is still in progress. The fact that the movement of these 10 loci is, like that of the origin, directed and rapid, and occurs at a similar rate, suggests that the same molecular machinery serves to partition and place many, if not most, chromosomal loci at defined subcellular sites.

  View details for DOI 10.1073/pnas.0402606101

  View details for Web of Science ID 000222278600018

  View details for PubMedID 15178755

  View details for PubMedCentralID PMC438963

• The topoisornerase IV ParC subunit colocalizes with the Caulobacter replisome and is required for polar localization of replication origins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, S. C., Shapiro, L. 2004; 101 (25): 9251-9256

  Abstract

  The process of bacterial DNA replication generates chromosomal topological constraints that are further confounded by simultaneous transcription. Topoisomerases play a key role in ensuring orderly replication and partition of DNA in the face of a continuously changing DNA tertiary structure. In addition to topological constraints, the cellular position of the replication origin is strictly controlled during the cell cycle. In Caulobacter crescentus, the origin of DNA replication is located at the cell pole. Upon initiation of DNA replication, one copy of the duplicated origin sequence rapidly appears at the opposite cell pole. To determine whether the maintenance of DNA topology contributes to the dynamic positioning of a specific DNA region within the cell, we examined origin localization in cells that express temperature-sensitive forms of either the ParC or ParE subunit of topoisomerase (Topo) IV. We found that in the absence of active Topo IV, replication initiation can occur but a significant percent of replication origins are either no longer moved to or maintained at the cell poles. During the replication process, the ParC subunit colocalizes with the replisome, whereas the ParE subunit is dispersed throughout the cell. However, an active ParE subunit is required for ParC localization to the replisome as it moves from the cell pole to the division plane during chromosome replication. We propose that the maintenance of DNA topology throughout the cell cycle contributes to the dynamic positioning of the origin sequence within the cell.

  View details for DOI 10.1073/pnas.0402567101

  View details for Web of Science ID 000222278600017

  View details for PubMedID 15178756

  View details for PubMedCentralID PMC438962

• An actin-like gene can determine cell polarity in bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gitai, Z., Dye, N., Shapiro, L. 2004; 101 (23): 8643-8648

  Abstract

  Achieving proper polarity is essential for cellular function. In bacteria, cell polarity has been observed by using both morphological and molecular markers; however, no general regulators of bacterial cell polarity have been identified. Here we investigate the effect on cell polarity of two cytoskeletal elements previously implicated in cell shape determination. We find that the actin-like MreB protein mediates global cell polarity in Caulobacter crescentus, although the intermediate filament-like CreS protein influences cell shape without affecting cell polarity. MreB is organized in an axial spiral that is dynamically rearranged during the cell cycle, and MreB dynamics may be critical for the determination of cell polarity. By examining depletion and overexpression strains, we demonstrate that MreB is required both for the polar localization of the chromosomal origin sequence and the dynamic localization of regulatory proteins to the correct cell pole. We propose that the molecular polarity inherent in an actin-like filament is translated into a mechanism for directing global cell polarity.

  View details for DOI 10.1073/pnas.0402638101

  View details for Web of Science ID 000222037000028

  View details for PubMedID 15159537

  View details for PubMedCentralID PMC423248

• Oscillating global regulators control the genetic circuit driving a bacterial cell cycle SCIENCE Holtzendorff, J., Hung, D., Brende, P., Reisenauer, A., Viollier, P. H., McAdams, H. H., Shapiro, L. 2004; 304 (5673): 983-987

  Abstract

  A newly identified cell-cycle master regulator protein, GcrA, together with the CtrA master regulator, are key components of a genetic circuit that drives cell-cycle progression and asymmetric polar morphogenesis in Caulobacter crescentus. The circuit drives out-of-phase temporal and spatial oscillation of GcrA and CtrA concentrations, producing time- and space-dependent transcriptional regulation of modular functions that implement cell-cycle processes. The CtrA/GcrA regulatory circuit controls expression of polar differentiation factors and the timing of DNA replication. CtrA functions as a silencer of the replication origin and GcrA as an activator of components of the replisome and the segregation machinery.

  View details for DOI 10.1126/science.1095191

  View details for Web of Science ID 000221383300040

  View details for PubMedID 15087506

• Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycle-regulated proteolysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ryan, K. R., Huntwork, S., Shapiro, L. 2004; 101 (19): 7415-7420

  Abstract

  The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. At the swarmer-to-stalked cell transition and in the stalked compartment of the predivisional cell, CtrA is localized to the cell pole just before its degradation. Analysis of the requirements for CtrA polar localization and CtrA proteolysis revealed that both processes require a motif within amino acids 1-56 of the CtrA receiver domain, and neither process requires CtrA phosphorylation. These results strongly suggest that CtrA polar localization is coupled to its cell cycle-regulated proteolysis. The polarly localized DivK response regulator promotes CtrA localization and proteolysis, but it does not directly recruit CtrA to the cell pole. Mutations in the divJ and pleC histidine kinases perturb the characteristic asymmetry of CtrA localization and proteolysis in the predivisional cell. We propose that polar recruitment of CtrA evolved to ensure that CtrA is degraded only in the stalked half of the predivisional cell, perhaps by localizing a proteolytic adaptor protein to the stalked pole. This is an example of controlled proteolysis of a cytoplasmic protein that is associated with its active recruitment to a specific subcellular address.

  View details for DOI 10.1073/pnas.0402153101

  View details for PubMedID 15123835

• Codon usage between genomes is constrained by genome-wide mutational processes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, S. L., Lee, W., Hottes, A. K., Shapiro, L., McAdams, H. H. 2004; 101 (10): 3480-3485

  Abstract

  Analysis of genome-wide codon bias shows that only two parameters effectively differentiate the genome-wide codon bias of 100 eubacterial and archaeal organisms. The first parameter correlates with genome GC content, and the second parameter correlates with context-dependent nucleotide bias. Both of these parameters may be calculated from intergenic sequences. Therefore, genome-wide codon bias in eubacteria and archaea may be predicted from intergenic sequences that are not translated. When these two parameters are calculated for genes from nonmammalian eukaryotic organisms, genes from the same organism again have similar values, and genome-wide codon bias may also be predicted from intergenic sequences. In mammals, genes from the same organism are similar only in the second parameter, because GC content varies widely among isochores. Our results suggest that, in general, genome-wide codon bias is determined primarily by mutational processes that act throughout the genome, and only secondarily by selective forces acting on translated sequences.

  View details for DOI 10.1073/pnas.0307827100

  View details for Web of Science ID 000220163800029

  View details for PubMedID 14990797

  View details for PubMedCentralID PMC373487

• A bacterial cell-cycle regulatory network operating in time and space SCIENCE McAdams, H. H., Shapiro, L. 2003; 301 (5641): 1874-1877

  Abstract

  Transcriptional regulatory circuits provide only a fraction of the signaling pathways and regulatory mechanisms that control the bacterial cell cycle. The CtrA regulatory network, important in control of the Caulobacter cell cycle, illustrates the critical role of nontranscriptional pathways and temporally and spatially localized regulatory proteins. The system architecture of Caulobacter cell-cycle control involves top-down control of modular functions by a small number of master regulatory proteins with cross-module signaling coordinating the overall process. Modeling the cell cycle probably requires a top-down modeling approach and a hybrid control system modeling paradigm to treat its combined discrete and continuous characteristics.

  View details for Web of Science ID 000185536700040

  View details for PubMedID 14512618

• Identification of long intergenic repeat sequences associated with DNA methylation sites in Caulobacter crescentus and other alpha-proteobacteria JOURNAL OF BACTERIOLOGY Chen, S. L., Shapiro, L. 2003; 185 (16): 4997-5002

  Abstract

  A systematic search for motifs associated with CcrM DNA methylation sites revealed four long (>100-bp) motifs (CIR sequences) present in up to 21 copies in Caulobacter crescentus. The CIR1 and CIR2 motifs exhibit a conserved inverted repeat organization, with a CcrM site in the center of one of the repeats.

  View details for DOI 10.1128/JB.185.16.4997-5002.2003

  View details for Web of Science ID 000184692800037

  View details for PubMedID 12897020

  View details for PubMedCentralID PMC166474

• Fluorescence bleaching reveals asymmetric compartment formation prior to cell division in Caulobacter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Judd, E. M., Ryan, K. R., Moerner, W. E., Shapiro, L., McAdams, H. H. 2003; 100 (14): 8235-8240

  Abstract

  Asymmetric cell division in Caulobacter crescentus yields daughter cells that have different cell fates. Compartmentalization of the predivisional cell is a critical event in the establishment of the differential distribution of regulatory factors that specify cell fate. To determine when during the cell cycle the cytoplasm is compartmentalized so that cytoplasmic proteins can no longer diffuse between the two nascent progeny cell compartments, we designed a fluorescence loss in photobleaching assay. Individual cells containing enhanced GFP were exposed to a bleaching laser pulse tightly focused at one cell pole. In compartmentalized cells, fluorescence disappears only in the compartment receiving the bleaching beam; in noncompartmentalized cells, fluorescence disappears from the entire cell. In a 135-min cell cycle, the cells were compartmentalized 18 +/- 5 min before the progeny cells separated. Clearance of the 22000 CtrA master transcriptional regulator molecules from the stalked portion of the predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a fluorescent marker for CtrA showed that the differential degradation of CtrA in the nascent stalk cell compartment occurs only after the cytoplasm is compartmentalized.

  View details for DOI 10.1073/pnas.1433105100

  View details for PubMedID 12824468

• A lytic transglycosylase homologue, PleA, is required for the assembly of pili and the flagellum at the Caulobacter crescentus cell pole MOLECULAR MICROBIOLOGY Viollier, P. H., Shapiro, L. 2003; 49 (2): 331-345

  Abstract

  Two distinct protein complexes, the flagellum and the pilus biogenesis machinery, are asymmetrically assembled at one pole of the Caulobacter predivisional cell. Cell division yields dissimilar daughter cells: a stalked cell and a swarmer cell that assembles several pili at the flagellated cell pole. Strains bearing mutations in the pleA gene are pililess and non-flagellated. The PleA protein contains a region that is similar to a peptidoglycan-hydrolytic active site, and a point mutation at this site in PleA results in the loss of flagellum and pili biogenesis. PleA was found to be required for the insertion of the outer membrane pilus secretion channel at the cell pole and for the accumulation of the PilA pilin subunit. PleA is also required for the assembly of substructures of the flagellar basal body hook complex that are located in or traverse the peptidoglycan layer. These results argue that PleA facilitates the assembly of envelope-spanning structures at the cell pole. In support of this, PleA was found to be present only during a short interval in the cell cycle that coincides with the assembly of the flagellum and the pilus secretion apparatus.

  View details for DOI 10.1046/j.1365-2958.2003.03576.x

  View details for Web of Science ID 000184224700005

  View details for PubMedID 12828633

• Bacterial cell division spirals into control PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gitai, Z., Shapiro, L. 2003; 100 (13): 7423-7424

  View details for DOI 10.1073/pnas.1332806100

  View details for Web of Science ID 000183845800003

  View details for PubMedID 12810947

  View details for PubMedCentralID PMC164599

• Polar localization of replicon origins in the multipartite Genomes of Agrobacterium tumefaciens and Sinorhizobium meliloti JOURNAL OF BACTERIOLOGY Kahng, L. S., Shapiro, L. 2003; 185 (11): 3384-3391

  Abstract

  The origins of replication of many different bacteria have been shown to reside at specific subcellular locations, but the mechanisms underlying their positioning and segregation are still being elucidated. In particular, little is known about the replication of multipartite genomes in bacteria. We determined the cellular positions of the origins of the replicons in the alpha proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti and found that they are located at the poles of the cells. Our work demonstrates the conserved extreme polar localization of circular chromosome origins in these alpha proteobacteria and is also the first to specify the cellular location of origin regions from the repABC family. The cellular location of a derivative of the RK2 plasmid is distinct from that of the alpha proteobacterium genomic replicon origins but is conserved across bacteria. Colocalization experiments with the genomic replicons of A. tumefaciens revealed that the repABC replicons, although preferentially positioned at the cell pole, colocalize only rarely. For the repABC replicons in this organism, occupying discrete spatial locations may contribute to their coexistence and stable inheritance.

  View details for DOI 10.1128/JB.185.11.3384-3391.2003

  View details for Web of Science ID 000183100900016

  View details for PubMedID 12754237

  View details for PubMedCentralID PMC155372

• Cell-cycle-regulated expression and subcellular localization of the Caulobacter crescentus SMC chromosome structural protein JOURNAL OF BACTERIOLOGY Jensen, R. B., Shapiro, L. 2003; 185 (10): 3068-3075

  Abstract

  Structural maintenance of chromosomes proteins (SMCs) bind to DNA and function to ensure proper chromosome organization in both eukaryotes and bacteria. Caulobacter crescentus possesses a single SMC homolog that plays a role in organizing and segregating daughter chromosomes. Approximately 1,500 to 2,000 SMC molecules are present per cell during active growth, corresponding to one SMC complex per 6,000 to 8,000 bp of chromosomal DNA. Although transcription from the smc promoter is induced during early S phase, a cell cycle transcription pattern previously observed with multiple DNA replication and repair genes, the SMC protein is present throughout the entire cell cycle. Examination of the intracellular location of SMC showed that in swarmer cells, which do not replicate DNA, the protein forms two or three foci. Stalked cells, which are actively engaged in DNA replication, have three or four SMC foci per cell. The SMC foci appear randomly distributed in the cell. Many predivisional cells have bright polar SMC foci, which are lost upon cell division. Thus, chromosome compaction likely involves dynamic aggregates of SMC bound to DNA. The aggregation pattern changes as a function of the cell cycle both during and upon completion of chromosome replication.

  View details for DOI 10.1128/JB.185.10.3068-3075.2003

  View details for Web of Science ID 000182686900012

  View details for PubMedID 12730166

  View details for PubMedCentralID PMC154060

• tmRNA in Caulobacter crescentus is cell cycle regulated by temporally controlled transcription and RNA degradation JOURNAL OF BACTERIOLOGY Keiler, K. C., Shapiro, L. 2003; 185 (6): 1825-1830

  Abstract

  SsrA, or tmRNA, is a small RNA found in all bacteria that intervenes in selected translation reactions to target the nascent polypeptide for rapid proteolysis. We have found that the abundance of SsrA RNA in Caulobacter crescentus is regulated with respect to the cell cycle. SsrA RNA abundance increases in late G(1) phase, peaks during the G(1)-S transition, and declines in early S phase, in keeping with the reported role for SsrA in the timing of DNA replication initiation. Cell cycle regulation of SsrA RNA is accomplished by a combination of temporally controlled transcription and regulated RNA degradation. Transcription from the ssrA promoter peaks late in G(1), just before the peak in SsrA RNA abundance. SsrA RNA is stable in G(1)-phase cells and late S-phase cells but is degraded with a half-life of 4 to 5 min at the onset of S phase. This degradation is surprising, since SsrA RNA is both highly structured and highly abundant. This is the first observation of a structural RNA that is cell cycle regulated.

  View details for DOI 10.1128/JB.185.6.1825-1830.2003

  View details for Web of Science ID 000181448900009

  View details for PubMedID 12618446

  View details for PubMedCentralID PMC150134

• Functions of the CckA histidine kinase in Caulobacter cell cycle control MOLECULAR MICROBIOLOGY Jacobs, C., Ausmees, N., Cordwell, S. J., Shapiro, L., Laub, M. T. 2003; 47 (5): 1279-1290

  Abstract

  The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. Because of its critical role, CtrA activity is temporally regulated by multiple mechanisms including phosphorylation and ClpXP-dependent degradation of CtrA. The CckA histidine kinase is known to contribute to CtrA phosphorylation. We show here that genes differentially expressed in a ctrA temperature-sensitive (ts) mutant are similarly affected in a cckA ts mutant, that the phosphorylation of CckA coincides temporally with CtrA phosphorylation during the cell cycle, and that CckA is essential for viability because it is required for CtrA phosphorylation. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. CckA also positively regulates CtrA activity by a mechanism that is independent of CtrA phosphorylation. CtrA is more stable in the presence of CckA than it is absence, suggesting that CckA may also be involved, directly or indirectly, in the regulation of CtrA proteolysis.

  View details for Web of Science ID 000181056400008

  View details for PubMedID 12603734

• Temporal and spatial regulation in prokaryotic cell cycle progression and development ANNUAL REVIEW OF BIOCHEMISTRY Ryan, K. R., Shapiro, L. 2003; 72: 367-394

  Abstract

  Bacteria exhibit a high degree of intracellular organization, both in the timing of essential processes and in the placement of the chromosome, the division site, and individual structural and regulatory proteins. We examine the temporal and spatial regulation of the Caulobacter cell cycle, bacterial chromosome segregation and cytokinesis, and Bacillus subtilis sporulation. Mechanisms that control timing of cell cycle and developmental events include transcriptional cascades, regulated phosphorylation and proteolysis of signal transduction proteins, transient genetic asymmetry, and intercellular communication. Surprisingly, many signal transduction proteins are dynamically localized to specific subcellular addresses during the cell division cycle and sporulation, and proper localization is essential for their function. The Min proteins that govern division site selection in Escherichia coli may be the first example of a system that generates positional information de novo.

  View details for DOI 10.1146/annurev.biochem.72.121801.161824

  View details for PubMedID 12651741

• tmRNA is required for correct timing of DNA replication in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Keiler, K. C., Shapiro, L. 2003; 185 (2): 573-580

  Abstract

  SsrA, or tmRNA, is a small RNA that interacts with selected translating ribosomes to target the nascent polypeptides for degradation. Here we report that SsrA activity is required for normal timing of the G(1)-to-S transition in Caulobacter crescentus. A deletion of the ssrA gene, or of the gene encoding SmpB, a protein required for SsrA activity, results in a specific delay in the cell cycle during the G(1)-to-S transition. The ssrA deletion phenotype is not due to accumulation of stalled ribosomes, because the deletion is not complemented by a mutated version of SsrA that releases ribosomes but does not target proteins for degradation. Degradation of the CtrA response regulator normally coincides with initiation of DNA replication, but in strains lacking SsrA activity there is a 40-min delay between the degradation of CtrA and replication initiation. This uncoupling of initiation of replication from CtrA degradation indicates that there is an SsrA-dependent pathway required for correct timing of DNA replication.

  View details for DOI 10.1128/JB.185.2.573-580.2003

  View details for Web of Science ID 000180272600023

  View details for PubMedID 12511504

  View details for PubMedCentralID PMC145339

• Generating and exploiting polarity in bacteria SCIENCE Shapiro, L., McAdams, H. H., Losick, R. 2002; 298 (5600): 1942-1946

  Abstract

  Bacteria are often highly polarized, exhibiting specialized structures at or near the ends of the cell. Among such structures are actin-organizing centers, which mediate the movement of certain pathogenic bacteria within the cytoplasm of an animal host cell; organized arrays of membrane receptors, which govern chemosensory behavior in swimming bacteria; and asymmetrically positioned septa, which generate specialized progeny in differentiating bacteria. This polarization is orchestrated by complex and dynamic changes in the subcellular localization of signal transduction and cytoskeleton proteins as well as of specific regions of the chromosome. Recent work has provided information on how dynamic subcellular localization occurs and how it is exploited by the bacterial cell. The main task of a bacterial cell is to survive and duplicate itself. The bacterium must replicate its genetic material and divide at the correct site in the cell and at the correct time in the cell cycle with high precision. Each kind of bacterium also executes its own strategy to find nutrients in its habitat and to cope with conditions of stress from its environment. This involves moving toward food, adapting to environmental extremes, and, in many cases, entering and exploiting a eukaryotic host. These activities often involve processes that take place at or near the poles of the cell. Here we explore some of the schemes bacteria use to orchestrate dynamic changes at their poles and how these polar events execute cellular functions. In spite of their small size, bacteria have a remarkably complex internal organization and external architecture. Bacterial cells are inherently asymmetric, some more obviously so than others. The most easily recognized asymmetries involve surface structures, e.g., flagella, pili, and stalks that are preferentially assembled at one pole by many bacteria. "New" poles generated at the cell division plane differ from old poles from the previous round of cell division. Even in Escherichia coli, which is generally thought to be symmetrical, old poles are more static than new poles with respect to cell wall assembly (1), and they differ in the deposition of phospholipid domains (2). There are many instances of differential polar functions; among these is the preferential use of old poles when attaching to host cells as in the interaction of Bradyrhizobium with plant root hairs (3) or the polar pili-mediated attachment of the Pseudomonas aeruginosa pathogen to tracheal epithelia (4). An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus. The gliding motility of this bacterium is propelled by a nozzle-like structure that squirts a polysaccharide-containing slime from the pole of the cell (5). Interestingly, M. xanthus, which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells.

  View details for Web of Science ID 000179629200032

  View details for PubMedID 12471245

• The CtrA response regulator essential for Caulobacter crescentus cell-cycle progression requires a bipartite degradation signal for temporally controlled proteolysis JOURNAL OF MOLECULAR BIOLOGY Ryan, K. R., Judd, E. M., Shapiro, L. 2002; 324 (3): 443-455

  Abstract

  The two-component signaling protein CtrA activates or represses the expression of one-quarter of the cell-cycle-regulated genes in Caulobacter crescentus, integrating DNA replication, morphogenesis, and cell division. The activity of this essential protein is controlled by a positive transcriptional feedback loop, cell-cycle-regulated phosphorylation, and rapid proteolysis as cells enter S-phase at the swarmer-to-stalked cell transition and in the stalked portion of the asymmetric predivisional cell. CtrA activity must be removed from cells at the onset of DNA replication, because phosphorylated CtrA binds to and silences the origin of replication. The ClpXP protease is required for CtrA proteolysis but is present throughout the cell-cycle, so the mechanism for activating and deactivating CtrA proteolysis is unknown. Here, we identify a bipartite proteolytic signal in the CtrA response regulator consisting of two determinants that are each necessary but not sufficient for regulated degradation. One determinant is present in the last 15 amino acid residues of CtrA, particularly the terminal Ala-Ala residues, and another is located within the first 56 residues of the CtrA receiver domain. A fusion of the receiver domain and last 15 residues of CtrA to YFP is properly degraded in living cells. Although the N-terminal 56 residues contain the conserved Asp51 phosphorylation site, mutant analyses show that cell-cycle-controlled CtrA proteolysis is insensitive to the CtrA phosphorylation state. The N-terminal proteolytic determinant is predicted to reside on the surface of the receiver domain in beta-sheet 2 and alpha-helix 2.

  View details for DOI 10.1016/S0022-2836(02)01042-2

  View details for PubMedID 12445780

• Identification of a localization factor for the polar positioning of bacterial structural and regulatory proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Viollier, P. H., Sternheim, N., Shapiro, L. 2002; 99 (21): 13831-13836

  Abstract

  Polar pili biogenesis in Caulobacter involves the asymmetric localization of the CpaE and CpaC components of the pili-specific secretion apparatus to one pole of the predivisional cell followed by the biosynthesis of the pili filaments in the daughter swarmer cell. The histidine kinase signaling protein, PleC, that controls the temporal accumulation of the PilA pilin subunit is asymmetrically localized to the pole at which pili are assembled. Here we identify a protein, PodJ, that provides the positional information for the polar localization of both PleC and CpaE. The PodJ protein was found to exist in two forms, a truncated 90-kDa and a full-length 110-kDa form, each controlling a different aspect of polar development and each localizing to the cell poles at a specific time in the cell cycle. When active PleC is delocalized in a DeltapodJ mutant, the accumulation of PilA, the downstream target of PleC signaling, is impaired, providing evidence that the polar localization of this histidine kinase stimulates the response signaled by a two-component system.

  View details for DOI 10.1073/pnas.182411999

  View details for Web of Science ID 000178635700088

  View details for PubMedID 12370432

  View details for PubMedCentralID PMC129783

• A signal transduction protein cues proteolytic events critical to Caulobacter cell cycle progression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hung, D. Y., Shapiro, L. 2002; 99 (20): 13160-13165

  Abstract

  Temporally controlled proteolysis of the essential response regulator, CtrA, is critical for cell cycle progression in Caulobacter crescentus. CtrA binds to and silences the origin of replication in swarmer cells. The initiation of replication depends on the proteolysis of CtrA. We present evidence that DivK, an essential single-domain response regulator, contributes to the control of the G(1)-S transition by signaling the temporally controlled proteolysis of CtrA. In a divK-cs mutant at the restrictive temperature, the initiation of DNA replication is blocked because of the retention of CtrA. A shift of cells from restrictive to permissive temperature results in rapid degradation of CtrA, initiation of DNA replication, and the resumption of cell cycle progression, including the ordered expression of genes involved in chromosome replication and polar organelle biogenesis. CtrA binds to and regulates the promoters of two genes critical to its temporally controlled proteolysis, divK and clpP, providing a transcriptional feedback loop for the control of cell cycle progression.

  View details for DOI 10.1073/pnas.202495099

  View details for Web of Science ID 000178391700119

  View details for PubMedID 12237413

  View details for PubMedCentralID PMC130603

• DNA methylation affects the cell cycle transcription of the CtrA global regulator in Caulobacter EMBO JOURNAL Reisenauer, A., Shapiro, L. 2002; 21 (18): 4969-4977

  Abstract

  The Caulobacter chromosome changes progressively from the fully methylated to the hemimethylated state during DNA replication. These changes in DNA methylation could signal differential binding of regulatory proteins to activate or repress transcription. The gene encoding CtrA, a key cell cycle regulatory protein, is transcribed from two promoters. The P1 promoter fires early in S phase and contains a GAnTC sequence that is recognized by the CcrM DNA methyltransferase. Using analysis of CcrM mutant strains, transcriptional reporters integrated at different sites on the chromosome, and a ctrA P1 mutant, we demonstrate that transcription of the P1 promoter is repressed by DNA methylation. Moreover moving the native ctrA gene to a position near the chromosomal terminus, which delays the conversion of the ctrA promoter from the fully to the hemimethylated state until late in the cell cycle, inhibited ctrA P1 transcription, and altered the time of accumulation of the CtrA protein and the size distribution of swarmer cells. Together, these results show that CcrM-catalyzed methylation adds another layer of control to the regulation of ctrA expression.

  View details for Web of Science ID 000178123100022

  View details for PubMedID 12234936

• A dynamically localized histidine kinase controls the asymmetric distribution of polar pili proteins EMBO JOURNAL Viollier, P. H., Sternheim, N., Shapiro, L. 2002; 21 (17): 4420-4428

  Abstract

  Each cell division in Caulobacter crescentus is asymmetric, yielding a swarmer cell with several polar pili and a non-piliated stalked cell. To identify factors contributing to the asymmetric biogenesis of polar pili, cytological studies of pilus assembly components were performed. We show here that the CpaC protein, which is thought to form the outer membrane pilus secretion channel, and its assembly factor, CpaE, are localized to the cell pole prior to the polymerization of the pilus filament. We demonstrate that the PleC histidine kinase, a two-component signal transduction protein shown previously to localize to the piliated cell pole before and during pilus assembly, controls the accumulation of the pilin subunit, PilA. Using an inactive form of PleC (PleCH610A) that lacks the catalytic histidine residue, we provide evidence that PleC activity is responsible for the asymmetric distribution of CpaE and itself to only one of the two cell poles. Thus, a polar signal transduction protein controls its own asymmetric location as well as that of a factor assembling a polar organelle.

  View details for Web of Science ID 000177770100004

  View details for PubMedID 12198144

• Biomolecular screening with encoded porous-silicon photonic crystals NATURE MATERIALS Cunin, F., Schmedake, T. A., Link, J. R., Li, Y. Y., Koh, J., Bhatia, S. N., Sailor, M. J. 2002; 1 (1): 39-41

  Abstract

  Strategies to encode or label small particles or beads for use in high-throughput screening and bioassay applications focus on either spatially differentiated, on-chip arrays or random distributions of encoded beads. Attempts to encode large numbers of polymeric, metallic or glass beads in random arrays or in fluid suspension have used a variety of entities to provide coded elements (bits)--fluorescent molecules, molecules with specific vibrational signatures, quantum dots, or discrete metallic layers. Here we report a method for optically encoding micrometre-sized nanostructured particles of porous silicon. We generate multilayered porous films in crystalline silicon using a periodic electrochemical etch. This results in photonic crystals with well-resolved and narrow optical reflectivity features, whose wavelengths are determined by the etching parameters. Millions of possible codes can be prepared this way. Micrometre-sized particles are then produced by ultrasonic fracture, mechanical grinding or by lithographic means. A simple antibody-based bioassay using fluorescently tagged proteins demonstrates the encoding strategy in biologically relevant media.

  View details for DOI 10.1038/nmat702

  View details for Web of Science ID 000181498400021

  View details for PubMedID 12618846

• Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H. 2002; 99 (7): 4632-4637

  Abstract

  Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression.

  View details for DOI 10.1073/pnas.062065699

  View details for Web of Science ID 000174856000089

  View details for PubMedID 11930012

  View details for PubMedCentralID PMC123699

• Dynamic localization of proteins and DNA during a bacterial cell cycle NATURE REVIEWS MOLECULAR CELL BIOLOGY Jensen, R. B., Wang, S. C., Shapiro, L. 2002; 3 (3): 167-176

  Abstract

  A cellular differentiation programme that culminates in an asymmetric cell division is an integral part of the cell cycle in the bacterium Caulobacter crescentus. Recent work has uncovered mechanisms that ensure the execution of many events at different times during the cell cycle and at specific places in the cell. Surprisingly, in this one-micron bacterial cell, the dynamic spatial disposition of regulatory proteins, structural proteins and specific regions of the chromosome are important components of both cell-cycle progression and the generation of daughter cells with different cell fates.

  View details for DOI 10.1038/nrm758

  View details for Web of Science ID 000174229800021

  View details for PubMedID 11994737

• Control of chromosome replication in Caulobacter crescentus ANNUAL REVIEW OF MICROBIOLOGY Marczynski, G. T., Shapiro, L. 2002; 56: 625-656

  Abstract

  Caulobacter crescentus permits detailed analysis of chromosome replication control during a developmental cell cycle. Its chromosome replication origin (Cori) may be prototypical of the large and diverse class of alpha-proteobacteria. Cori has features that both affiliate and distinguish it from the Escherichia coli chromosome replication origin. For example, requirements for DnaA protein and RNA transcription affiliate both origins. However, Cori is distinguished by several features, and especially by five binding sites for the CtrA response regulator protein. To selectively repress and limit chromosome replication, CtrA receives both protein degradation and protein phosphorylation signals. The signal mediators, proteases, response regulators, and kinases, as well as Cori DNA and the replisome, all show distinct patterns of temporal and spatial organization during cell cycle progression. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria.

  View details for DOI 10.1146/annurev.micro.56.012302.161103

  View details for Web of Science ID 000179054200025

  View details for PubMedID 12142494

• A moving DNA replication factory in Caulobacter crescentus EMBO JOURNAL Jensen, R. B., Wang, S. C., Shapiro, L. 2001; 20 (17): 4952-4963

  Abstract

  The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.

  View details for Web of Science ID 000170907900034

  View details for PubMedID 11532959

• Conserved promoter motif is required for cell cycle timing of dnaX transcription in Caulobacter JOURNAL OF BACTERIOLOGY Keiler, K. C., Shapiro, L. 2001; 183 (16): 4860-4865

  Abstract

  Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including dnaX, dnaA, dnaN, gyrB, and dnaK, are induced at the G(1)/S-phase transition. By analyzing mutations in the dnaX promoter, we identified a motif between the -10 and -35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro.

  View details for Web of Science ID 000170118600021

  View details for PubMedID 11466289

  View details for PubMedCentralID PMC99540

• A homolog of the CtrA cell cycle regulator is present and essential in Sinorhizobium meliloti JOURNAL OF BACTERIOLOGY Barnett, M. J., Hung, D. Y., Reisenauer, A., Shapiro, L., Long, S. R. 2001; 183 (10): 3204-3210

  Abstract

  During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.

  View details for Web of Science ID 000168535000028

  View details for PubMedID 11325950

  View details for PubMedCentralID PMC95222

• The CcrM DNA methyltransferase of Agrobacterium tumefaciens is essential, and its activity is cell cycle regulated JOURNAL OF BACTERIOLOGY Kahng, L. S., Shapiro, L. 2001; 183 (10): 3065-3075

  Abstract

  DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in Escherichia coli and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog of Agrobacterium tumefaciens was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the -35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of Agrobacterium cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling ccrM expression.

  View details for Web of Science ID 000168535000012

  View details for PubMedID 11325934

  View details for PubMedCentralID PMC95206

• Single-molecule imaging in Caulobacter crescentus. Paige, M. F., Judd, E., Shapiro, L., Moerner, W. E. AMER CHEMICAL SOC. 2001: U285–U285

  View details for Web of Science ID 000168824801666

• Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jacobs, C., Hung, D., Shapiro, L. 2001; 98 (7): 4095-4100

  Abstract

  We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.

  View details for Web of Science ID 000167833700088

  View details for PubMedID 11274434

  View details for PubMedCentralID PMC31185

• Complete genome sequence of Caulobacter crescentus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., Heidelberg, J. F., Alley, M. R., Ohta, N., Maddock, J. R., Potocka, I., Nelson, W. C., Newton, A., Stephens, C., Phadke, N. D., Ely, B., DeBoy, R. T., Dodson, R. J., Durkin, A. S., Gwinn, M. L., Haft, D. H., Kolonay, J. F., Smit, J., Craven, M. B., Khouri, H., Shetty, J., Berry, K., Utterback, T., Tran, K., Wolf, A., Vamathevan, J., Ermolaeva, M., White, O., Salzberg, S. L., Venter, J. C., Shapiro, L., Fraser, C. M. 2001; 98 (7): 4136-4141

  Abstract

  The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

  View details for Web of Science ID 000167833700095

  View details for PubMedID 11259647

  View details for PubMedCentralID PMC31192

• Global analysis of the genetic network controlling a bacterial cell cycle SCIENCE Laub, M. T., McAdams, T. H., Feldblyum, T., Fraser, C. M., Shapiro, L. 2000; 290 (5499): 2144-2148

  Abstract

  This report presents full-genome evidence that bacterial cells use discrete transcription patterns to control cell cycle progression. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. We conclude that in bacteria, as in yeast, (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding proteins that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA member of the two-component signal transduction family, is directly or indirectly involved in the control of 26% of the cell cycle-regulated genes.

  View details for Web of Science ID 000165870600056

  View details for PubMedID 11118148

• Proteins on the move: dynamic protein localization in prokaryotes TRENDS IN CELL BIOLOGY Jensen, R. B., Shapiro, L. 2000; 10 (11): 483-488

  Abstract

  Despite their small size and lack of obvious intracellular structures, bacteria have a complex and dynamic intracellular organization. Recent work has shown that many proteins, and even regions of the chromosome, are localized to specific subcellular regions that can change over time, sometimes extraordinarily fast. Protein function can depend on cellular position, so the analysis of the intracellular location of a protein can be crucial for understanding its activity. Because regulatory proteins are among those that reside at specific cellular sites, it is now necessary to consider three-dimensional organization when describing the genetic networks that control bacterial cells.

  View details for Web of Science ID 000165066300004

  View details for PubMedID 11050420

• tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Keiler, K. C., Shapiro, L., Williams, K. P. 2000; 97 (14): 7778-7783

  Abstract

  A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. This RNA encodes a peptide tag that is incorporated at the end of the aberrant polypeptide and targets it for proteolysis. We have identified a circularly permuted version of the tmRNA gene in alpha-proteobacteria as well as in a lineage of cyanobacteria. The genes in these two groups seem to have arisen from two independent permutation events. As a result of the altered genetic structure, these tmRNAs are composed of two distinct RNA molecules. The mature two-piece tmRNAs are predicted to have a tRNA-like domain and an mRNA-like domain similar to those of standard one-piece tmRNAs, with a break located in the loop containing the tag reading frame. A related sequence was found in the mitochondrial genome of Reclinomonas americana, but only the tRNA-like portion is retained. Although several sequence and structural motifs that are conserved among one-piece tmRNAs have been lost, the alpha-proteobacterium Caulobacter crescentus produces a functional two-piece tmRNA.

  View details for Web of Science ID 000088048400024

  View details for PubMedID 10884408

  View details for PubMedCentralID PMC16621

• Identification and cell cycle control of a novel pilus system in Caulobacter crescentus EMBO JOURNAL Skerker, J. M., Shapiro, L. 2000; 19 (13): 3223-3234

  Abstract

  Pilus assembly in CAULOBACTER: crescentus occurs during a short period of the cell cycle and pili are only present at the flagellar pole of the swarmer cell. Here we report a novel assay to visualize pili by light microscopy that led to the purification of CAULOBACTER: pili and the isolation of a cluster of seven genes, including the major pilin subunit gene pilA. This gene cluster encodes a novel group of pilus assembly proteins. We have shown that the pilA promoter is activated late in the cell cycle and that transcription of the pilin subunit plays an important role in the timing of pilus assembly. pilA transcription is regulated by the global two-component response regulator CtrA, which is essential for the expression of multiple cell cycle events, providing a direct link between assembly of the pilus organelle and bacterial cell cycle control.

  View details for Web of Science ID 000088132600007

  View details for PubMedID 10880436

  View details for PubMedCentralID PMC313932

• The Brucella abortus CcrM DNA methyltransferase is essential for viability, and its overexpression attenuates intracellular replication in murine macrophages JOURNAL OF BACTERIOLOGY Robertson, G. T., Reisenauer, A., Wright, R., Jensen, R. B., Jensen, A., Shapiro, L., Roop, R. M. 2000; 182 (12): 3482-3489

  Abstract

  The CcrM DNA methyltransferase of the alpha-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes in B. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrM copy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.

  View details for Web of Science ID 000087307700023

  View details for PubMedID 10852881

  View details for PubMedCentralID PMC101938

• Dynamic spatial regulation in the bacterial cell CELL Shapiro, L., Losick, R. 2000; 100 (1): 89-98

  View details for Web of Science ID 000084722600008

  View details for PubMedID 10647934

• Chromosome segregation during the prokaryotic cell division cycle CURRENT OPINION IN CELL BIOLOGY Jensen, R. B., Shapiro, L. 1999; 11 (6): 726-731

  Abstract

  Recent work has dramatically changed our view of chromosome segregation in bacteria. Rather than being a passive process, it involves rapid movement of parts of the circular chromosome. Several genes involved in chromosome segregation have been identified, and the analysis of their functions and intracellular localization are beginning to shed light on the mechanisms that ensure efficient chromosome segregation.

  View details for Web of Science ID 000084010000013

  View details for PubMedID 10600703

• Differential localization of two histidine kinases controlling bacterial cell differentiation MOLECULAR CELL Wheeler, R. T., Shapiro, L. 1999; 4 (5): 683-694

  Abstract

  The bacterium C. crescentus coordinates cellular differentiation and cell cycle progression via a network of signal transduction proteins. Here, we demonstrate that the antagonistic DivJ and PleC histidine kinases that regulate polar differentiation are differentially localized as a function of the cell cycle. The DivJ kinase localizes to the stalked pole in response to a signal at the G1-to-S transition, while the PleC kinase is localized to the flagellar pole in swarmer and predivisional cells but is dispersed throughout the cell in the stalked cell. PleC, which is required for DivJ localization, may provide the cue at the G1-to-S transition that directs the polar positioning of DivJ. The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle.

  View details for Web of Science ID 000083885400003

  View details for PubMedID 10619016

• The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jensen, R. B., Shapiro, L. 1999; 96 (19): 10661-10666

  Abstract

  The highly conserved SMC (Structural Maintenance of Chromosomes) proteins function in chromosome condensation, segregation, and other aspects of chromosome dynamics in both eukaryotes and prokaryotes. A null mutation in the Caulobacter crescentus smc gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. Chromosome segregation in wild-type and smc null mutant cells was examined by monitoring the intracellular localization of the replication origin and terminus by using fluorescence in situ hybridization. In wild-type cells, the origin is located at the flagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. The terminus moves from the end of the swarmer cell opposite the origin to midcell. A subpopulation of the smc null mutant cells had mislocalized origins or termini, showing that the smc null mutation gives DNA segregation defects. Nucleoid morphology was also abnormal. Thus, we propose that the Caulobacter chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. The specific cell cycle arrest in the smc null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation.

  View details for Web of Science ID 000082574100028

  View details for PubMedID 10485882

  View details for PubMedCentralID PMC17939

• Bacterial DNA methylation: a cell cycle regulator? JOURNAL OF BACTERIOLOGY Reisenauer, A., Kahng, L. S., McCollum, S., Shapiro, L. 1999; 181 (17): 5135-5139

  View details for Web of Science ID 000082318000001

  View details for PubMedID 10464180

  View details for PubMedCentralID PMC94015

• Changing views on the nature of the bacterial cell: from biochemistry to cytology JOURNAL OF BACTERIOLOGY Losick, R., Shapiro, L. 1999; 181 (14): 4143-4145

  View details for Web of Science ID 000081360100001

  View details for PubMedID 10400568

  View details for PubMedCentralID PMC93912

• Feedback control of a master bacterial cell-cycle regulator PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Domian, I. J., Reisenauer, A., Shapiro, L. 1999; 96 (12): 6648-6653

  Abstract

  The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that ctrA expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.

  View details for Web of Science ID 000080842200015

  View details for PubMedID 10359766

  View details for PubMedCentralID PMC21969

• Bacterial cell division: A moveable feast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jacobs, C., Shapiro, L. 1999; 96 (11): 5891-5893

  View details for Web of Science ID 000080527100001

  View details for PubMedID 10339512

  View details for PubMedCentralID PMC34200

• Cell cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division CELL Jacobs, C., Domian, I. J., Maddock, J. R., Shapiro, L. 1999; 97 (1): 111-120

  Abstract

  The master CtrA response regulator functions in Caulobacter to repress replication initiation in different phases of the cell cycle. Here, we identify an essential histidine kinase, CckA, that is responsible for CtrA activation by phosphorylation. Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. Removal of the membrane-spanning region of CckA results in loss of polar localization and cell death. We propose that polar CckA functions to activate CtrA just after the initiation of DNA replication, thereby preventing premature reinitiations of chromosome replication. Thus, dynamic changes in cellular location of critical signal proteins provide a novel mechanism for the control of the prokaryote cell cycle.

  View details for Web of Science ID 000079843900013

  View details for PubMedID 10199407

• The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle JOURNAL OF BACTERIOLOGY Reisenauer, A., Quon, K., Shapiro, L. 1999; 181 (8): 2430-2439

  Abstract

  In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in the Caulobacter crescentus cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. PfliQ is activated earlier than PccrM. Phosphorylated CtrA (CtrA approximately P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in PccrM significantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrM expression. Our data indicate that differences in the affinity of CtrA approximately P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. However, the timing of fliQ transcription but not of ccrM transcription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA approximately P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA approximately P and its interaction with accessory proteins influence the temporal expression of fliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle.

  View details for Web of Science ID 000079706600016

  View details for PubMedID 10198005

  View details for PubMedCentralID PMC93667

• Protein localization during the Caulobacter crescentus cell cycle CURRENT OPINION IN MICROBIOLOGY Wheeler, R. T., Gober, J. W., Shapiro, L. 1998; 1 (6): 636-642

  Abstract

  New research on bacterial cells has demonstrated that they have a dynamic and complex subcellular organization. Work in Caulobacter crescentus shows that essential and nonessential proteins localize to discrete positions in the cell as a function of cell-cycle progression. The flagellum and chemotaxis receptor are asymmetrically localized to a single pole in the predivisional cell by coordinated proteolysis and transcriptional regulation. Cell type- and compartment-specific localization of the CtrA global transcriptional regulator is essential for proper cell-cycle progression, and subcellular localization of key chromosome partitioning proteins is correlated with proper nucleoid segregation. Given this structural complexity, we are driven to ask how localization is achieved, and to what end.

  View details for Web of Science ID 000077377300004

  View details for PubMedID 10066543

• DNA replication - Bringing the mountain to mohammed SCIENCE Losick, R., Shapiro, L. 1998; 282 (5393): 1430-1431

  View details for Web of Science ID 000077110800030

  View details for PubMedID 9867650

• Microbial asymmetric cell division: Localization of cell fate determinants CURRENT OPINION IN GENETICS & DEVELOPMENT Jacobs, C., Shapiro, L. 1998; 8 (4): 386-391

  Abstract

  The genetic mechanisms that control asymmetric cell divisions--yielding progeny cells that differ from one another--have been conserved among prokaryotes, eukaryotic microbes, and higher organisms. All use the paradigm of regulatory protein localization as a way of translating genetic information into three-dimensional space.

  View details for Web of Science ID 000075603800002

  View details for PubMedID 9729712

• A membrane-associated protein, FliX, is required for an early step in Caulobacter flagellar assembly JOURNAL OF BACTERIOLOGY Mohr, C. D., MacKichan, J. K., Shapiro, L. 1998; 180 (8): 2175-2185

  Abstract

  The ordered assembly of the Caulobacter crescentus flagellum is accomplished in part through the organization of the flagellar structural genes in a regulatory hierarchy of four classes. Class II genes are the earliest to be expressed and are activated at a specific time in the cell cycle by the CtrA response regulator. In order to identify gene products required for early events in flagellar assembly, we used the known phenotypes of class II mutants to identify new class II flagellar genes. In this report we describe the isolation and characterization of a flagellar gene, fliX. A fliX null mutant is nonmotile, lacks a flagellum, and exhibits a marked cell division defect. Epistasis experiments placed fliX within class II of the flagellar regulatory hierarchy, suggesting that FliX functions at an early stage in flagellar assembly. The fliX gene encodes a 15-kDa protein with a putative N-terminal signal sequence. Expression of fliX is under cell cycle control, with transcription beginning relatively early in the cell cycle and peaking in Caulobacter predivisional cells. Full expression of fliX was found to be dependent on ctrA, and DNase I footprinting analysis demonstrated a direct interaction between CtrA and the fliX promoter. The fliX gene is located upstream and is divergently transcribed from the class III flagellar gene flgI, which encodes the basal body P-ring monomer. Analysis of the fliX-flgI intergenic region revealed an arrangement of cis-acting elements similar to that of another set of Caulobacter class II and class III flagellar genes, fliL-flgF, that is also divergently transcribed. In parallel with the FliL protein, FliX copurifies with the membrane fraction, and although its expression is cell cycle controlled, the protein is present throughout the cell cycle.

  View details for Web of Science ID 000073041000027

  View details for PubMedID 9555902

  View details for PubMedCentralID PMC107146

• A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Berdis, A. J., Lee, I., Coward, J. K., Stephens, C., Wright, R., Shapiro, L., Benkovic, S. J. 1998; 95 (6): 2874-2879

  Abstract

  The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated DNA (DNAHM) substrates. Catalytic efficiency is significantly enhanced with a DNAHM substrate. Biphasic kinetic behavior during methyl incorporation is observed when unmethylated or DNAHM substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the release of enzyme from methylated DNA product. The enzyme is thermally inactivated at 30 degrees C within 20 min; this process is substantially decreased in the presence of saturating concentrations of DNAHM, suggesting that the enzyme preferentially binds DNA before S-adenosylmethionine. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order of product release in which S-adenosylhomocysteine is released from enzyme before fully methylated DNA. The kinetic behavior and activity of the enzyme are consistent with the temporal constraints during the cell cycle-regulated methylation of newly replicated chromosomal DNA.

  View details for Web of Science ID 000072596200032

  View details for PubMedID 9501183

  View details for PubMedCentralID PMC19662

• Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Quon, K. C., Yang, B., Domian, I. J., Shapiro, L., Marczynski, G. T. 1998; 95 (1): 120-125

  Abstract

  Caulobacter crescentus divides asymmetrically generating two distinct cell types at each cell division: a stalked cell competent for DNA replication, and a swarmer cell that is unable to initiate DNA replication until it differentiates into a stalked cell later in the cell cycle. The CtrA protein, a member of the response regulator family of the two-component signal transduction system, controls multiple cell cycle processes in Caulobacter and is present in swarmer cells but absent from stalked cells. We report that CtrA binds five sites within the chromosome replication origin in vitro. These sites overlap an essential DnaA box and a promoter in the origin that is essential for replication initiation. Analysis of mutant alleles of ctrA and point mutations in one of the CtrA binding sites in the origin demonstrate that CtrA represses replication in vivo. CtrA-mediated repression at the origin thus restricts replication to the stalked cell type. Thus, the direct coupling of chromosome replication with the cell cycle is mediated by the ubiquitous two-component signaling proteins.

  View details for Web of Science ID 000071429500027

  View details for PubMedID 9419339

  View details for PubMedCentralID PMC18146

• Transcriptional analysis of the Caulobacter 4.5 S RNA ffs gene and the physiological basis of an ffs mutant with a Ts phenotype JOURNAL OF MOLECULAR BIOLOGY Winzeler, E., Wheeler, R., Shapiro, L. 1997; 272 (5): 665-676

  Abstract

  A temperature-sensitive (ts) mutation in the ffs gene, encoding 4.5 S RNA, gives rise to cell division and DNA replication defects in Caulobacter crescentus. The ffs gene is transcribed throughout the cell-cycle and is transcribed at similar rates in mutant (ffs36) and wild-type strains, but in the mutant the 4.5 S RNA is unstable leading to lower 4.5 S RNA levels. The ffs36 phenotype results from a single base change in one of the non-conserved stems of the mature RNA, and is completely rescued by a compensating mutation in the opposite strand, providing confirmation of the predicted secondary structure of the 4.5 S RNA. The Caulobacter ffs gene was shown to be functionally comparable to the Escherichia coli ffs gene by complementation. Comparison of the ffs36 strain to a ts secA strain of Caulobacter, also having cell-cycle and DNA replication phenotypes, showed that both exhibit a permanent induction of a heat shock response at the restrictive temperature. To explain the phenotype of both the secA and ffs36 strains, we propose that a cell-cycle checkpoint prevents further progression through the cell-cycle in response to increased intracellular levels of heat shock and misfolded proteins.

  View details for Web of Science ID A1997YB26700002

  View details for PubMedID 9368649

• Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system JOURNAL OF BACTERIOLOGY Stephens, C., Mohr, C., Boyd, C., Maddock, J., Gober, J., Shapiro, L. 1997; 179 (17): 5355-5365

  Abstract

  Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly.

  View details for Web of Science ID A1997XT77200016

  View details for PubMedID 9286988

  View details for PubMedCentralID PMC179404

• Bacterial protein secretion - a target for new antibiotics? CHEMISTRY & BIOLOGY Stephens, C., Shapiro, L. 1997; 4 (9): 637-641

  Abstract

  The heavy use of antibiotics over recent decades has resulted in widespread resistance of bacteria to many drugs. Overcoming resistance requires new approaches to antibiotic development, including the exploitation of new targets in the bacterial cell. Protein secretion is essential for bacterial cell growth and virulence, so it could be a suitable target for new therapeutic agents.

  View details for Web of Science ID A1997YA84900001

  View details for PubMedID 9331405

• The CcrM DNA methyltransferase is widespread in the alpha subdivision of proteobacteria, and its essential functions are conserved in Rhizobium meliloti and Caulobacter crescentus JOURNAL OF BACTERIOLOGY Wright, R., Stephens, C., Shapiro, L. 1997; 179 (18): 5869-5877

  Abstract

  The Caulobacter crescentus DNA methyltransferase CcrM (M.CcrMI) methylates the adenine residue in the sequence GANTC. The CcrM DNA methyltransferase is essential for viability, but it does not appear to be part of a DNA restriction-modification system. CcrM homologs are widespread in the alpha subdivision of gram-negative bacteria. We have amplified and sequenced a 258-bp region of the cerM gene from several of these bacteria, including Rhizobium meliloti, Brucella abortus, Agrobacterium tumefaciens, and Rhodobacter capsulatus. Alignment of the deduced amino acid sequences revealed that these proteins constitute a highly conserved DNA methyltransferase family. Isolation of the full-length ccrM genes from the aquatic bacterium C. crescentus, the soil bacterium R. meliloti, and the intracellular pathogen B. abortus showed that this sequence conservation extends over the entire protein. In at least two alpha subdivision bacteria, R. meliloti and C. crescentus, CcrM-mediated methylation has important cellular functions. In both organisms, CcrM is essential for viability. Overexpression of CcrM in either bacterium results in defects in cell division and cell morphology and in the initiation of DNA replication. Finally, the C. crescentus and R. meliloti ccrM genes are functionally interchangeable, as the complemented strains are viable and the chromosomes are methylated. Thus, in both R. meliloti and C. crescentus, CcrM methylation is an integral component of the cell cycle. We speculate that CcrM-mediated DNA methylation is likely to have similar roles among alpha subdivision bacteria.

  View details for Web of Science ID A1997XV69900030

  View details for PubMedID 9294447

  View details for PubMedCentralID PMC179479

• Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle CELL Domian, I. J., Quon, K. C., Shapiro, L. 1997; 90 (3): 415-424

  Abstract

  The global transcriptional regulator CtrA controls multiple events in the Caulobacter cell cycle, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems and is activated by phosphorylation. We report here that this phosphorylation signal enters the cell cycle at mid S phase. In addition, CtrA function is modulated by temporally and spatially controlled proteolysis. When an active CtrA protein is present at the wrong time in the cell cycle, owing to expression of a mutant CtrA derivative that is active in the absence of phosphorylation and is not turned over during the cell cycle, the G1-to-S transition is blocked and the cell cycle aborts. Thus, both phosphorylation and proteolysis are critical determinants of bacterial cell cycle control in a manner that is analogous to the control of the eukaryotic cell cycle.

  View details for Web of Science ID A1997XQ06300006

  View details for PubMedID 9267022

• Translation of the leaderless Caulobacter dnaX mRNA JOURNAL OF BACTERIOLOGY Winzeler, E., Shapiro, L. 1997; 179 (12): 3981-3988

  Abstract

  The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.

  View details for Web of Science ID A1997XE30000021

  View details for PubMedID 9190815

  View details for PubMedCentralID PMC179208

• Protein localization and cell fate in bacteria SCIENCE Shapiro, L., Losick, R. 1997; 276 (5313): 712-718

  Abstract

  A major breakthrough in understanding the bacterial cell is the discovery that the cell is highly organized at the level of protein localization. Proteins are positioned at particular sites in bacteria, including the cell pole, the incipient division plane, and the septum. Differential protein localization can control DNA replication, chromosome segregation, and cytokinesis and is responsible for generating daughter cells with different fates upon cell division. Recent discoveries have revealed that progression through the cell cycle and communication between cellular compartments are mediated by two-component signal transduction systems and signaling pathways involving transcription factor activation by proteolytic processing. Asymmetric cell division in Caulobacter crescentus and sporulation in Bacillus subtilis are used as paradigms for the control of the cell cycle and cellular morphogenesis in bacterial cells.

  View details for Web of Science ID A1997WW90000037

  View details for PubMedID 9115191

• Transcription of genes encoding DNA replication proteins is coincident with cell cycle control of DNA replication in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Roberts, R. C., Shapiro, L. 1997; 179 (7): 2319-2330

  Abstract

  DNA replication in the dimorphic bacterium Caulobacter crescentus is tightly linked to its developmental cell cycle. The initiation of chromosomal replication occurs concomitantly with the transition of the motile swarmer cell to the sessile stalked cell. To identify the signals responsible for the cell cycle control of DNA replication initiation, we have characterized a region of the C. crescentus chromosome containing genes that are all involved in DNA replication or recombination, including dnaN, recF, and gyrB. The essential dnaN gene encodes a homolog of the Escherichia coli beta subunit of DNA polymerase III. It is transcribed from three promoters; one is heat inducible, and the other two are induced at the transition from swarmer to stalked cell, coincident with the initiation of DNA replication. The single gyrB promoter is induced at the same time point in the cell cycle. These promoters, as well as those for several other genes encoding DNA replication proteins that are induced at the same time in the cell cycle, share two sequence motifs, suggesting that they represent a family whose transcription is coordinately regulated.

  View details for Web of Science ID A1997WQ86300029

  View details for PubMedID 9079919

  View details for PubMedCentralID PMC178970

• Bacterial chromosome segregation: Is there a mitotic apparatus? CELL Wheeler, R. T., Shapiro, L. 1997; 88 (5): 577-579

  View details for Web of Science ID A1997WM41300002

  View details for PubMedID 9054496

• Isolation and characterization of a xylose-dependent promoter from Caulobacter crescentus JOURNAL OF BACTERIOLOGY Meisenzahl, A. C., Shapiro, L., Jenal, U. 1997; 179 (3): 592-600

  Abstract

  An inducible promoter is a useful tool for the controlled expression of a given gene. Accordingly, we identified, cloned, and sequenced a chromosomal locus, xylX, from Caulobacter crescentus which is required for growth on xylose as the sole carbon source and showed that transcription from a single site is dependent on the presence of xylose in the growth medium. P(xylX) promoter activity was determined as a function of the composition of the growth medium both in single copy and on a plasmid using different reporter genes. One hundred micromolar exogenously added xylose was required for maximal induction of P(xylX) in a strain that is unable to metabolize xylose. P(xylX) activity was induced immediately after the addition of xylose and repressed almost completely when xylose was removed from the growth medium. In addition to the strong transcriptional control, the expression of xylX is also regulated on the translational level.

  View details for Web of Science ID A1997WE44000004

  View details for PubMedID 9006009

  View details for PubMedCentralID PMC178736

• A novel promoter motif for Caulobacter cell cycle-controlled DNA replication genes JOURNAL OF MOLECULAR BIOLOGY Winzeler, E., Shapiro, L. 1996; 264 (3): 412-425

  Abstract

  Caulobacter crescentus contains a single chromosome that is replicated once during a defined period in the cell cycle. The onset of replication coincides with the stimulation of transcription of several genes involved in the replication process. Analysis of the C. crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, identified the dnaX transcription start site and showed that activity from the dnaX promoter is stimulated fourfold at the onset of DNA replication. We have identified a conserved sequence motif that is present in the promoter of dnaX and several other genes involved in the replication of DNA, all of which show an induction of transcription at the onset of chromosome replication. Independent mutations in the conserved sequence that lies between the -10 and -35 regions increased transcription, suggesting that a repressor may bind at this site. We propose that the coincident transcriptional activation of several dna genes at the swarmer to stalked cell transition occurs in response to cell cycle regulatory factors, in a manner analogous to the transient transcriptional regulation of flagellar and DNA methylation genes later in the cell cycle.

  View details for Web of Science ID A1996VW70900002

  View details for PubMedID 8969294

• The control of temporal and spatial organization during the Caulobacter cell cycle CURRENT OPINION IN GENETICS & DEVELOPMENT Domian, I. J., Quon, K. C., Shapiro, L. 1996; 6 (5): 538-544

  Abstract

  The Caulobacter cell cycle exhibits time-dependent expression of differentiation events. These include the morphological transition of a swarmer cell to a replication-competent stalked cell and the subsequent polarized distribution of specific gene products that results in an asymmetric predivisional cell. Cell division then yields a new swarmer cell and a stem-cell-like stalked cell. Two-component signal transduction proteins involved in cell cycle control and proteins required for cell division and flagellar biogenesis have been shown to be regulated temporally and spatially during the cell cycle. The mechanisms underlying this regulation include protein phosphorylation and proteolysis.

  View details for Web of Science ID A1996VP61500004

  View details for PubMedID 8939718

• Bacterial pathogenesis: Delivering the payload CURRENT BIOLOGY Stephens, C., Shapiro, L. 1996; 6 (8): 927-930

  Abstract

  A specialized protein secretion pathway is used by some Gram-negative bacterial pathogens for delivery of virulence factors directly into mammalian host cells. This pathway is parallel to, and probably evolved from, a system used for construction of the bacterial flagellum.

  View details for Web of Science ID A1996VB22500010

• Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation GENES & DEVELOPMENT Wright, R., Stephens, C., Zweiger, G., Shapiro, L., Alley, M. R. 1996; 10 (12): 1532-1542

  Abstract

  CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional. cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development.

  View details for Web of Science ID A1996UU11700009

  View details for PubMedID 8666236

• Cell cycle-controlled proteolysis of a flagellar motor protein that is asymmetrically distributed in the Caulobacter predivisional cell EMBO JOURNAL Jenal, U., Shapiro, L. 1996; 15 (10): 2393-2406

  Abstract

  Flagellar biogenesis and release are developmental events tightly coupled to the cell cycle of Caulobacter crescentus. A single flagellum is assembled at the swarmer pole of the predivisional cell and is released later in the cell cycle. Here we show that the MS-ring monomer FliF, a central motor component that anchors the flagellum in the cell membrane, is synthesized only in the predivisional cell and is integrated into the membrane at the incipient swarmer cell pole, where it initiates flagellar assembly. FliF is proteolytically turned over during swarmer-to-stalked cell differentiation, coinciding with the loss of the flagellum, suggesting that its degradation is coupled to flagellar release. The membrane topology of FliF was determined and a region of the cytoplasmic C-terminal domain was shown to be required for the interaction with a component of the motor switch. The very C-terminal end of FliF contains a turnover determinant, required for the cell cycle-dependent degradation of the MS-ring. The cell cycle-dependent proteolysis of FliF and the targeting of FliF to the swarmer pole together contribute to the asymmetric localization of the MS-ring in the predivisional cell.

  View details for Web of Science ID A1996UM40600009

  View details for PubMedID 8665847

  View details for PubMedCentralID PMC450171

• Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE JOURNAL OF BACTERIOLOGY Roberts, R. C., Toochinda, C., Avedissian, M., Baldini, R. L., Gomes, S. L., Shapiro, L. 1996; 178 (7): 1829-1841

  Abstract

  In response to elevated temperature, both prokaryotic and eukaryotic cells increase expression of a small family of chaperones. The regulatory network that functions to control the transcription of the heat shock genes in bacteria includes unique structural motifs in the promoter region of these genes and the expression of alternate sigma factors. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. We report the identification of another C. crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog. Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element. This suggests a role for HrcA in negative regulation of heat shock gene expression. We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE. Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock. Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions. C. crescentus GrpE, shown to be essential for viability at low and high temperatures, complemented an Escherichia coli delta grpE strain in spite of significant differences in the N- and C-terminal regions of these two proteins, demonstrating functional conservation of this important stress protein.

  View details for Web of Science ID A1996UD48400009

  View details for PubMedID 8606155

  View details for PubMedCentralID PMC177876

• Regulation of a heat shock sigma(32) homolog in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Reisenauer, A., Mohr, C. D., Shapiro, L. 1996; 178 (7): 1919-1927

  Abstract

  High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase sigma32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus sigma32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli sigma32 and cross-reacts with a monoclonal antibody to E. coli sigma32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in sigma32 levels after a temperature shift from 30 to 42 degrees C similar to that described for E. coli. In addition, increasing the cellular content of sigma32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30 degrees C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and sigma32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to sigma32 expression in this organism. Both the rpoH gene and sigma32 protein were expressed constitutively throughout the cell cycle at 30 degrees C. The isolation of rpoH provides an important tool for future studies of the role of sigma32 in the normal physiology of C. crescentus.

  View details for Web of Science ID A1996UD48400020

  View details for PubMedID 8606166

  View details for PubMedCentralID PMC177887

• A cell cycle-regulated bacterial DNA methyltransferase is essential for viability PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stephens, C., Reisenauer, A., Wright, R., Shapiro, L. 1996; 93 (3): 1210-1214

  Abstract

  The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well. Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA. CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

  View details for Web of Science ID A1996TU64000047

  View details for PubMedID 8577742

  View details for PubMedCentralID PMC40058

• Flagellar assembly in Caulobacter crescentus: A basal body P-ring null mutation affects stability of the L-ring protein JOURNAL OF BACTERIOLOGY Mohr, C. D., Jenal, U., Shapiro, L. 1996; 178 (3): 675-682

  Abstract

  The P- and L-rings are structural components of the flagellar basal body that are positioned in the periplasmic space and outer membrane, respectively. In order to explore the mechanism of P- and L-ring assembly, we examined the effect of a null mutation in the gene encoding the P-ring subunit, FlgI, on the expression, stability, and subcellular localization of the L-ring subunit, FlgH, in Caulobacter crescentus. Transcription of the L-ring gene and synthesis of the L-ring protein were both increased in the P-ring null mutant. However, steady-state L-ring protein levels were dramatically reduced compared with those of wild type. This reduction, which was not observed in flagellar hook mutants, was due to a decreased stability of the L-ring protein. The instability of the L-ring protein was apparent throughout the cell cycle of the P-ring mutant and contrasted with the fairly constant level of L-ring protein during the cell cycle of wild-type cells. Low levels of the L-ring protein were detected exclusively in the cell envelope of cells lacking the P-ring, suggesting that, in the absence of P-ring assembly, L-ring monomers are unable to form multimeric rings and are thus subject to proteolysis in the periplasm.

  View details for Web of Science ID A1996TR39200015

  View details for PubMedID 8550499

  View details for PubMedCentralID PMC177711

• Cell cycle control by an essential bacterial two-component signal transduction protein CELL Quon, K. C., Marczynski, G. T., Shapiro, L. 1996; 84 (1): 83-93

  Abstract

  Dividing cells must coordinate cell cycle events to ensure genetic stability. Here we identify an essential two-component signal transduction protein that controls multiple events in the Caulobacter cell cycle, including cell division, stalk synthesis, and cell cycle-specific transcription. This protein, CtrA, is homologous to response regulator transcription factors and controls transcription from a group of cell cycle-regulated promoters critical for DNA replication, DNA methylation, and flagellar biogenesis. CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. As purified CtrA binds an essential DNA sequence motif found within its target promoters, we propose that CtrA acts in a phosphorelay signal transduction system to control bacterial cell cycle events directly at the transcriptional level.

  View details for Web of Science ID A1996TQ17000011

  View details for PubMedID 8548829

• Developmental programs in bacteria CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 34 Roberts, R. C., Mohr, C. D., Shapiro, L. 1996; 34: 207-257

  View details for Web of Science ID A1996BG26T00006

  View details for PubMedID 8787575

• USE OF FLOW-CYTOMETRY TO IDENTIFY A CAULOBACTER 4.5 S RNA TEMPERATURE-SENSITIVE MUTANT DEFECTIVE IN THE CELL-CYCLE JOURNAL OF MOLECULAR BIOLOGY Winzeler, E., Shapiro, L. 1995; 251 (3): 346-365

  Abstract

  Flow cytometry was used to screen a collection of temperature-sensitive mutants for those blocked at discrete points in the cell cycle with respect to the replicative status of the chromosome. At the non-permissive temperature, one such mutant, LS439, could not initiate new rounds of DNA replication and arrested primarily as cells with two completed chromosomes Extended incubation at the restrictive temperature resulted in filament formation. Following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. A 0.2 kb fragment of DNA located immediately upstream of the Caulobacter homolog of the Escherichia coli dnaX gene was able to completely rescue the temperature-sensitive phenotype of LS439. The 0.2 kb fragment contained a homolog of the bacterial gene encoding 4.5 S RNA. The original point mutation is predicted to disrupt the stem structure in the 4.5 S RNA thus providing a rationale for the genetic basis of the LS439 phenotype.

  View details for Web of Science ID A1995RP99400003

  View details for PubMedID 7544413

• CIRCUIT SIMULATION OF GENETIC NETWORKS SCIENCE McAdams, H. H., Shapiro, L. 1995; 269 (5224): 650-656

  Abstract

  Genetic networks with tens to hundreds of genes are difficult to analyze with currently available techniques. Because of the many parallels in the function of these biochemically based genetic circuits and electrical circuits, a hybrid modeling approach is proposed that integrates conventional biochemical kinetic modeling within the framework of a circuit simulation. The circuit diagram of the bacteriophage lambda lysislysogeny decision circuit represents connectivity in signal paths of the biochemical components. A key feature of the lambda genetic circuit is that operons function as active integrated logic components and introduce signal time delays essential for the in vivo behavior of phage lambda.

  View details for Web of Science ID A1995RM70200022

  View details for PubMedID 7624793

• A DEVELOPMENTALLY-REGULATED CHROMOSOMAL ORIGIN OF REPLICATION USES ESSENTIAL TRANSCRIPTION ELEMENTS GENES & DEVELOPMENT Marczynski, G. T., LENTINE, K., Shapiro, L. 1995; 9 (12): 1543-1557

  Abstract

  Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells. Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell. This regulated promoter has an unusual sequence organization, and transcription from this promoter is essential for regulated (cell type-specific) replication. Our analysis defines a new class of bacterial origins and suggests a coupling between transcription and replication that is consistent with the phylogenetic relationship of Caulobacter to the ancestral mitochondrion.

  View details for Web of Science ID A1995RG64300010

  View details for PubMedID 7601356

• REGULATION OF THE CAULOBACTER-CRESCENTUS DNAKJ OPERON JOURNAL OF BACTERIOLOGY Avedissian, M., Lessing, D., Gober, J. W., Shapiro, L., Gomes, S. L. 1995; 177 (12): 3479-3484

  Abstract

  The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.

  View details for Web of Science ID A1995RC46100016

  View details for PubMedID 7768857

  View details for PubMedCentralID PMC177052

• THE CONTROL OF ASYMMETRIC GENE-EXPRESSION DURING CAULOBACTER CELL-DIFFERENTIATION ARCHIVES OF MICROBIOLOGY Marczynski, G. T., Shapiro, L. 1995; 163 (5): 313-321

  Abstract

  The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

  View details for Web of Science ID A1995QY55500001

  View details for PubMedID 7794099

• IDENTIFICATION OF AN ESSENTIAL SIGMA-32 HOMOLOG IN COULOBACTER-CRESCENIUS Reisenauer, A., Mohr, C., Shapiro, L. FEDERATION AMER SOC EXP BIOL. 1995: A1275–A1275

  View details for Web of Science ID A1995QV27400206

• COORDINATE CELL-CYCLE CONTROL OF A CAULOBACTER DNA METHYLTRANSFERASE AND THE FLAGELLAR GENETIC HIERARCHY JOURNAL OF BACTERIOLOGY Stephens, C. M., Zweiger, G., Shapiro, L. 1995; 177 (7): 1662-1669

  Abstract

  The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Biol. 235:472-485, 1994). Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. Mutational analysis of two M.Ccr II methylation sites located 3' to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. An additional parallel between the ccrM and class II flagellar promoters is that their transcription responds to a cell cycle DNA replication checkpoint. We propose that a common regulatory system coordinates the expression of functionally diverse genes during the Caulobacter cell cycle.

  View details for Web of Science ID A1995QP81000003

  View details for PubMedID 7896686

  View details for PubMedCentralID PMC176791

• THE BACTERIAL FLAGELLUM - FROM GENETIC NETWORK TO COMPLEX ARCHITECTURE CELL Shapiro, L. 1995; 80 (4): 525-527

  View details for Web of Science ID A1995QJ60200003

  View details for PubMedID 7867059

• IDENTIFICATION OF A NOVEL PROTEIN OR PROTEIN DOMAIN INVOLVED IN INITIATION OF DNA-REPLICATION IN CAULOBACTER Winzeler, E., Shapiro, L. WILEY-BLACKWELL. 1995: 121–121

  View details for Web of Science ID A1995QQ99700450

• TEMPORAL AND SPATIAL CONTROL OF CELL-DIFFERENTIATION DURING A BACTERIAL-CELL CYCLE Shapiro, L., Quon, K., Marczynski, G., Stephens, C. WILEY-BLACKWELL. 1995: 331–331

  View details for Web of Science ID A1995QQ99701144

• Regulation of asymmetry and polarity during the Caulobacter cell cycle ADVANCES IN ENZYMOLOGY & RELATED AREAS OF MOLECULAR BIOLOGY, VOL 71 Jenal, U., Stephens, C., Shapiro, L. 1995; 71: 1-39

  View details for Web of Science ID A1995BG35H00001

  View details for PubMedID 8644489

• CAULOBACTER FLIQ AND FLIR MEMBRANE-PROTEINS, REQUIRED FOR FLAGELLAR BIOGENESIS AND CELL-DIVISION, BELONG TO A FAMILY OF VIRULENCE FACTOR EXPORT PROTEINS JOURNAL OF BACTERIOLOGY Zhuang, W. Y., Shapiro, L. 1995; 177 (2): 343-356

  Abstract

  The Caulobacter crescentus fliQ and fliR genes encode membrane proteins that have a role in an early step of flagellar biogenesis and belong to a family of proteins implicated in the export of virulence factors. These include the MopD and MopE proteins from Erwinia carotovora, the Spa9 and Spa29 proteins from Shigella flexneri, and the YscS protein from Yersinia pestis. Inclusion in this family of proteins suggests that FliQ and FliR may participate in an export pathway required for flagellum assembly. In addition, mutations in either fliQ or fliR exhibit defects in cell division and thus may participate directly or indirectly in the division process. fliQ and fliR are class II flagellar genes residing near the top of the regulatory hierarchy that determines the order of flagellar gene transcription. The promoter sequence of the fliQR operon differs from most known bacterial promoter sequences but is similar to other Caulobacter class II flagellar gene promoter sequences. The conserved nucleotides in the promoter region are clustered in the -10, -20 to -30, and -35 regions. The importance of the conserved bases for promoter activity was demonstrated by mutational analysis. Transcription of the fliQR operon is initiated at a specific time in the cell cycle, and deletion analysis revealed that the minimal sequence required for transcriptional activation resides within 59 bp of the start site.

  View details for Web of Science ID A1995QB30700010

  View details for PubMedID 7814323

  View details for PubMedCentralID PMC176597

• CAULOBACTER FLAGELLAR FUNCTION, BUT NOT ASSEMBLY, REQUIRES FLIL, A NON-POLARLY LOCALIZED MEMBRANE-PROTEIN PRESENT IN ALL CELL-TYPES JOURNAL OF MOLECULAR BIOLOGY Jenal, U., WHITE, J., Shapiro, L. 1994; 243 (2): 227-244

  Abstract

  Caulobacter crescentus has a single polar flagellum, which is assembled in the predivisional cell. Known flagellar genes encode structural and regulatory components that are required for flagellar assembly and function. These genes are organized in several classes which form a transcriptional regulatory hierarchy. A member of the Class II genes, the fliLM operon, encodes homologs of the Escherichia coli flagellar switch protein, FliM, and a protein with a hitherto unknown function, FliL. We report here that flagellar rotation requires the FliL protein. In-frame deletions in the chromosomal copy of the fliL gene result in cells that form a flagellum but are non-motile. The FliL protein was found to be associated with the inner membrane and to be present in all cell types. This is the first report of a Caulobacter crescentus protein that is essential for motility but is not spatially restricted to the region of the flagellar basal body. Although FliL is required for flagellar function, it is not part of the transcriptional hierarchy, supporting the hypothesis that, as is the case for the enterics, the regulatory hierarchy responds to assembly cues rather than directly to the expression of flagellar proteins.

  View details for Web of Science ID A1994PM98800011

  View details for PubMedID 7932752

• CELL-CYCLE ARREST OF A CAULOBACTER-CRESCENTUS SECA MUTANT JOURNAL OF BACTERIOLOGY Kang, P. J., Shapiro, L. 1994; 176 (16): 4958-4965

  Abstract

  Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.

  View details for Web of Science ID A1994PA42600022

  View details for PubMedID 8051008

• BACTERIAL SPORULATION - AN ATP/ADP SWITCH CURRENT BIOLOGY Stephens, C., Shapiro, L., Singer, M. 1994; 4 (7): 630-632

  Abstract

  Regulatory factors that initiate forespore-specific transcription during Bacillus subtilis sporulation respond to adenosine nucleotide ratios.

  View details for Web of Science ID A1994NX67800011

  View details for PubMedID 7953541

• A CAULOBACTER DNA METHYLTRANSFERASE THAT FUNCTIONS ONLY IN THE PREDIVISIONAL CELL JOURNAL OF MOLECULAR BIOLOGY Zweiger, G., MARCYNSKI, G., Shapiro, L. 1994; 235 (2): 472-485

  Abstract

  Caulobacter crescentus was found to have a DNA methyltransferase, CcrM, that methylates the adenine base of the HinfI recognition sequence, GANTC. The ccrM gene was cloned, and DNA sequence analysis revealed that the predicted amino acid sequence has 49% identity with the Haemophilus influenzae methyltransferase HinfM. Expression of the ccrM gene was found to be restricted to the portion of the cell cycle immediately prior to cell division. At three separate chromosomal sites the CcrM recognition sequence is fully methylated in swarmer cells, becomes hemimethylated upon DNA replication in stalked cells, and does not become remethylated until just prior to cell division. The time of methyltransferase expression coincides with the time of methylation of these three chromosomal sites and of plasmid DNA in the predivisional cell. When ccrM gene expression is placed under control of a constitutive promoter, these chromosomal sites are fully methylated throughout the cell cycle. A high proportion of morphologically aberrant cells, and cells that have undergone an additional chromosome replication initiation, are found in this population. Thus, the temporal control of this methyltransferase appears to contribute to the accurate cell-cycle control of DNA replication and cellular morphology.

  View details for Web of Science ID A1994MQ86600007

  View details for PubMedID 8289276

• EXPRESSION OF CAULOBACTER-DNAA AS A FUNCTION OF THE CELL-CYCLE JOURNAL OF BACTERIOLOGY Zweiger, G., Shapiro, L. 1994; 176 (2): 401-408

  Abstract

  The initiation of DNA replication is under differential control in Caulobacter crescentus. Following cell division, only the chromosome in the progeny stalked cell is able to initiate DNA replication, while the chromosome in the progeny swarmer cell does not replicate until later in the cell cycle. We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C. crescentus. Analysis of the cloned C. crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli. The gene is located 2 kb from the origin of replication. Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter. Immunoprecipitation of a DnaA'-beta-lactamase fusion protein showed that although expression occurs throughout the cell cycle, there is a doubling in the rate of expression just prior to the initiation of replication.

  View details for Web of Science ID A1994MQ78200018

  View details for PubMedID 8288535

• THE EXPRESSION OF ASYMMETRY DURING CAULOBACTER CELL-DIFFERENTIATION ANNUAL REVIEW OF BIOCHEMISTRY Brun, Y. V., Marczynski, G., Shapiro, L. 1994; 63: 419-450

  View details for Web of Science ID A1994NV05900013

  View details for PubMedID 7979244

• CHECKPOINTS THAT COUPLE GENE-EXPRESSION TO MORPHOGENESIS SCIENCE Losick, R., Shapiro, L. 1993; 262 (5137): 1227-1228

  View details for Web of Science ID A1993MH32400028

  View details for PubMedID 8235653

• POLARIZED CELLS, POLAR ACTIONS JOURNAL OF BACTERIOLOGY Maddock, J. R., Alley, M. R., Shapiro, L. 1993; 175 (22): 7125-7129

  Abstract

  The recognition of polar bacterial organization is just emerging. The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions. A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C. crescentus and E. coli, the maltose-binding protein in E. coli, the B. japonicum surface attachment proteins, and the actin tail of L. monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host. The significance of this observation remains unclear. Polarity in bacteria poses many problems, including the necessity for a mechanism for asymmetrically distributing proteins as well as a mechanism by which polar localization is maintained. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole. Perhaps the periplasmic proteins are retained at the pole by the presence of the periseptal annulus (35). The constraining features for membrane components are not known. For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement. In most cases of cellular asymmetry, bacteria are able to discriminate between the new pole and the old pole and to utilize this information for localization specificity. The maturation of new pole to old pole appears to be a common theme as well. Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized.

  View details for Web of Science ID A1993MG71100001

  View details for PubMedID 8226658

• ASYMMETRIC EXPRESSION OF THE GYRASE-B GENE FROM THE REPLICATION-COMPETENT CHROMOSOME IN THE CAULOBACTER-CRESCENTUS PREDIVISIONAL CELL JOURNAL OF BACTERIOLOGY Rizzo, M. F., Shapiro, L., Gober, J. 1993; 175 (21): 6970-6981

  Abstract

  The bacterium Caulobacter crescentus undergoes an asymmetric cell division resulting in the formation of two different daughter cells, a motile swarmer cell and a nonmotile stalked cell. These two cell types differ in their program of gene expression, their ability to replicate DNA, and the physical properties of their nucleoids. We show here that two genes, gyrB (encoding the gyrase B subunit) and orf-1, are specifically transcribed from the chromosome in the portion of the predivisional cell destined for the progeny stalked cell. This is in contrast to a subset of flagellar genes which are transcribed from the chromosome in the incipient swarmer portion of the predivisional cell. gyrB and orf-1 are within a newly identified cluster of genes involved in DNA replication and recombination, including dnaN and recF. The transcription of gyrB and orf1 occurs from the replication-competent chromosome in stalked and predivisional cells and is silenced in swarmer cells. We hypothesize that selective silencing of groups of genes in the chromosomes at the swarmer and stalked poles of the predivisional cell results in the different developmental programs and the difference in replicative ability of the two progeny cells.

  View details for Web of Science ID A1993ME00800030

  View details for PubMedID 8226640

• Bacterial chromosome origins of replication. Current opinion in genetics & development Marczynski, G. T., Shapiro, L. 1993; 3 (5): 775-782

  Abstract

  Bacteria regulate chromosomal replication from one specific origin. We compare the regulatory requirements, DNA structures, and biochemical properties of the prototypic Escherichia coli origin with those of evolutionarily distant Bacillus subtilis and Caulobacter crescentus origins. The ubiquitous DnaA protein is a major regulator of all three bacterial origins. Unique features of these origins, however, may reflect specific regulatory requirements placed on them.

  View details for PubMedID 8274862

• AN UNUSUAL PROMOTER CONTROLS CELL-CYCLE REGULATION AND DEPENDENCE ON DNA-REPLICATION OF THE CAULOBACTER-FLILM EARLY FLAGELLAR OPERON MOLECULAR MICROBIOLOGY Stephens, C. M., Shapiro, L. 1993; 9 (6): 1169-1179

  Abstract

  Transcription of flagellar genes in Caulobacter crecentus is programmed to occur during the predivisional stage of the cell cycle. The mechanism of activation of Class II flagellar genes, the highest identified genes in the Caulobacter flagellar hierarchy, is unknown. As a step toward understanding this process, we have defined cis-acting sequences necessary for expression of a Class II flagellar operon, fliLM. Deletion analysis indicated that a 55 bp DNA fragment was sufficient for normal, temporally regulated promoter activity. Transcription from this promoter-containing fragment was severely reduced when chromosomal DNA replication was inhibited. Extensive mutational analysis of the promoter region from -42 to -5 identified functionally important nucleotides at -36 and -35, between -29 and -22, and at -12, which correlates well with sequences conserved between fliLM and the analogous regions of two other Class II flagellar operons. The promoter sequence does not resemble that recognized by any known bacterial sigma factor. Models for regulation of Caulobacter early flagellar promoters are discussed in which RNA polymerase containing a novel sigma subunit interacts with an activation factor bound to the central region of the promoter.

  View details for Web of Science ID A1993LX92900005

  View details for PubMedID 7934930

• PROTEIN LOCALIZATION AND ASYMMETRY IN THE BACTERIAL-CELL CELL Shapiro, L. 1993; 73 (5): 841-855

  View details for Web of Science ID A1993LF06100004

  View details for PubMedID 8500178

• DEVELOPMENT AND BEHAVIOR IN BACTERIA CELL Shapiro, L., Kaiser, D., Losick, R. 1993; 73 (5): 835-836

  View details for Web of Science ID A1993LF06100002

  View details for PubMedID 8500176

• FLOW-CYTOMETRY OF CAULOBACTER-CRESCENTUS - IDENTIFICATION AND CHARACTERIZATION OF A CELL-CYCLE MUTANT Winzeler, E. A., Kang, P. J., Shapiro, L. WILEY-BLACKWELL. 1993: 307–307

  View details for Web of Science ID A1993KX96501075

• ISOLATION AND CHARACTERIZATION OF TRANS-ACTING MUTATIONS WHICH CAUSE OVEREXPRESSION OF A CELL-CYCLE REGULATED OPERON Quon, K. C., Shapiro, L. WILEY-BLACKWELL. 1993: 303–303

  View details for Web of Science ID A1993KX96501060

• POLAR LOCATION OF THE CHEMORECEPTOR COMPLEX IN THE ESCHERICHIA-COLI CELL SCIENCE Maddock, J. R., Shapiro, L. 1993; 259 (5102): 1717-1723

  Abstract

  The eukaryotic cell exhibits compartmentalization of functions to various membrane-bound organelles and to specific domains within each membrane. The spatial distribution of the membrane chemoreceptors and associated cytoplasmic chemotaxis proteins in Escherichia coli were examined as a prototypic functional aggregate in bacterial cells. Bacterial chemotaxis involves a phospho-relay system brought about by ligand association with a membrane receptor, culminating in a switch in the direction of flagellar rotation. The transduction of the chemotaxis signal is initiated by a chemoreceptor-CheW-CheA ternary complex at the inner membrane. These ternary complexes aggregate predominantly at the cell poles. Polar localization of the cytoplasmic CheA and CheW proteins is dependent on membrane-bound chemoreceptor. Chemoreceptors are not confined to the cell poles in strains lacking both CheA and CheW. The chemoreceptor-CheW binary complex is polarly localized in the absence of CheA, whereas the chemoreceptor-CheA binary complex is not confined to the cell poles in strains lacking CheW. The subcellular localization of the chemotaxis proteins may reflect a general mechanism by which the bacterial cell sequesters different regions of the cell for specialized functions.

  View details for Web of Science ID A1993KT81000027

  View details for PubMedID 8456299

• REQUIREMENT OF THE CARBOXYL TERMINUS OF A BACTERIAL CHEMORECEPTOR FOR ITS TARGETED PROTEOLYSIS SCIENCE Alley, M. R., Maddock, J. R., Shapiro, L. 1993; 259 (5102): 1754-1757

  Abstract

  The bacterium Caulobacter crescentus yields two different progeny at each cell division; a chemotactically competent swarmer cell and a sessile stalked cell. The chemotaxis proteins are synthesized in the predivisional cell and then partition only to the swarmer cell upon division. The chemoreceptors that were newly synthesized were located at the nascent swarmer pole of the predivisional cell, an indication that asymmetry was established prior to cell division. When the swarmer cell differentiated into a stalked cell, the chemoreceptor was specifically degraded by virtue of an amino acid sequence located at its carboxyl terminus. Thus, a temporally and spatially restricted proteolytic event was a component of this differentiation process.

  View details for Web of Science ID A1993KT81000037

  View details for PubMedID 8456303

• THE CHEMORECEPTORS AND CHEMOTAXIS SIGNAL TRANSDUCTION PROTEINS ARE CLUSTERED AT THE POLE OF THE ESCHERICHIA-COLI CELL Maddock, J., Shapiro, L. WILEY-BLACKWELL. 1993: 136–136

  View details for Web of Science ID A1993KN46600471

• REGULATION AND LOCALIZATION OF THE FTSZ PROTEIN AND IDENTIFICATION OF THE FTSZ GENE OF CAULOBACTER-CRESCENTUS Brun, Y. V., Shapiro, L. WILEY-BLACKWELL. 1993: 134–134

  View details for Web of Science ID A1993KN46600465

• DEVELOPMENTAL CONTROL OF DNA-REPLICATION IN C-CRESCENTUS Marczynski, G. T., LENTINE, K., Shapiro, L. WILEY-BLACKWELL. 1993: 137–137

  View details for Web of Science ID A1993KN46600478

• ORGANIZATION AND ORDERED EXPRESSION OF CAULOBACTER GENES ENCODING FLAGELLAR BASAL BODY ROD AND RING PROTEINS JOURNAL OF MOLECULAR BIOLOGY Dingwall, A., Garman, J. D., Shapiro, L. 1992; 228 (4): 1147-1162

  Abstract

  The biogenesis of the polar flagellum in Caulobacter crescentus is limited to a specific time in the cell cycle and to a specific site on the cell. The basal body is the first part of the flagellum to be assembled. In this report we identify a cluster of genes encoding basal body components and describe their transcriptional regulation. The genes in this cluster form an operon whose expression is controlled temporally. The first two genes encode homologs of FlgF and FlgG, which are the proximal and distal rod proteins, respectively. The sequences of the N and C termini of the Salmonella typhimurium flagellar axial proteins, rod, hook and HAP-1, known to be highly conserved, share a high degree of sequence identity with the FlgF and FlgG rod proteins of the distantly related, C. crescentus. Two additional genes in the flgF, flgG operon, flaD and flgH, both encode proteins with potentially cleavable signal sequences. The flgH gene, encoding the L-ring protein, is also transcribed from an internal promoter. Transcription from the flgF promoter initiates prior to initiation at the internal flgH promoter. The internal promoter and its activator site reside within the C-terminal coding sequence of the upstream flaD gene. This type of gene overlap is also observed in bacterial genes involved in cell division. Flagellum biogenesis, like cell division, is a morphogenic event that requires the orderly assembly of component proteins and the overlapping gene organization may affect this "ordering" of assembly. The promoters for the flgF operon and the flgH gene use sigma 54 to initiate transcription. The use of sigma 54 promoters, known to require cognate binding proteins, could allow the fine-tuning that provides the temporal ordering of flagellar gene transcription. In this context, we have found that the flgF operon and the distal flgI gene encoding the P-ring, share a sigma 54 activator sequence (class IIA) that differs from the flgH L-ring gene sigma 54 activator site (class IIB) and the hook cluster (class IIC) sigma 54 activator site. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum.

  View details for Web of Science ID A1992KE60200013

  View details for PubMedID 1474584

• A TEMPORALLY CONTROLLED SIGMA-FACTOR IS REQUIRED FOR POLAR MORPHOGENESIS AND NORMAL-CELL DIVISION IN CAULOBACTER GENES & DEVELOPMENT Brun, Y. V., Shapiro, L. 1992; 6 (12A): 2395-2408

  Abstract

  The transcription of many spatially and temporally controlled flagellar structural genes in Caulobacter requires the RNA polymerase sigma 54 subunit. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. Surprisingly, the transcription of rpoN is temporally regulated during the cell cycle; it increases 10-fold commensurate with stalk formation and just before the onset of flagellar gene expression. These results suggest that sigma 54 abundance responds to cell cycle cues and is involved in the global timing of the central events of Caulobacter development, whereas the transcriptional activators of sigma 54-dependent promoters are responsible for the refined control of the expression of individual or small groups of genes required for each specific event.

  View details for Web of Science ID A1992KB97700015

  View details for PubMedID 1459461

• CELL-CYCLE CONTROL OF A CLONED CHROMOSOMAL ORIGIN OF REPLICATION FROM CAULOBACTER-CRESCENTUS JOURNAL OF MOLECULAR BIOLOGY Marczynski, G. T., Shapiro, L. 1992; 226 (4): 959-977

  Abstract

  Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

  View details for Web of Science ID A1992JK69700007

  View details for PubMedID 1518064

• A DEVELOPMENTALLY REGULATED CAULOBACTER FLAGELLAR PROMOTER IS ACTIVATED BY 3' ENHANCER AND IHF BINDING-ELEMENTS MOLECULAR BIOLOGY OF THE CELL Gober, J. W., Shapiro, L. 1992; 3 (8): 913-926

  Abstract

  The transcription of a group of flagellar genes is temporally and spatially regulated during the Caulobacter crescentus cell cycle. These genes all share the same 5' cis-regulatory elements: a sigma 54 promoter, a binding site for integration host factor (IHF), and an enhancer sequence, known as the ftr element. We have partially purified the ftr-binding proteins, and we show that they require the same enhancer sequences for binding as are required for transcriptional activation. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Using site-directed mutagenesis, we provide the first demonstration that natural enhancer sequences and IHF binding elements that reside 3' to the sigma 54 promoter of a bacterial gene, flaNQ, are required for transcription of the operon, in vivo. The IHF protein and the ftr-binding protein is primarily restricted to the predivisional cell, the cell type in which these promoters are transcribed. flaNQ promoter expression is localized to the swarmer pole of the predivisional cell, as are other flagellar promoters that possess these regulatory sequences 5' to the start site. The requirement for an IHF binding site and an ftr-enhancer element in spatially transcribed flagellar promoters indicates that a common mechanism may be responsible for both temporal and polar transcription.

  View details for Web of Science ID A1992JM38600007

  View details for PubMedID 1392079

• POLAR LOCALIZATION OF A BACTERIAL CHEMORECEPTOR GENES & DEVELOPMENT Alley, M. R., Maddock, J. R., Shapiro, L. 1992; 6 (5): 825-836

  Abstract

  The bacterial chemotaxis signal transducer MCP is an integral membrane receptor protein. The chemoreceptor is localized at the flagellum-bearing pole of Caulobacter crescentus swarmer cells. Amino-terminal sequences of the MCP target the protein to the membrane while the carboxy-terminal portion of the protein is responsible for polar localization. The C. crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E. coli MCP is expressed in C. crescentus, it is targeted to the swarmer cell progeny. These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus.

  View details for Web of Science ID A1992HT80400011

  View details for PubMedID 1577276

• EARLY CAULOBACTER-CRESCENTUS GENES FLIL AND FLIM ARE REQUIRED FOR FLAGELLAR GENE-EXPRESSION AND NORMAL-CELL DIVISION JOURNAL OF BACTERIOLOGY Yu, J., Shapiro, L. 1992; 174 (10): 3327-3338

  Abstract

  The biogenesis of the Caulobacter crescentus polar flagellum requires the expression of more than 48 genes, which are organized in a regulatory hierarchy. The flbO locus is near the top of the hierarchy, and consequently strains with mutations in this locus are nonmotile and lack the flagellar basal body complex. In addition to the motility phenotype, mutations in this locus also cause abnormal cell division. Complementing clones restore both motility and normal cell division. Sequence analysis of a complementing subclone revealed that this locus encodes at least two proteins that are homologs of the Salmonella typhimurium and Escherichia coli flagellar proteins FliL and FliM. FliM is thought to be a switch protein and to interface with the flagellum motor. The C. crescentus fliL and fliM genes form an operon that is expressed early in the cell cycle. Tn5 insertions in the fliM gene prevent the transcription of class II and class III flagellar genes, which are lower in the regulatory hierarchy. The start site of the fliLM operon lies 166 bp from the divergently transcribed flaCBD operon that encodes several basal body genes. Sequence comparison of the fliL transcription start site with those of other class I genes, flaS and flaO, revealed a highly conserved 29-bp sequence in a potential promoter region that differs from sigma 70, sigma 54, sigma 32, and sigma 28 promoter sequences, suggesting that at least three class I genes share a unique 5' regulatory region.

  View details for Web of Science ID A1992HU93800030

  View details for PubMedID 1315735

• EXPRESSION OF AN EARLY GENE IN THE FLAGELLAR REGULATORY HIERARCHY IS SENSITIVE TO AN INTERRUPTION IN DNA-REPLICATION JOURNAL OF BACTERIOLOGY Dingwall, A., Zhuang, W. Y., Quon, K., Shapiro, L. 1992; 174 (6): 1760-1768

  Abstract

  Genes involved in the biogenesis of the flagellum in Caulobacter crescentus are expressed in a temporal order and are controlled by a trans-acting regulatory hierarchy. Strains with mutations in one of these genes, flaS, cannot transcribe flagellar structural genes and divide abnormally. This gene was cloned, and it was found that its transcription is initiated early in the cell cycle. Subclones that restored motility to FlaS mutants also restored normal cell division. Although transcription of flaS was not dependent on any other known gene in the flagellar hierarchy, it was autoregulated and subject to mild negative control by other genes at the same level of the hierarchy. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition of flaS transcription. The flaS transcript initiation site was identified, and an apparently unique promoter sequence was found to be highly conserved among the genes at the same level of the hierarchy. The flagellar genes with this conserved 5' region all initiate transcription early in the cell cycle and are all sensitive to a disruption in DNA replication. Mutations in these genes also cause an aberrant cell division phenotype. Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division.

  View details for Web of Science ID A1992HJ50200007

  View details for PubMedID 1372311

• The control of timing and spatial organization during Caulobacter cell differentiation. Harvey lectures Shapiro, L. 1992; 88: 23-48

  View details for PubMedID 1365874

• Positional information during Caulobacter cell differentiation. Current opinion in genetics & development Gober, J. W., Alley, M. R., Shapiro, L. 1991; 1 (3): 324-329

  Abstract

  The formation of two distinct daughter cells upon division of the bacterium Caulobacter crescentus is the result of asymmetry in the predivisional cell, in part due to localization of both flagellar and chemotaxis proteins to the swarmer cell pole. Recent evidence suggests that both localized transcription and protein targeting directed by specific amino acid sequence are involved in the localization.

  View details for PubMedID 1840888

• Molecular genetics of simple developmental systems CURRENT OPINION IN GENETICS & DEVELOPMENT Blackburn, E., Firtel, R. A., Shapiro, L. 1991; 1 (3): 305–6

  View details for DOI 10.1016/S0959-437X(05)80291-6

  View details for Web of Science ID 000450884800001

• GENETIC-ANALYSIS OF A TEMPORALLY TRANSCRIBED CHEMOTAXIS GENE-CLUSTER IN CAULOBACTER-CRESCENTUS GENETICS Alley, M. R., Gomes, S. L., Alexander, W., Shapiro, L. 1991; 129 (2): 333-342

  Abstract

  Caulobacter crescentus performs chemotaxis by short intermittent reversals of rotation of its single polar flagellum. Tn5 insertions causing a general chemotaxis phenotype, an inability to reverse swimming direction and to form large swarm colonies, have been mapped to an 8-kb region of the C. crescentus genome. These Tn5 mutations had different effects on the methyl-accepting chemotaxis proteins (MCP), and the activities of methyltransferase and methylesterase. The Tn5 insertion mutant SC1130 had no cross-reacting MCP and had reduced levels of activity of the methyltransferase and methylesterase. Other mutants bearing Tn5 insertions retained cross-reacting MCP activity and were altered only in their methyltransferase and methylesterase activities. Using a cosmid library we isolated a clone that complemented SC1130. Complementation studies of the Tn5 mutants using derivatives of the cosmid clone showed that all the Tn5 insertions lie within a single operon that appears to encode many chemotaxis genes. The first gene in this operon was shown to encode an MCP by immuno-blot analysis of strains carrying beta-galactosidase protein fusions to portions of the operon. The promoter of this operon was located by chromosomal integration of subclones of this region and by identifying DNA fragments that were capable of expressing lacZ transcriptional fusions. The transcription of the che operon occurred at a defined time in the cell cycle, prior to cell division.

  View details for Web of Science ID A1991GH44500004

  View details for PubMedID 1660425

• TEMPORAL AND SPATIAL REGULATION OF DEVELOPMENTALLY EXPRESSED GENES IN CAULOBACTER BIOESSAYS Gober, J. W., Shapiro, L. 1991; 13 (6): 277-283

  View details for Web of Science ID A1991FT97800003

• EXPRESSION OF POSITIONAL INFORMATION DURING CELL-DIFFERENTIATION IN CAULOBACTER CELL Gober, J. W., CHAMPER, R., Reuter, S., Shapiro, L. 1991; 64 (2): 381-391

  Abstract

  The asymmetric targeting of proteins to the Caulobacter predivisional cell poles yields dissimilar progeny. We show that the products of transcriptional reporter gene fusions to a flagellin gene and to the flagellar hook operon are segregated to the progeny swarmer cell. This segregation does not depend on sequences within the mRNA, but on the upstream regulatory region. The subset of developmentally regulated flagellar genes that exhibit mRNA segregation has the same upstream cis-acting elements: an activator-binding site known as the ftr sequence and an IHF-binding site. We propose that these genes are preferentially transcribed from the chromosome in the incipient swarmer cell pole of the predivisional cell.

  View details for Web of Science ID A1991EV33600014

  View details for PubMedID 1988153

• IDENTIFICATION OF CIS AND TRANS-ELEMENTS INVOLVED IN THE TIMED CONTROL OF A CAULOBACTER FLAGELLAR GENE JOURNAL OF MOLECULAR BIOLOGY Gober, J. W., Xu, H., Dingwall, A. K., Shapiro, L. 1991; 217 (2): 247-257

  Abstract

  The genes encoding the structural components of the Caulobacter crescentus flagellum are temporally controlled and their order of expression reflects the sequence of assembly. Transcription of the operon containing the structural gene for the flagellar hook protein occurs at a defined time in the cell cycle, and information necessary for transcription is contained within a region between -81 and -120 base-pairs from the transcription start site. To identify the sequence elements that contribute to the temporal control of hook operon transcription, we constructed deletions and base changes in the 5' region and fused the mutagenized regulatory region to transcription reporter genes. We demonstrate that sequences 3' to the transcription start site do not contribute to temporal control. We confirm that upstream sequences between -81 and -120 base-pairs are necessary for temporal activation, and that transcription also requires sequences at -26 to -46 base-pairs. A specific binding activity for the region between -81 and -122 base-pairs was shown to be temporally controlled, appearing prior to the activation of hook operon transcription. This binding activity was missing from strains containing mutations in flaO and flaW, two genes near the top of the flagellar hierarchy known to be required for hook operon transcription. Thus, the hook operon upstream region contains a sequence element that responds to a temporally controlled trans-acting factor(s), and in concert with a second sequence element causes the timed activation of transcription.

  View details for Web of Science ID A1991EW29800007

  View details for PubMedID 1992161

• Temporal and spatial regulation of developmentally expressed genes in Caulobacter Bio Essays Gober, J.W., Shapiro, L. 1991; 13 (6): 277-283
• IDENTIFICATION OF A CAULOBACTER BASAL BODY STRUCTURAL GENE AND A CIS-ACTING SITE REQUIRED FOR ACTIVATION OF TRANSCRIPTION JOURNAL OF BACTERIOLOGY Dingwall, A., Gober, J. W., Shapiro, L. 1990; 172 (10): 6066-6076

  Abstract

  The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. One of these genes, flbN, is required early in the flagellar assembly process. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH. Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.

  View details for Web of Science ID A1990EB36200070

  View details for PubMedID 2211524

• REGULATORY INTERACTIONS BETWEEN PHOSPHOLIPID-SYNTHESIS AND DNA-REPLICATION IN CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY LOEWY, B., Marczynski, G. T., Dingwall, A., Shapiro, L. 1990; 172 (10): 5523-5530

  Abstract

  Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome.

  View details for Web of Science ID A1990EB36200001

  View details for PubMedID 2211495

• INTEGRATION HOST FACTOR IS REQUIRED FOR THE ACTIVATION OF DEVELOPMENTALLY REGULATED GENES IN CAULOBACTER GENES & DEVELOPMENT Gober, J. W., Shapiro, L. 1990; 4 (9): 1494-1504

  Abstract

  Several temporally controlled flagellar genes in Caulobacter crescentus require a sigma 54 promoter and upstream sites for transcription activation. We demonstrate here that in some of these genes, an AT-rich region containing an integration host factor (IHF) consensus binding site lies between the activator and the promoter, and that this region binds IHF in vitro. Analysis of mutations in the IHF-binding region of the hook operon demonstrated that an intact IHF-binding site is necessary for transcription in vivo. An adjacent and divergent promoter also has an IHF consensus sequence that binds IHF. The IHF and enhancer sites are 3' to the transcription start site in this promoter. We postulate that IHF mediates the formation of a higher order structure between the divergent promoter regions in a manner analogous to the nucleosome-like structure generated for lambda-Escherichia coli DNA recombination and that this higher order structure modulates transcription.

  View details for Web of Science ID A1990DY49600006

  View details for PubMedID 2253876

• EXPRESSION OF THE CAULOBACTER HEAT-SHOCK GENE DNAK IS DEVELOPMENTALLY CONTROLLED DURING GROWTH AT NORMAL TEMPERATURES JOURNAL OF BACTERIOLOGY Gomes, S. L., Gober, J. W., Shapiro, L. 1990; 172 (6): 3051-3059

  Abstract

  Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.

  View details for Web of Science ID A1990DG18600034

  View details for PubMedID 2345134

• PLASMID AND CHROMOSOMAL DNA-REPLICATION AND PARTITIONING DURING THE CAULOBACTER-CRESCENTUS CELL-CYCLE JOURNAL OF MOLECULAR BIOLOGY Marczynski, G. T., Dingwall, A., Shapiro, L. 1990; 212 (4): 709-722

  Abstract

  Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.

  View details for Web of Science ID A1990DB07400012

  View details for PubMedID 2329579

• A CAULOBACTER GENE INVOLVED IN POLAR MORPHOGENESIS JOURNAL OF BACTERIOLOGY Driks, A., Schoenlein, P. V., DeRosier, D. J., Shapiro, L., Ely, B. 1990; 172 (4): 2113-2123

  Abstract

  At specific times in the cell cycle, the bacterium Caulobacter crescentus assembles two major polar organelles, the flagellum and the stalk. Previous studies have shown that flbT mutants overproduce flagellins and are unable to form chemotaxis swarm rings. In this paper, we report alterations in both the stalk and the flagellar structure that result from a mutation in the flagellar gene flbT. Mutant strains produce some stalks that have a flagellum, produce some stalks that have an extra lobe protruding from their sides, have filaments lacking the 29-kilodalton flagellin, and produce several unusual cell types, including filamentous cells as well as predivisional cells with two stalks and predivisional cells with no stalk at all. We propose that flagellated stalks arise as a consequence of a failure to eject the flagellum at the correct time in the cell cycle and that the extra stalk lobe is due to a second site for the initiation of stalk biogenesis. Thus, a step in the pathway that establishes the characteristic asymmetry of the C. crescentus cell appears to be disrupted in flbT mutants. We have also identified a new structural feature at the flagellated pole and the tip of the stalk: the 10-nm polar particle. The polar particles appear as a cluster of approximately 1 to 10 stain-excluding rings, visible in electron micrographs of negatively stained wild-type cells. This structure is absent at the flagellar pole but not in the stalks of flbT mutant predivisional cells.

  View details for Web of Science ID A1990CW01800056

  View details for PubMedID 2318810

• PURIFICATION AND CHARACTERIZATION OF FATTY-ACID BETA-OXIDATION ENZYMES FROM CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY OCONNELL, M. A., Orr, G., Shapiro, L. 1990; 172 (2): 997-1004

  Abstract

  Acetoacetyl coenzyme A (acetoacetyl-CoA) thiolase, an enzyme required for short-chain fatty acid degradation, has been purified to near homogeneity from Caulobacter crescentus. The relative heat stability of this enzyme allowed it to be separated from beta-ketoacyl-CoA thiolase. The purification scheme minus the heating step also permitted the copurification of crotonase and 3-hydroxyacyl-CoA dehydrogenase. These activities are in a multienzyme complex in Escherichia coli, but a similar complex was not observed in C. crescentus. Instead, separate proteins differing in enzymatic activity were detected, analogous to the beta-oxidation enzymes that have been isolated from Clostridium acetobutylicum and from mitochondria of higher eucaryotes. In these cells, as appears to be the case with C. crescentus, the individual enzymes form multimers of identical subunits.

  View details for Web of Science ID A1990CL74300058

  View details for PubMedID 1967602

• Genetic regulatory hierarchy in Caulobacter development. Advances in genetics Bryan, R., Glaser, D., Shapiro, L. 1990; 27: 1-31

  View details for PubMedID 2112299

• Analysis of bacterial genome organization and replication using pulsed field gel electrophoresis Methods Dingwall, A., Shapiro, L., Ely, B 1990; 1 (2): 160-168
• THE MOLECULAR-GENETICS OF DIFFERENTIATION GENETICS Ely, B., Shapiro, L. 1989; 123 (3): 427-429

  View details for Web of Science ID A1989AX26700001

  View details for PubMedID 2574695

• NEGATIVE TRANSCRIPTIONAL REGULATION IN THE CAULOBACTER FLAGELLAR HIERARCHY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xu, H., Dingwall, A., Shapiro, L. 1989; 86 (17): 6656-6660

  Abstract

  The Caulobacter crescentus flagellum is formed at a specific time in the cell cycle and its assembly requires the ordered expression of a large number of genes. These genes are controlled in a positive trans-acting hierarchy that reflects the order of assembly of the flagellum. Using plasmids carrying transcriptional fusions of either a neo or a lux reporter gene to the promoters of three flagellar genes representing different ranks in the hierarchy (the hook operon, a basal body gene flbN, and the flaO gene), we have measured the level of chimeric gene expression in 13 flagellar mutant backgrounds. Mutants in the hook operon or in basal body genes caused overproduction of both hook operon and basal body gene chimeric mRNAs, suggesting that negative regulation is superimposed on the positive trans-acting control for these early events in the flagellar hierarchy. Mutants in the structural genes and in genes involved in flagellar assembly had no effect on flaO expression, placing the flaO gene near the top of the hierarchy. However, flaO expression appears to be under negative control by two regulatory genes flaS and flaW. Negative control, as a response to the completion of specific steps in the assembly process, may be an important mechanism used by the cell to turn off flagellar gene expression once the gene product is no longer needed.

  View details for Web of Science ID A1989AP19100046

  View details for PubMedID 2771950

• AN ESCHERICHIA-COLI CHEMORECEPTOR GENE IS TEMPORALLY CONTROLLED IN CAULOBACTER PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Frederikse, P. H., Shapiro, L. 1989; 86 (11): 4061-4065

  Abstract

  Flagellar and chemotaxis genes are transcribed at a discrete time in the Caulobacter cell cycle. We demonstrate here that the expression of the Escherichia coli chemoreceptor gene tsr, with 2.6 kilobases of its upstream sequence, is temporally controlled in Caulobacter crescentus. The tsr gene was placed on the chromosome in single copy or on a low-copy-number plasmid. It was found that the Tsr protein appeared at the same point in the cell cycle as an endogenous C. crescentus methyl-accepting chemotaxis protein. Nuclease S1 mapping experiments showed that the tsr transcript was also controlled by the cell cycle, suggesting that the E. coli tsr gene is regulated by C. crescentus factors that mediate the timing of transcription initiation. The apparent transcription start site of the E. coli tsr gene was determined in both E. coli and C. crescentus, and we found that in both backgrounds the promoter used conforms to the consensus sequence for the promoters of the flagellar and chemosensory genes of Bacillus subtilis and E. coli. The use of this promoter suggests that C. crescentus has a cognate sigma factor and predicts that other C. crescentus genes are expressed from this consensus promoter.

  View details for Web of Science ID A1989U940700029

  View details for PubMedID 2657737

• THE ORGANIZATION OF THE CAULOBACTER-CRESCENTUS FLAGELLAR FILAMENT JOURNAL OF MOLECULAR BIOLOGY Driks, A., Bryan, R., Shapiro, L., DeRosier, D. J. 1989; 206 (4): 627-636

  Abstract

  The structural organization of the flagellar filament of Caulobacter crescentus, as revealed by immunoelectron microscopy, shows five antigenically distinct regions within the hook-filament complex. The first region is the hook. The second region is adjacent to the hook and is approximately 10 nm in length. On the basis of its location in the hook-filament complex, this region may contain hook-associated proteins. Next to this is the third region, which is approximately 60 nm in length. Antibody decoration experiments using mutant strains with deletions of the structural gene for the 29 x 10(3) Mr flagellin (flgJ) showed that the presence of this region is correlated with the expression of the 29 x 10(3) Mr flagellin gene. The next region (region IV), of length approximately 1 to 2 microns, appears to contain the 27.5 x 10(3) Mr flagellin, but at its distal end includes, in gradually increasing amounts, the 25 x 10(3) Mr flagellin. The rest of the filament (region V) is made up predominantly, if not completely, of the 25 x 10(3) Mr flagellin. Except for the hook, there are no morphological features that would otherwise distinguish these regions. A functional flagellum, having the wild-type length and morphology, is assembled by mutant strains deficient in the 29 x 10(3) Mr flagellin and 27.5 x 10(3) Mr flagellin.

  View details for Web of Science ID A1989U499000006

  View details for PubMedID 2738912

• IMAGE-RECONSTRUCTION OF THE FLAGELLAR BASAL BODY OF CAULOBACTER-CRESCENTUS JOURNAL OF MOLECULAR BIOLOGY Stallmeyer, M. J., Hahnenberger, K. M., Sosinsky, G. E., Shapiro, L., DeRosier, D. J. 1989; 205 (3): 511-518

  Abstract

  The bacterium Caulobacter crescentus has a single polar flagellum, which is present for only a portion of its cell cycle. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. The flagellum is composed of a transmembrane basal body, a hook and a filament. Single-particle averaging and image reconstruction methods were applied to the electron micrographs of negatively stained basal bodies from C. crescentus. These basal bodies have five rings threaded on a rod. The L and P rings are connected by a bridge of material at their outer radii. The E ring is a thin, flat disk. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. With the other method, the M ring is similar to that of S. typhimurium; that is, it contacts the S ring only at an outer radius and lacks the button. Averages of the rod-hook-filament subassembly ejected by swarmer cells reveal that the rod consists of two parts with the E ring marking the approximate position of the break. The structures of basal bodies from two mutants defective in the hook assembly were found to be indistinguishable from wild-type basal bodies, suggesting that the assembly of the basal body is independent of the hook or filament assembly.

  View details for Web of Science ID A1989T211600005

  View details for PubMedID 2926815

• TEMPORAL REGULATION AND OVERLAP ORGANIZATION OF 2 CAULOBACTER FLAGELLAR GENES JOURNAL OF MOLECULAR BIOLOGY Kaplan, J. B., Dingwall, A., Bryan, R., CHAMPER, R., Shapiro, L. 1989; 205 (1): 71-83

  Abstract

  The biogenesis of the bacterial flagellum and chemotaxis apparatus in both Escherichia coli and Caulobacter crescentus requires the ordered expression of over 40 genes whose expression is controlled by a trans-acting regulatory hierarchy. In C. crescentus, additional control mechanisms ensure that the transcription of these genes is initiated at the correct time in the cell cycle. We demonstrate here that two flagellar genes, flaE and flaY, whose products function in trans to modulate the level of transcription of other flagellar genes, are themselves temporally controlled. DNA sequence analysis of the 3413 base-pairs encompassing the flaE and flaY coding sequences and the 5' regulatory region showed that flaE encodes a protein of 16,000 Mr and flaY a protein of 17,000 Mr. Evidence that flaE and flaY are transcribed as a polycistronic message includes (1) the polar effect of Tn5 insertions; (2) deletion analysis showing that the flaE promoter is essential for complementation of both flaE and flaY alleles; and (3) nuclease S1 assays showing protection of a transcript spanning both genes. The transcript start site in front of flaE was determined and the -10 region conforms to the E. coli sigma 28 promoter consensus sequence. Nuclease S1 analysis also revealed a protected fragment whose size was consistent with a transcript initiating in vivo at a consensus "nif" promoter sequence in front of the flaY gene. The entire promoter region and an upstream consensus sequence that might be a regulatory element for the flaY gene lies within the carboxyl-terminal coding sequence of the flaE gene.

  View details for Web of Science ID A1989R914200005

  View details for PubMedID 2648000

• POSITIONING OF GENE-PRODUCTS DURING CAULOBACTER CELL-DIFFERENTIATION JOURNAL OF CELL SCIENCE Shapiro, L., Gober, J. W. 1989: 85-97

  Abstract

  Caulobacter crescentus has one of the simplest known developmental programs that exhibits both temporal and spatial organization. A hallmark of the Caulobacter cell cycle is that the progeny cells that result from each cell division differ from one another with respect to structure and developmental program. The process of establishing asymmetry prior to cell division requires that a number of gene products be targeted to a pole of the predivisional cell and consequently segregated to one of the two progeny. Several products involved in flagellar biogenesis and the chemotaxis machinery are segregated to the swarmer cell. Evidence suggests that the protein product of some fla and che genes is targeted to the incipient swarmer cell pole. In the case of other flagellar genes, it is the mRNA that is apparently segregated to the swarmer cell. Two heat shock proteins, DnaK and Lon are specifically segregated to the progeny stalked cell.

  View details for Web of Science ID A1989AK51300008

  View details for PubMedID 2693463

• RATE, ORIGIN, AND BIDIRECTIONALITY OF CAULOBACTER CHROMOSOME-REPLICATION AS DETERMINED BY PULSED-FIELD GEL-ELECTROPHORESIS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dingwall, A., Shapiro, L. 1989; 86 (1): 119-123

  Abstract

  Cell division in Caulobacter crescentus yields progeny cells that differ with respect to cell structure and developmental program. Chromosome replication initiates in the daughter stalked cell but is repressed in the daughter swarmer cell until later in the cell cycle. To study cell-type-specific DNA initiation, chromosome replication was directly analyzed by pulsed-field gel electrophoresis. Analysis of Dra I restriction fragments of DNA taken at various times from synchronized cell cultures labeled with 2'-deoxy[3H]guanosine has allowed us to determine the origin of DNA replication, the rate and direction of fork movement, and the order of gene replication. The first labeled Dra I fragment to appear contains the site of replication initiation. Based on the correlation of the physical and genetic maps derived by Ely and Gerardot [Ely, B. & Gerardot, C. J. (1988) Gene 68, 323-333], the origin was localized to a 305-kilobase fragment containing the rrnA gene. Furthermore, the sequential replication through unmapped Dra I fragments has enabled us to localize their positions on the genome. The order of appearance of labeled restriction fragments revealed that the chromosome replicates bidirectionally at a fork movement rate of 21 kilobases per minute.

  View details for Web of Science ID A1989R820200026

  View details for PubMedID 2911562

• ORGANIZATION AND TEMPORAL EXPRESSION OF A FLAGELLAR BASAL BODY GENE IN CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY Hahnenberger, K. M., Shapiro, L. 1988; 170 (9): 4119-4124

  Abstract

  Caulobacter crescentus assembles a single polar flagellum at a defined time in the cell cycle. The protein components of the flagellar hook and filament are synthesized just prior to their assembly. We demonstrated that the expression of a gene, flaD, that is involved in the formation of the flagellar basal body is under temporal control and is transcribed relatively early in the cell cycle, before the hook and flagellin genes are transcribed. Thus, the order of flagellar gene transcription reflects the order of assembly of the protein components. A mutation in the flaD gene results in the assembly of a partial basal body which is missing the outermost P and L rings as well as the external hook and filament (K.M. Hahnenberger and L. Shapiro, J. Mol. Biol. 194:91-103, 1987). The flaD gene was cloned and characterized by nucleotide sequencing and S1 nuclease protection assays. In contrast to the protein components of the hook and filament, the protein encoded by the flaD gene contains a hydrophobic leader peptide. The predicted amino acid sequence of the leader peptide of flaD is very similar to the leader peptide of the flagellar basal body P ring of Salmonella typhimurium (M. Homma, Y. Komeda, T. Iino, and R.M. Macnab, J. Bacteriol. 169:1493-1498, 1987).

  View details for Web of Science ID A1988P905300045

  View details for PubMedID 2842303

• CONTROL OF SYNTHESIS AND POSITIONING OF A CAULOBACTER-CRESCENTUS FLAGELLAR PROTEIN GENES & DEVELOPMENT LOEWY, Z. G., Bryan, R. A., REUTER, S. H., Shapiro, L. 1987; 1 (6): 626-635

  Abstract

  The Caulobacter crescentus flagellum is assembled during a defined time period in the cell cycle. Two genes encoding the major components of the flagellar filament, the 25K and the 27.5K flagellins, are expressed coincident with flagellar assembly. A third gene, flgJ, is also temporally regulated. The synthesis of the product of flgJ, the 29K flagellin, occurs prior to the synthesis of the other flagellin proteins. We demonstrate here that the time of initiation of flgJ expression is independent of chromosomal location but is dependent upon cis-acting sequences present upstream of the flgJ structural gene. Evidence that there is transcriptional control of flgJ expression includes the following: (1) The initial appearance of flgJ message was coincident with the onset of 29K flagellin protein synthesis, and (2) expression of an NPT II reporter gene driven by the flgJ promoter was temporally correct. Post-transcriptional regulation might contribute to the control of expression, because the flgJ mRNA persisted for a longer period of time than did the synthesis of the 29K protein. The 29K flagellin was found only in the progeny swarmer cell after cell division. In a mutant strain that failed to assemble a flagellum, the 29K flagellin still segregated to the presumptive swarmer cell, demonstrating that positioning of the protein is independent of filament assembly. Analysis of a chimeric flgJ-NPT II transcriptional fusion showed that the flgJ regulatory sequences do not control the segregation of the 29K flagellin to the swarmer cell progeny, suggesting that correct segregation depends on the protein product.

  View details for Web of Science ID A1987J718100012

  View details for PubMedID 3315855

• ASYMMETRIC SEGREGATION OF HEAT-SHOCK PROTEINS UPON CELL-DIVISION IN CAULOBACTER-CRESCENTUS JOURNAL OF MOLECULAR BIOLOGY REUTER, S. H., Shapiro, L. 1987; 194 (4): 653-662

  Abstract

  Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.

  View details for Web of Science ID A1987G981000008

  View details for PubMedID 3309328

• CASCADE REGULATION OF CAULOBACTER FLAGELLAR AND CHEMOTAXIS GENES JOURNAL OF MOLECULAR BIOLOGY CHAMPER, R., Dingwall, A., Shapiro, L. 1987; 194 (1): 71-80

  Abstract

  The assembly of a functional flagellum in the bacterium Caulobacter crescentus requires the protein products of approximately 30 genes expressed in a temporally discrete and spatially distinct manner. Our current understanding of this system has been limited by the fact that purified protein products are available for only about one-fifth of these genes. A genetically engineered transposon promoter probe, Tn5-VB32, containing a promoterless gene encoding neomycin phosphotransferase II (NPTase II) was used to generate a series of non-motile (fla-), kanamycin resistant strains of C. crescentus. These transcription-fusions allow the expression of NPTase II to be controlled by flagellar promoters, and thus questions of temporal regulation of flagellar genes can be addressed without the need to obtain purified protein products. The flagellar promoters accessed by Tn5-VB32 exhibited temporal regulation analogous to the known flagellar and chemotaxis gene products. The expression of NPTase II in these mutants is read from a chimeric mRNA that initiates in a chromosomal fla promoter and continues through the inserted NPTase II gene. Thus, temporal regulation is controlled by modulating either the initiation of transcription, or transcript turnover, at specific times in the cell cycle. Epistatic interactions between the genes accessed by the promoter probe and other flagellar loci were studied in double fla mutants generated by transducing the promoter-probe mutations into spontaneously derived second-site fla-mutant backgrounds. The synthesis of both natural fla gene products and the accessed NPTase II was assayed in these strains using antisera to purified components of the flagellum and to purified NPTase II. On the basis of these interactions, a trans-acting hierarchy of flagellar and chemotaxis gene expression is proposed.

  View details for Web of Science ID A1987G456800007

  View details for PubMedID 3039148

• IDENTIFICATION OF A GENE-CLUSTER INVOLVED IN FLAGELLAR BASAL BODY BIOGENESIS IN CAULOBACTER-CRESCENTUS JOURNAL OF MOLECULAR BIOLOGY Hahnenberger, K. M., Shapiro, L. 1987; 194 (1): 91-103

  Abstract

  The bacterial flagellum is a complex structure composed of a transmembrane basal body, a hook, and a filament. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. To help to define the order of assembly of the flagellar components and to identify the genes involved in the early steps of basal body construction, mutants defective in basal body formation have been analyzed. Mutants in the flaD flaB flaC gene cluster were found to be unable to assemble a complete basal body. The flaD BC motC region was cloned and the genes were localized by subcloning and complementation analysis. A series of Tn5 insertion mutations in the flaD BC region were mapped. Complementation analysis of the Tn5 insertion mutants indicated the existence of at least four transcriptional units in the region and identified the presence of two new genes designated flbN and flbO. Mutants in flbN, flaB, flaC and flbO were unable to assemble any basal body structure and are likely to be involved in the early steps of basal body formation. The flaD mutant, however, was found to contain a partially assembled basal body consisting of the rod and three hook-distal rings. All of the mutants in this cluster exhibited pleiotropic effects on the expression of other flagellar and chemotaxis functions, including the level of synthesis of flagellins, the hook protein and hook protein precursor, and the level of chemotaxis methylation.

  View details for Web of Science ID A1987G456800009

  View details for PubMedID 3039149

• SEPARATION OF TEMPORAL CONTROL AND TRANS-ACTING MODULATION OF FLAGELLIN AND CHEMOTAXIS GENES IN CAULOBACTER MOLECULAR AND GENERAL GENETICS Bryan, R., CHAMPER, R., Gomes, S., Ely, B., Shapiro, L. 1987; 206 (2): 300-306

  Abstract

  The genes involved in the biogenesis of the flagellum and the chemotaxis machinery are temporally regulated during the Caulobacter crescentus cell cycle. Using plasmid complementation, we have mapped the extent of the flaY and flaE genes. These genes function in trans to regulate the expression of the flagellin genes and the chemotaxis genes. We have found that the trans regulation that modulates the amount of the flagellins and the chemotaxis proteins can be separated from the temporal control of fla and che gene expression. This conclusion is based on two observations: the low level of synthesis of flagellins and chemotaxis proteins in flaY and flaE mutant strains occurred at the correct time in the cell cycle, and complementation with plasmids containing intact flaY and flaE genes resulted in the synthesis of normal levels of flagellins and chemotaxis gene products with the maintenance of temporal cell cycle control.

  View details for Web of Science ID A1987G196300016

  View details for PubMedID 3473275

• GENERAL NONCHEMOTACTIC MUTANTS OF CAULOBACTER-CRESCENTUS GENETICS Ely, B., GERARDOT, C. J., Fleming, D. L., Gomes, S. L., Frederikse, P., Shapiro, L. 1986; 114 (3): 717-730

  Abstract

  We have examined 35 mutants that have defects in general chemotaxis. Genetic analysis of these mutants resulted in the identification of at least eight che genes located at six different positions on the Caulobacter crescentus chromosome. The cheR, cheB and cheT genes appeared to be located in a three-gene cluster. Mutations in these three genes resulted in the inability of the flagellum to reverse the direction of rotation. Defects in the cheR gene resulted in a loss of the ability to methylate the methyl-accepting chemotaxis proteins. In vitro experiments showed that the lack of in vivo methylation in cheR mutants was due to the absence of methyltransferase activity. Defects in the cheB gene resulted in greatly reduced chemotaxis-associated methylation in vivo and a loss of methylesterase activity in vitro. The specific defects responsible for the lack of a chemotactic response have not been determined for the other identified che genes.

  View details for Web of Science ID A1986E866400004

  View details for PubMedID 3792824

• DIFFERENTIAL LOCALIZATION OF MEMBRANE-RECEPTOR CHEMOTAXIS PROTEINS IN THE CAULOBACTER PREDIVISIONAL CELL JOURNAL OF MOLECULAR BIOLOGY Nathan, P., Gomes, S. L., Hahnenberger, K., Newton, A., Shapiro, L. 1986; 191 (3): 433-440

  Abstract

  The methyl-accepting chemotaxis proteins (MCPs) are membrane receptors that initiate signal transduction to the flagellar rotor upon ligand binding. The synthesis of these proteins occurs only in the Caulobacter crescentus predivisional cell coincident with the biosynthesis of the polar flagellum. Both the flagellum and the MCPs are partitioned to only one daughter cell, the swarmer cell, upon division. We report the results of experiments designed to determine the distribution of these MCPs within swarmer cells and predivisional cells. Flagellated and non-flagellated vesicles were prepared from these cells by immunoaffinity chromatography and the level of MCPs that had been labeled either in vivo or in vitro with methyl-3H was determined. Small membrane vesicles from swarmer cells contained [methyl-3H]MCPs both in the flagellated and non-flagellated vesicles, which indicates that the region immediately surrounding the flagellum, as well as the rest of the surface of the swarmer cell, contains [methyl-3H]MCP. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. The distribution of MCPs was examined in flagellated and non-flagellated vesicles isolated from predivisional cells. The analysis of small predivisional vesicles showed that the MCP content is higher in the flagellated vesicles, and analysis of large flagellated vesicles showed that the MCPs are positioned preferentially in the swarmer cell portion of the predivisional cell. This positional bias of MCPs within predivisional cells could reflect either a large compartment or membrane domain within the incipient swarmer cell, or a gradient of MCPs, with the highest concentration in the vicinity of the flagellum.

  View details for Web of Science ID A1986E317300012

  View details for PubMedID 3820292

• FATTY-ACID DEGRADATION IN CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY OCONNELL, M., Henry, S., Shapiro, L. 1986; 168 (1): 49-54

  Abstract

  Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.

  View details for Web of Science ID A1986E228900007

  View details for PubMedID 2875991

• TRANSCRIPTION INITIATION INVITRO AND INVIVO AT A HIGHLY CONSERVED PROMOTER WITHIN A 16 S-RIBOSOMAL RNA GENE JOURNAL OF MOLECULAR BIOLOGY Amemiya, K., Bellofatto, V., Shapiro, L., Feingold, J. 1986; 187 (1): 1-14

  Abstract

  Transcription initiation has been shown to occur in vitro at several sites within a cloned Caulobacter crescentus ribosomal RNA gene cluster that lacks the major promoter region 5' to the 16 S rRNA gene. The predominant transcription start site in vitro was located near the 3' end of the 16 S rRNA gene. Transcription initiation from this region was also detected in vivo, when the cloned rRNA gene cluster was present on a multi-copy plasmid. The transcription start sites in vitro and in vivo were shown to be identical by S1 nuclease mapping and were found to be located approximately 300 nucleotides upstream from the 3' end of the 16 S rRNA gene. The transcript synthesized in vitro was shown to be cleaved by C. crescentus RNase III and to release the transfer RNA genes from the downstream 16 S/23 S intergenic spacer region. Analysis of the nucleotide sequence near the internal 16 S rRNA transcription start site revealed the presence of a consensus promoter sequence followed by the beginning of an open reading frame approximately 90 nucleotides downstream. Examination of the 16 S rRNA genes from other bacterial species and chloroplasts and 18 S rRNA genes from Xenopus and yeast revealed that the nucleotide sequence of this internal 16 S rRNA promoter region was highly conserved. Although the length of these 16 S and 18 S rRNA genes is slightly variable, the distance of the conserved promoter sequence from the 3' end of these genes has been conserved.

  View details for Web of Science ID A1986AYB3000001

  View details for PubMedID 2420995

• GENERATION OF POLARITY DURING CAULOBACTER CELL-DIFFERENTIATION ANNUAL REVIEW OF CELL BIOLOGY Shapiro, L. 1985; 1: 173-207

  View details for Web of Science ID A1985E667900007

  View details for PubMedID 2881560

• PHOSPHORYLATION OF THE BETA'-SUBUNIT OF RNA-POLYMERASE AND OTHER HOST PROTEINS UPON PHI-CD1 INFECTION OF CAULOBACTER-CRESCENTUS JOURNAL OF VIROLOGY Hodgson, D., Shapiro, L., Amemiya, K. 1985; 55 (1): 238-241

  Abstract

  A protein kinase activity is induced early after infection of Caulobacter crescentus by the DNA phage phiCd1. After phage infection at least 40 proteins are phosphorylated; these include DNA-binding proteins, a membrane-associated protein, and several ribosomal proteins. One of the phosphorylated DNA-binding proteins was identified as the beta' subunit of the host RNA polymerase.

  View details for Web of Science ID A1985AKH8700031

  View details for PubMedID 16789252

• ORGANIZATION AND NUCLEOTIDE-SEQUENCE ANALYSIS OF AN RIBOSOMAL-RNA AND TRANSFER-RNA GENE-CLUSTER FROM CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY Feingold, J., Bellofatto, V., Shapiro, L., Amemiya, K. 1985; 163 (1): 155-166

  Abstract

  rRNA genes of Caulobacter crescentus CB13 were isolated and shown to be present in two gene clusters in the genome. The organization of each rRNA gene cluster was found to be 5'-16S-tRNA spacer-23S-5S-3'. The DNA sequence of 40% of the 16S rRNA gene, the entire 16S/23S intergenic spacer region, and portions of the 23S rRNA gene were determined. Analysis of the nucleotide sequence in the 16S-23S intergenic spacer region revealed the presence of tRNAIle and tRNAAla genes. Large invert repeat sequences were found surrounding the 16S rRNA gene. These inverted repeat sequences are analogous to the RNase III-processing sites in the E. coli rRNA precursor. Small invert repeat sequences were also found flanking the individual tRNA genes. RNA polymerase-binding studies with restriction fragments of the rRNA gene cluster revealed three regions which bound enzyme, and these regions were shown to contain transcription initiation sites. One of these sites was located within the 16S gene near its 3' end, and the other two were found at the 5' end of the 23S gene.

  View details for Web of Science ID A1985ALK8400022

  View details for PubMedID 4008439

• TEMPORAL AND SPATIAL CONTROL OF FLAGELLAR AND CHEMOTAXIS GENE-EXPRESSION DURING CAULOBACTER CELL-DIFFERENTIATION COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY CHAMPER, R., Bryan, R., Gomes, S. L., Purucker, M., Shapiro, L. 1985; 50: 831-840

  Abstract

  Each Caulobacter cell division yields daughter cells that differ from one another both structurally and functionally. By focusing on the biogenesis of the polar flagellum and the proteins of the chemosensory system, several laboratories have now defined an extensive network of genes whose temporal expression is controlled in the predivisional cell. The differential turn-on of these genes contributes to the generation of asymmetry in the predivisional cell in that the products of these genes are targeted to specific cellular locations. To define the mechanisms that mediate this temporal and spatial control, fla genes whose products are not known were accessed by the insertion of transposon-carried drug resistance markers. The transposons were altered so that upon insertion into the chromosome, transcription fusions are formed in which the promoter regions of fla genes drive the expression of the downstream promoter-less drug resistance genes. Assays of the differential placement of the promoter-less drug resistance proteins (encoded within the interrupted fla genes) allow us to determine whether the positioning of the fla gene products is controlled by signal sequences in their proteins, by specific mRNA-targeting sequences in the 5'-regulatory regions of these genes, or by specific transcription from only one of the two newly replicated chromosomes in the predivisional cell.

  View details for Web of Science ID A1985C628800100

  View details for PubMedID 3938370

• ANALYSIS OF THE PLEIOTROPIC REGULATION OF FLAGELLAR AND CHEMOTAXIS GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS BY USING PLASMID COMPLEMENTATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Bryan, R., Purucker, M., Gomes, S. L., Alexander, W., Shapiro, L. 1984; 81 (5): 1341-1345

  Abstract

  The biosynthesis of the single polar flagellum and the proteins that comprise the chemotaxis methylation machinery are both temporally and spacially regulated during the Caulobacter crescentus cell-division cycle. The genes involved in these processes are widely separated on the chromosome. The region of the chromosome defined by flaE mutations contains at least one flagellin structural gene and appears to regulate flagellin synthesis and flagellar assembly. The protein product of the adjacent flaY gene was found to be required to regulate the expression of several flagellin proteins and the assembly of a functional flagellum. We demonstrate here that each of these genes is also required for the expression of chemotaxis methylation genes known to map elsewhere on the chromosome. In order to study the regulation of these genes, plasmids were constructed that contain either an intact flaYE region or deletions in the region of flaY. These plasmids were mated into a wild-type strain and into strains containing various Tn5 insertion and deletion mutations and a temperature-sensitive mutation in the flaYE region. The presence of a plasmid containing the flaYE region allowed the mutant strains to swim and to exhibit chemotaxis, to synthesize increased amounts of the flagellins, to methylate their "methyl-accepting chemotaxis proteins" (MCPs), and to regain wild-type levels of methyltransferase activity. Chromosomal deletions that extend beyond the cloned region were not complemented by this plasmid. Plasmids containing small deletions in the flaY region failed to restore to any flaY or flaE mutants the ability to swim or to assemble a flagellar filament. When mated into a wild-type strain, plasmids bearing deletions in the flaY region were found to be recessive. The pleiotropic regulation of flagellin synthesis, assembly, and chemotaxis methylation functions exhibited by both the flaY and flaE genes suggest that their gene products function in a regulatory hierarchy that controls both flagellar and chemotaxis gene expression.

  View details for Web of Science ID A1984SJ69300012

  View details for PubMedID 6324186

• GENETIC-ANALYSIS AND CHARACTERIZATION OF A CAULOBACTER-CRESCENTUS MUTANT DEFECTIVE IN MEMBRANE BIOGENESIS JOURNAL OF BACTERIOLOGY Hodgson, D., Shaw, P., Letts, V., Henry, S., Shapiro, L. 1984; 158 (2): 430-440

  Abstract

  A mutant of Caulobacter crescentus has been isolated which has an auxotrophic requirement for unsaturated fatty acids or biotin for growth on medium containing glucose as the carbon source. This mutant exhibits a pleiotropic phenotype which includes (i) the auxotrophic requirement, (ii) cell death in cultures attempting to grow on glucose in the absence of fatty acids or biotin, and (iii) a major change in the outer membrane protein composition before cell death. This genetic lesion did not appear to affect directly a fatty acid biosynthetic reaction because fatty acid and phospholipid syntheses were found to continue in the absence of supplement. Oleic acid repressed fatty acid biosynthesis and induced fatty acid degradation in the wild-type parent, AE5000 . The mutant strain, AE6000 , was altered in both of these regulatory functions. The AE6000 mutant also showed specific inhibition of the synthesis of outer membrane and flagellar proteins. Total phospholipid, DNA, RNA, and protein syntheses were unaffected. The multiple phenotypes of the AE6000 mutant were found to cosegregate and to map between hclA and lacA on the C. crescentus chromosome. The defect in this mutant appears to be associated with a regulatory function in membrane biogenesis and provides evidence for a direct coordination of membrane protein synthesis and lipid metabolism in C. crescentus.

  View details for Web of Science ID A1984SP90900006

  View details for PubMedID 6202671

• ISOLATION AND GENETIC-ANALYSIS OF CAULOBACTER MUTANTS DEFECTIVE IN CELL-SHAPE AND MEMBRANE LIPID-SYNTHESIS GENETICS Hodgson, D. A., Shaw, P., Shapiro, L. 1984; 108 (4): 809-826

  Abstract

  In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. One of these, a fatty acid bradytroph, AE6002, was shown to be due to a mutation in the fatA gene. In addition to the presence of the fatA506 mutation, this strain was found to contain two other mutations, one of which caused the production of a water-soluble brown-orange pigment (pigA) and another which caused formation of helical cells (hclA). Expression of the latter two phenotypes required complex media and both were repressed by glucose. However, the lesions were mapped to loci that are separated by a substantial distance. The hclA and the fatA genes mapped close together, possibly implying that comutation had occurred in AE6002. Data are presented that allow the unambiguous identification of a second Fat gene (fatB) in C. crescentus. The map position of another mutation in membrane lipid biogenesis, the glycerol-3-PO4 auxotroph gpsA505, was also determined. During this study the flaZ gene was fine-mapped and the positions of proC and rif changed from the previously reported location.

  View details for Web of Science ID A1984TT35100004

  View details for PubMedID 6510708

• GENERATION OF A TN5 PROMOTER PROBE AND ITS USE IN THE STUDY OF GENE-EXPRESSION IN CAULOBACTER-CRESCENTUS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Bellofatto, V., Shapiro, L., Hodgson, D. A. 1984; 81 (4): 1035-1039

  Abstract

  A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.

  View details for Web of Science ID A1984SH73800012

  View details for PubMedID 6322183

• DIFFERENTIAL EXPRESSION AND POSITIONING OF CHEMOTAXIS METHYLATION PROTEINS IN CAULOBACTER JOURNAL OF MOLECULAR BIOLOGY Gomes, S. L., Shapiro, L. 1984; 178 (3): 551-568

  Abstract

  Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.

  View details for Web of Science ID A1984TP18100004

  View details for PubMedID 6492158

• CAULOBACTER-CRESCENTUS FATTY ACID-DEPENDENT CELL-CYCLE MUTANT JOURNAL OF BACTERIOLOGY Hodgson, D., Shaw, P., OCONNELL, M., Henry, S., Shapiro, L. 1984; 158 (1): 156-162

  Abstract

  A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Starvation of AE6001 for unsaturated fatty acids resulted in a block in the cell cycle. Starved cultures accumulated at the predivisional cell stage after a round of DNA replication had been completed and after a flagellum had been assembled at the pole of the cell. Cell division and cell growth failed to occur probably because the mutant was unable to synthesize a membrane. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. The coincident block in both the initiation of DNA replication and membrane assembly, exhibited by starved cultures of this mutant, suggests that the fatB503 gene product may be involved in the coordination of these events.

  View details for Web of Science ID A1984SL14200024

  View details for PubMedID 6201473

• PURIFICATION AND CHARACTERIZATION OF AN RNA PROCESSING ENZYME FROM CAULOBACTER-CRESCENTUS JOURNAL OF BIOLOGICAL CHEMISTRY Bellofatto, V., Amemiya, K., Shapiro, L. 1983; 258 (9): 5467-5476

  Abstract

  An RNA processing enzyme has been isolated from Caulobacter crescentus which is specific for double-stranded RNA, has an absolute requirement for monovalent cations, and can be eluted from a poly I:C agarose affinity column in pure form. This enzyme, like RNase III isolated from Escherichia coli, processes precursor ribosomal RNAs and polycistronic phage mRNAs and has a monomeric Mr of approximately 20,000. The two enzymes differ, however, in the recognition of specific cleavage sites and yield different digestion products when either coliphage T7 or C. crescentus phage phi Cdl early mRNA is used as substrate. Two lines of evidence are presented which show that an RNase III activity functions as a processing enzyme in C. crescentus. (a) In an in vitro reaction, C. crescentus phage phi Cdl major early mRNA synthesized in vitro by host RNA polymerase was processed by RNase III to yield RNA species which co-migrated with phage RNA synthesized in vivo in phi Cdl-infected cells, and (b) an in vitro transcript of a C. crescentus DNA clone containing the entire 16 S gene and part of the 23 S gene was processed by C. crescentus RNase III to yield an RNA product which co-migrated with 16 S RNA. The RNase III activity isolated from C. crescentus cell extracts has potential use in the analysis of specific RNA species because it was found to be more stringent in the recognition of cleavage sites than the E. coli enzyme.

  View details for Web of Science ID A1983QR08100025

  View details for PubMedID 6343387

• DIFFERENTIAL TEMPLATE RECOGNITION BY THE CAULOBACTER-CRESCENTUS AND THE ESCHERICHIA-COLI RNA-POLYMERASES JOURNAL OF BIOLOGICAL CHEMISTRY Amemiya, K., Shapiro, L. 1983; 258 (14): 8984-8992

  Abstract

  Transcription of Escherichia coli and Caulobacter crescentus phage DNAs by their respective host RNA polymerase was examined to determine their ability to recognize specific transcription signals on the heterologous template. Analysis of coliphage T7 in vitro transcripts showed that, like the E. coli enzyme, the C. crescentus RNA polymerase initiated transcription from the three major T7 early promoters and recognized the terminator at the end of the early region. On the other hand, several differences were found between the C. crescentus and E. coli RNA polymerases with respect to their interaction with Caulobacter phage phiCdl DNA. The rates of open complex formation and RNA elongation were slower when phiCdl DNA was transcribed by the E. coli RNA polymerase. In addition, transcription of phiCdl DNA by the E. coli enzyme produced a subset of transcripts not synthesized by the C. crescentus enzyme. The production of these different transcripts by the E. coli enzyme was dependent on salt concentration and, in at least one case, appeared to be the result of differential termination. Although both enzymes protected the same sites on phiCdl DNA from cleavage with HincII, the E. coli enzyme was unable to form stable complexes with some phiCdl restriction fragments that formed stable complexes with the C. crescentus RNA polymerase. These results indicate that although the C. crescentus RNA polymerase can accurately recognize transcription signals on a heterologous phage template, the E. coli enzyme exhibits altered specificity with a heterologous phage template of higher G + C content.

  View details for Web of Science ID A1983RA96700072

  View details for PubMedID 6306009

• METHYLATION INVOLVED IN CHEMOTAXIS IS REGULATED DURING CAULOBACTER DIFFERENTIATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Shaw, P., Gomes, S. L., Sweeney, K., Ely, B., Shapiro, L. 1983; 80 (17): 5261-5265

  Abstract

  Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We have asked if the biochemical machinery that mediates chemotaxis exists coincident with the cell's structural ability to respond to a chemotactic signal. We first demonstrated that one function of the chemotaxis machinery, the ability to methylate the carboxyl side chains of a specific set of membrane proteins (methyl-accepting chemotaxis proteins, MCPs), is present in C. crescentus. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from S-adenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. In vivo methylation reappeared coincident with the biogenesis of the flagellum just prior to cell division. In vitro reconstitution experiments with heterologous cell fractions from different cell types showed that swarmer cells contain methyltransferase and their membranes can be methylated. However, newly differentiated stalked cells lack methyltransferase activity and membranes from these cells cannot accept methyl groups. These results demonstrate that MCP methylation is confined to that portion of the cell cycle when flagella are present.

  View details for Web of Science ID A1983RE89100021

  View details for PubMedID 6577421

• ISOLATION OF A CAULOBACTER GENE-CLUSTER SPECIFYING FLAGELLUM PRODUCTION BY USING NON-MOTILE TN5 INSERTION MUTANTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Purucker, M., Bryan, R., Amemiya, K., Ely, B., Shapiro, L. 1982; 79 (22): 6797-6801

  Abstract

  Caulobacter crescentus assembles a single polar flagellum from protein components synthesized at a specific time in the cell cycle. Of the 26 genes required for flagellum production, at least 4 of them-flaY, E, F, and G-map together in a single cluster. We have isolated DNA from this region of the chromosome by using a nonmotile mutant with a Tn5 insertion into flaE. C. crescentus DNA carrying the Tn5-flaE region and adjacent sequences was cloned into pBR325 and selected by transposon-encoded kanamycin resistance. The resulting plasmid was used as a probe to isolate the flaE region from a wild-type gene bank and to determine the chromosomal location of several deletion and insertion mutations within the flaY/E/F/G cluster. At least three promotors and three major transcripts were shown to originate from the cloned gene cluster. The role of these genes in flagellar biogenesis was examined by immunoprecipitation of mutant cell extracts with antiflagellin antibody. Deletions extending rightward into this gene cluster eliminated one of the two flagellin proteins normally synthesized by C. crescentus. Mutations mapping to the left permitted synthesis of both normal flagellins but at significantly decreased levels. These results suggest that the leftward end of this cluster contains a region that may function in a regulatory capacity whereas the rightward end may contain sequences overlapping a flagellin structural gene.

  View details for Web of Science ID A1982PR09400010

  View details for PubMedID 16593248

• CHARACTERIZATION OF THE PROTEINS OF THE CAULOBACTER-CRESCENTUS FLAGELLAR FILAMENT - PEPTIDE ANALYSIS AND FILAMENT ORGANIZATION JOURNAL OF BIOLOGICAL CHEMISTRY WEISSBORN, A., Steinman, H. M., Shapiro, L. 1982; 257 (4): 2066-2074

  View details for Web of Science ID A1982NC62300080

  View details for PubMedID 7056757

• SYNTHESIS AND UTILIZATION OF FATTY-ACIDS BY WILD-TYPE AND FATTY-ACID AUXOTROPHS OF CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY Letts, V., Shaw, P., Shapiro, L., Henry, S. 1982; 151 (3): 1269-1278

  Abstract

  The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.

  View details for Web of Science ID A1982PF59600027

  View details for PubMedID 7107555

• INVITRO TRANSCRIPTION OF THE EARLY REGION OF CAULOBACTER PHAGE PHI-CD1 DEOXYRIBONUCLEIC-ACID BY HOST RNA-POLYMERASE BIOCHEMISTRY Amemiya, K., Shapiro, L. 1982; 21 (19): 4707-4713

  Abstract

  Transcription of the Caulobacter crescentus phage phi Cd1 genome requires both the host RNA polymerase and a phage-encoded, rifampicin-resistant RNA polymerase. Transcription of the early region of the phi Cd1 genome was examined in vitro with C. crescentus RNA polymerase. Four transcripts, A, B, C, and D, which ranged in size from 2.9 X 10(6) to 0.53 X 10(6) daltons, were synthesized in vitro by the holoenzyme. Transcript A appeared to be the major transcript since (a) it was the size of the entire 20% of the genome shown in vivo to code for the early phage mRNA, (b) it was one of the first transcripts synthesized at low enzyme-to-DNA molar ratios, and (c) it was synthesized in approximately 3 times the molar equivalent observed for the other transcripts. The A transcript initiated primarily with GTP although a portion was also labeled with ATP. The B, C, and D transcripts were present in equivalent molar ratios, were all smaller than transcript A, and were found to yield RNase III digestion products that were subsets of each other as well as of transcript A. Each of these transcripts proved to be a de novo transcript since (a) each could be pulse labeled during the initial 20 s of the reaction and (b) each transcript contained a triphosphate at its 5' terminus. Evidence is presented that suggests that the B and C transcripts initiate at or near the major A promoter but terminate at different termination or pause sites within the early region of the phage genome. Transcript D appears to initiate at a minor promoter within the terminally redundant region of the genome preceding the A promoter.

  View details for Web of Science ID A1982PG49500029

  View details for PubMedID 6291589

• SYNTHESIS OF SPECIFIC MEMBRANE-PROTEINS IS A FUNCTION OF DNA-REPLICATION AND PHOSPHOLIPID-SYNTHESIS IN CAULOBACTER-CRESCENTUS JOURNAL OF MOLECULAR BIOLOGY Shapiro, L., MANSOUR, J., Shaw, P., Henry, S. 1982; 159 (2): 303-322

  View details for Web of Science ID A1982PA28400005

  View details for PubMedID 7143443

• 3-DIMENSIONAL RECONSTRUCTION OF THE FLAGELLAR HOOK FROM CAULOBACTER-CRESCENTUS JOURNAL OF MOLECULAR BIOLOGY Wagenknecht, T., DeRosier, D., Shapiro, L., WEISSBORN, A. 1981; 151 (3): 439-465

  View details for Web of Science ID A1981MJ92600005

  View details for PubMedID 7338902

• PHOSPHOLIPID BIOSYNTHESIS IS REQUIRED FOR STALK ELONGATION IN CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY MANSOUR, J. D., Henry, S., Shapiro, L. 1981; 145 (3): 1404-1409

  Abstract

  Membrane phospholipid synthesis was inhibited in Caulobacter crescentus by growth of a glycerol auxotroph in the absence of glycerol or by treatment with the antibiotic cerulenin. It was observed that the final step in the swarmer cell-to-stalked cell transition, stalk elongation, was inhibited under these conditions. Since an early effect of inhibiting phospholipid synthesis in C. crescentus is the termination of deoxyribonucleic acid (DNA) replication (I. Contreras, R. Bender, A. Weissborn, K. Amemiya J. D. Mansour, S. Henry, and L. Shapiro, J. Mol. Biol. 138:401-410, 1980), we questioned whether the inhibition of stalk formation was due directly to the inhibition phospholipid synthesis or secondarily to the inhibition of DNA synthesis. Under conditions which inhibited DNA synthesis but permitted phospholipid synthesis, i.e., growth of a temperature-sensitive DNA elongation mutant at the restrictive temperature or treatment with hydroxy-urea, stalk elongation occurred normally. Therefore phospholipid synthesis is required for stalk elongation in C. crescentus.

  View details for Web of Science ID A1981LG93700035

  View details for PubMedID 7204344

• Inverted-repeat nucleotide sequences in Escherichia coli and Caulobacter crescentus. Cold Spring Harbor symposia on quantitative biology Nisen, P., Shapiro, L. 1981; 45: 81-85

  View details for PubMedID 6271494

• PHYSICAL MAP OF CAULOBACTER-CRESCENTUS BACTERIOPHAGE PHI-CD1 DNA JOURNAL OF VIROLOGY Raboy, B., Shapiro, L., Amemiya, K. 1980; 34 (2): 542-549

  Abstract

  A restriction map of the Caulobacter crescentus bacteriophage phi Cd1 genome was constructed by using the restriction endonucleases HindIII and HpaI. A total of 12 fragments, ranging in molecular weight from 7.7 X 10(6) to 0.25 X 10(6), were produced by HindIII, and 7 fragments, ranging in molecular weight from 9.0 X 10(6) to 0.24 X 10(6), were generated by HpaI. The molecular weight of the genome was estimated to be approximately 28.8 X 10(6) on the basis of the relative electrophoretic mobilities of the restriction fragments. The relative order of the cleavage fragments was determined by specific cleavage of isolated restriction fragments, terminal labeling of both the whole genome and isolated fragments, and hybridization of isolated fragments to restriction fragments generated by other restriction enzymes. The genome of phi Cd1 was found to be terminally repetitive, and analysis of previously determined in vivo and in vitro RNA transcripts showed that the restriction map could be oriented such that transcription began on the left and proceeded to the right end of the genome.

  View details for Web of Science ID A1980JS45300028

  View details for PubMedID 6246279

• INVOLVEMENT OF THE HOST RNA-POLYMERASE IN THE EARLY TRANSCRIPTION PROGRAM OF CAULOBACTER-CRESCENTUS BACTERIOPHAGE PHI-CDL DNA VIROLOGY Amemiya, K., Raboy, B., Shapiro, L. 1980; 104 (1): 109-116

  Abstract

  The host RNA polymerase appears to be involved in the early transcription program of the Caulobacter crescentus bacteriophage phiCdl. The addition of rifampicin early after infection inhibited the production of phage, whereas phiCdl production was not inhibited by the addition of rifampicin at any time after infection of a rifampicin-resistant host. In addition, we found that a rifampicin-resistant RNA polymerase activity dependent on de novo protein synthesis is required for late transcription. The region of early phiCdl was mapped by hybridizing labeled RNA extracted from phiCdl-infected cells grown in the presence or absence of chloramphenicol to HindIII and HpaI restriction fragments of the phiCdl genome.

  View details for Web of Science ID A1980JX98100009

  View details for PubMedID 18631659

• DIFFERENTIAL MEMBRANE PHOSPHOLIPID-SYNTHESIS DURING THE CELL-CYCLE OF CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY MANSOUR, J. D., Henry, S., Shapiro, L. 1980; 141 (1): 262-269

  Abstract

  The pattern of phospholipid synthesis during the cell cycle of Caulobacter crescentus has been determined. Although the phospholipid composition of swarmer and stalked cells was indistinguishable in continuously labeled cultures if the two cell types were pulse-labeled for a short time period, marked differences in the pattern of phospholipid synthesis were detected. Pulse-labeled swarmer cells exhibited a higher proportion of phosphatidic acid and a lower proportion of phosphatidylglycerol. In addition, minor phospholipids were detected in the swarmer cells that were not detected in stalked cells. Stalked cells that developed directly from swarmer cells showed that same phospholipid profile as the swarmer cells. The switch to the second phospholipid profile was observed to occur at the predivisional cell stage. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis.

  View details for Web of Science ID A1980JF34600034

  View details for PubMedID 7353999

• INVERTED-REPEAT NUCLEOTIDE-SEQUENCES IN ESCHERICHIA-COLI AND CAULOBACTER-CRESCENTUS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Nisen, P., Shapiro, L. 1980; 45: 81-85

  View details for Web of Science ID A1980LV23700014

• THE EFFECT OF TERMINATION OF MEMBRANE PHOSPHOLIPID-SYNTHESIS ON CELL-DEPENDENT EVENTS IN CAULOBACTER JOURNAL OF MOLECULAR BIOLOGY Contreras, I., WEISSBORN, A., Amemiya, K., MANSOUR, J., Henry, S., Shapiro, L., Bender, R. 1980; 138 (2): 401-409

  View details for Web of Science ID A1980JN48100014

  View details for PubMedID 6157828

• E. coli ribosomal RNA contains sequences homologous to insertion sequences IS1 and IS2. Nature Nisen, P., Shapiro, L. 1979; 282 (5741): 872-874

  Abstract

  The insertion sequence (IS) elements, IS1 and IS2, present in multiple copies in the Escherichia coli chromosome, are transposable genetic elements of known nucleotide sequence. These elements can modulate gene expression, but it is not known whether they normally function in genetic control. To determine whether IS elements could exert control through specific RNA transcripts, we hybridised lambda NNC1857 r14 (carrying IS1) and pBR322 (carrying a portion of IS2) to Northern blots of E. coli RNA. Regions of homology between the IS elements and ribosomal RNA were observed. Computer analysis of reported nucleotide sequences detected large segments of homology between the IS elements and both 23S and 16S rRNA. Additional homologous sequences in phi X174 and a leader region of a ribosomal protein gene cluster were also detected. The homologous sequence between IS2 and 16S rTNA is the same sequence in phi X174 DNA which codes for the ends of the E and D gene and the start of J. The partial IS sequences may represent silent evolutionary remnants or they could modulate the expression of genes carrying these sequences.

  View details for PubMedID 390405

• DEOXYRIBONUCLEIC-ACID SEQUENCE HOMOLOGIES AMONG BACTERIAL INSERTION SEQUENCE ELEMENTS AND GENOMES OF VARIOUS ORGANISMS JOURNAL OF BACTERIOLOGY Nisen, P., Purucker, M., Shapiro, L. 1979; 140 (2): 588-596

  Abstract

  Plasmid and phage deoxyribonucleic acid (DNA) harboring bacterial insertion sequence (IS) elements IS1, IS2, and IS5 were characterized and used as probes to detect homologous sequences in various procaryotic and eucaryotic genomes. The hybridization method used permits the detection of sequences partially homologous to the elements. Hybridization of the IS-containing probes to each other revealed a region of limited homology between IS1 and IS2. Homologous sequences were then detected by computer analysis of the published IS1 and IS2 nucleotide sequences. The homologous sequence contains a tandemly repeated tetranucleotide sequence which resembles the repeated sequence at the hot spot for spontaneous mutations in the lacI gene (P. J. Farabaugh, U. Schmeissner, M. Hofer, and J. Miller, J. Mol. Biol. 126:847-863, 1978). Homology between the IS elements and various genomes was determined by hybridizing labeled DNA containing IS1, IS2, and IS5 sequences to Southern blots of chromosomal DNA cleaved with restriction endonucleases. IS1 and IS5 appear limited to the enteric bacteria, whereas IS2 sequences can also be detected in Pseudomonas putida, Pseudomonas aeruginosa, and Serratia marcescens. Bacteria which appear not to possess extrachromosomal elements, e.g., Caulobacter crescentus, did not show homology with any insertion sequences tested. In addition, sequences homologous to IS1, IS2, or IS5 were not detected in Saccharomyces cerevisiae, Dictyostelium discoideum, or calf thymus DNA.

  View details for Web of Science ID A1979HV87000036

  View details for PubMedID 159291

• CELL-CYCLE-ASSOCIATED REARRANGEMENT OF INVERTED REPEAT DNA-SEQUENCES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nisen, P., Medford, R., MANSOUR, J., Purucker, M., SKALKA, A., Shapiro, L. 1979; 76 (12): 6240-6244

  Abstract

  Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage lambda vector. Both whole populations and individual clones of these sequences were hybridized to restriction endonuclease-generated fragments of chromosomal DNA isolated from cells that were in different stages of the cell cycle. Some inverted repeat DNA sequences were observed to hybridize to different regions of the chromosomal DNA isolated from the morphologically and biochemically distinct swarmer cell and stalked cell populations. These results suggest that the inverted repeat sequences have the capacity to rearrange and thus be located at different sites on the genomes of the different cell types.

  View details for Web of Science ID A1979JA38200049

  View details for PubMedID 293718

• FLAGELLAR HOOK AND BASAL COMPLEX OF CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY Johnson, R. C., Walsh, M. P., Ely, B., Shapiro, L. 1979; 138 (3): 984-989

  Abstract

  Intact bacterial flagella possessing a membrane-free hook and basal complex were purified from Caulobacter crescentus CB15, as well as from mutants which synthesize incomplete flagella. The basal body consisted of five rings mounted on a rod. Two rings were in the hook-proximal upper set, and three rings (two narrow and one wide) were in the lower set. The diameters of the two upper rings differed, being 32 and 21 nm, respectively. The lower rings were all approximately 21 nm in diameter, although they varied significantly in width. During the normal course of the C. crescentus cell cycle, the polar flagellum with hook and rod was shed into the culture medium without the basal rings. Similarly, hooks with attached rods were shed from nonflagellate mutants, and these structures also lacked the basal rings. The hook structure was purified from nonflagellated mutants and found to be composed of a 70,000-molecular-weight protein component.

  View details for Web of Science ID A1979HA45300045

  View details for PubMedID 457596

• CAULOBACTER-CRESCENTUS MUTANT DEFECTIVE IN MEMBRANE PHOSPHOLIPID-SYNTHESIS JOURNAL OF BACTERIOLOGY Contreras, I., Bender, R. A., MANSOUR, J., Henry, S., Shapiro, L. 1979; 140 (2): 612-619

  Abstract

  To study the relationship between phospholipid synthesis and organelle biogenesis in the dimorphic bacterium Caulobacter crescentus, auxotrophs have been isolated which require exogenous glycerol or glycerol 3-phosphate for growth when glucose is used as the carbon source. Upon glycerol deprivation, net phospholipid synthesis ceased immediately in a glycerol 3-phosphate auxotroph which was shown to have levels of biosynthetic sn-glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) activity 10 times lower than that of the wild type. In the absence of glycerol, the optical density of the culture continued to increase for the equivalent of one generation, although the cells did not divide. After the equivalent of one generation time, rapid cell death occurred. Cell death also occurred when phospholipid synthesis was inhibited by cerulenin. Although ribonucleic acid and protein syntheses continued at a reduced rate for the equivalent of one generation in mutant strains, a substantial decrease in the rate of deoxyribonucleic acid synthesis occurred immediately upon glycerol deprivation. Revertant strains had wild-type levels of glycerol 3-phosphate dehydrogenase activity and normal rates of phospholipid and macromolecular synthesis.

  View details for Web of Science ID A1979HV87000039

  View details for PubMedID 500564

• In situ immunoassays for translation products. Methods in enzymology Anderson, D., Shapiro, L., SKALKA, A. M. 1979; 68: 428-436

  View details for PubMedID 161606

• GALACTOSE CATABOLISM IN CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY Kurn, N., Contreras, I., Shapiro, L. 1978; 135 (2): 517-520

  Abstract

  Caulobacter crescentus wild-type strain CB13 is unable to utilize galactose as the sole carbon source unless derivatives of cyclic AMP are present. Spontaneous mutants have been isolated which are able to grow on galactose in the absence of exogenous cyclic nucleotides. These mutants and the wild-type strain were used to determine the pathway of galactose catabolism in this organism. It is shown here that C. crescentus catabolizes galactose by the Entner-Duodoroff pathway. Galactose is initially converted to galactonate by galactose dehydrogenase and then 2-keto-3-deoxy-6-phosphogalactonate aldolase catalyzes the hydrolysis of 2-keto-3-deoxy-6-phosphogalactonic acid to yield triose phosphate and pyruvate. Two enzymes of galactose catabolism, galactose dehydrogenase and 2-keto-3-deoxy-6-phosphogalactonate aldolase, were shown to be inducible and independently regulated. Furthermore, galactose uptake was observed to be regulated independently of the galactose catabolic enzymes.

  View details for Web of Science ID A1978FL46900030

  View details for PubMedID 210153

• MEMBRANE PHOSPHOLIPID COMPOSITION OF CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY Contreras, I., Shapiro, L., Henry, S. 1978; 135 (3): 1130-1136

  Abstract

  The phospholipid composition of Caulobacter crescentus CB13 and CB15 was determined. The acidic phospholipids, phosphatidylglycerol and cardiolipin, comprise approximately 87% of the total phospholipids. Neither phosphatidylethanolamine nor its precursor phosphatidylserine was detected. The outer and inner membranes of C. crescentus CB13 were separated, and phospholipid analysis revealed heterogeneity with respect to the relative amounts of phosphatidylglycerol and cardiolipin in the two membranes. As has been shown to be the case for other bacterial membranes, the concentration of cardiolipin increases and phosphatidylglycerol decreases as cell cultures enter stationary phase.

  View details for Web of Science ID A1978FP55600049

  View details for PubMedID 690071

• SPONTANEOUS FREQUENCY OF A DEVELOPMENTAL MUTANT IN VOLVOX DEVELOPMENTAL BIOLOGY Kurn, N., COLB, M., Shapiro, L. 1978; 66 (1): 266-269

  View details for Web of Science ID A1978FP11300023

  View details for PubMedID 751840

• CAULOBACTER-CRESCENTUS RNA-POLYMERASE - PURIFICATION AND CHARACTERIZATION OF HOLOENZYME AND CORE POLYMERASE JOURNAL OF BIOLOGICAL CHEMISTRY Amemiya, K., Wu, C. W., Shapiro, L. 1977; 252 (12): 4157-4165

  View details for Web of Science ID A1977DL60800021

  View details for PubMedID 325002

• PH-CONDITIONAL MUTANT OF ESCHERICHIA-COLI PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA COLB, M., Shapiro, L. 1977; 74 (12): 5637-5641

  Abstract

  Mutants of Escherichia coli have been isolated that are able to grow on lactose at pH 7.0 but not at pH 8.1. One of these mutants was analyzed and shown to map in the Z region of the lactose operon. beta-Galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) activity in toluenized mutant cells at pH 8.0 was one-tenth that at pH 7.0. Enzyme purified to near homogeneity from the pH-conditional mutant similarly exhibited pH-conditional activity under conditions where wild-type enzyme was unaffected over a pH range of 6.0-8.0. The pH-conditional beta-galactosidase was used in vivo as a probe for intracellular pH. We show that an internal pH of approximately 7.8-8.0 is maintained through an external pH range of 5.9-7.8. The phenotype of pH-conditional mutants was defined on medium with lactose as the sole carbon source. Under such conditions the gene product itself, beta-galactosidase, is required to maintain intracellular pH, since such maintenance is clearly energy-dependent. Therefore, we were able to recover a pH-conditional mutant in a cytoplasmic gene product. We predict that with any phenotype independent of energy production, however, pH-sensitive mutants will be recovered only in surface elements.

  View details for Web of Science ID A1977EH42100096

  View details for PubMedID 23535

• EFFECT OF CARBON SOURCE AND ROLE OF CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE ON CAULOBACTER CELL-CYCLE JOURNAL OF BACTERIOLOGY Kurn, N., Shapiro, L., Agabian, N. 1977; 131 (3): 951-959

  Abstract

  The expression of cell cycle events in Caulobacter crescentus CB13 has been shown to be associated with regulation of carbohydrate utilization. Growth on lactose and galactose depends on induction of specific enzymes. Prior growth on glucose results in a delay in enzyme expression and cell cycle arrest at the nonmotile, predivisional stage. Dibutyryl cyclic adenosine 3',5'-monophosphate (AMP) was shown to stimulate expression of the inducible enzymes and, thus, the initiation of the cell cycle. beta-Galactosidase-constitutive mutants did not exhibit a cell cycle arrest upon transfer of cultures from glucose to lactose. Furthermore, carbon source starvation results in accumulation of the cells at the predivisional stage. The cell cycle arrest therefore results from nutritional deprivation and is analogous to the general control system exhibited by yeast (Hartwell, Bacteriol. Rev. 38:164-198, 1974; Wolfner et al., J. Mol. Biol. 96:273-290, 1975), which coordinates cell cycle initiation with metabolic state. Transfer of C. crescentus CB13 from glucose to mannose did not result in a cell cycle arrest, and it was demonstrated that this carbon source is metabolized by constitutive enzymes. Growth on mannose, however, is stimulated by exogenous dibutyryl cyclic AMP without a concomitant increase in the specific activity of the mannose catabolic enzymes. The effect of cyclic AMP on growth on sugars metabolized by inducible enzymes, as well as on sugars metabolized by constitutive enzymes, may represent a regulatory system common to both types of sugar utilization, since they share features that differ from glucose utilization, namely, temperature-sensitive growth and low intracellular concentrations of cyclic guanosine 3',5'-monophosphate.

  View details for Web of Science ID A1977DU20100033

  View details for PubMedID 197060

• DIFFERENTIATION IN CAULOBACTER CELL-CYCLE ANNUAL REVIEW OF MICROBIOLOGY Shapiro, L. 1976; 30: 377-407

  View details for Web of Science ID A1976CH91600017

  View details for PubMedID 185940

• INSITU IMMUNOASSAYS FOR GENE TRANSLATION PRODUCTS IN PHAGE PLAQUES AND BACTERIAL COLONIES GENE SKALKA, A., Shapiro, L. 1976; 1 (1): 65-79

  Abstract

  A series of simple, in situ immunoassays have been developed which can be used in screening for translation products of genes cloned in vitro recombination experiments with either phage or plasmid vectors. Antigen-antibody complex formation occurring within a vector-phage plaque can be used to detect the production of a specific protein from an amplified gene. Immunoassays of colonies lysed in situ either by lambda prophage induction or by biochemical means afford a much higher level of sensitivity than the plaque assay probably adequate to detect the production of a few molecules of protein per cell.

  View details for Web of Science ID A1976CU45400006

  View details for PubMedID 829886

• EFFECT OF 3'-5'-CYCLIC GMP DERIVATIVES ON FORMATION OF CAULOBACTER SURFACE-STRUCTURES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kurn, N., Shapiro, L. 1976; 73 (9): 3303-3307

  Abstract

  Exogenous derivatives of 3':5'-cyclic GMP, 8-bromo- and N2,O2'-dibutyryl cyclic GMP, coordinately repress surface structure differentiation in Caulobacter crescentus. Growth in the presence of cyclic GMP derivatives resulted in the loss of flagella and pili formation and concomitant resistance to both DNA phage phiCbK and RNA phage phiCb5 infection without affecting growth rate, stalk formation, and equatorial cell division. The effect of cyclic GMP derivatives was shown to be the repression of synthesis of specific structural proteins. This effect could be reversed by exogenous N6,O2'-dibutyryl 3':5'-cyclic AMP, and mutants resistant to repression by cyclic GMP derivatives exhibited a pleiotropic phenotype affecting a cyclic AMP-mediated event.

  View details for Web of Science ID A1976CE95700078

  View details for PubMedID 184471

• CONDITIONAL SURFACE-STRUCTURE MUTANTS OF CAULOBACTER-CRESCENTUS TEMPERATURE-SENSITIVE FLAGELLA FORMATION DUE TO AN ALTERED FLAGELLIN MONOMER JOURNAL OF MOLECULAR BIOLOGY Marino, W., Ammer, S., Shapiro, L. 1976; 107 (2): 115-130

  View details for Web of Science ID A1976CK72400002

  View details for PubMedID 1003462

• STRUCTURE OF CAULOBACTER DEOXYRIBONUCLEIC-ACID JOURNAL OF BACTERIOLOGY Wood, N. B., RAKE, A. V., Shapiro, L. 1976; 126 (3): 1305-1315

  Abstract

  The deoxyribonucleic acid of the dimorphic bacterium Caulobacter crescentus contains a component that renatures with rapid, unimolecular kinetics. This component was present in both swarmer and stalked cells and exhibited the sensitivity to endonuclease S1 expected for hairpin loops. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. No extrachromosomal elements were found in spite of systematic attempts to detect their presence. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria.

  View details for Web of Science ID A1976BU75500037

  View details for PubMedID 947891

• SPECIFIC CYCLIC GUANOSINE 3'-5'-MONOPHOSPHATE-BINDING PROTEIN IN CAULOBACTER-CRESCENTUS JOURNAL OF BIOLOGICAL CHEMISTRY Sun, I. Y., Shapiro, L., Rosen, O. M. 1975; 250 (15): 6181-6184

  Abstract

  A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity. Binding of cyclic GMP is not affected by the addition of cyclic AMP or 5'-GMP, but is inhibited about 50 percent by a 50-fold molar excess of dibutyryl cyclic GMP or cyclic hypoxanthine 3':5'-monophosphate. The apparent dissociation constant for the cyclic GMP-binding protein complex is 1.1 X 10(-6) M.

  View details for Web of Science ID A1975AM69800061

  View details for PubMedID 168209

• Regulation of the Caulobacter cell cycle. Current topics in cellular regulation Kurn, N., Shapiro, L. 1975; 9: 41-64

  View details for PubMedID 164329

• PURIFICATION AND CHARACTERIZATION OF GUANYLATE CYCLASE FROM CAULOBACTER-CRESCENTUS BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Sun, I. C., Shapiro, L., Rosen, O. M. 1974; 61 (1): 193-203

  View details for Web of Science ID A1974U579000028

  View details for PubMedID 4155295

• PLEIOTROPIC MUTATION AFFECTING EXPRESSION OF POLAR DEVELOPMENT EVENTS IN CAULOBACTER-CRESCENTUS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kurn, N., Ammer, S., Shapiro, L. 1974; 71 (8): 3157-3161

  Abstract

  A developmental mutant of C. crescentus with altered polar surface structures has been isolated. The mutant lacks a flagellum and pili, and may have an abnormal DNA phage receptor site. A revertant regains the normal structures simultaneously. This point mutation allows normal flagellin synthesis, stalk formation, equatorial cell division, and rate of growth. The mutant phenotype indicates that the assembly of the polar surface structures is coordinately regulated and independent of mechanisms regulating cell polarity and division.

  View details for Web of Science ID A1974U269600052

  View details for PubMedID 4212892

• SYNTHESIS AND STRUCTURE OF CAULOBACTER-CRESCENTUS FLAGELLA JOURNAL OF BACTERIOLOGY Shapiro, L., Maizel, J. V. 1973; 113 (1): 478-485

  Abstract

  During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.

  View details for Web of Science ID A1973O437500058

  View details for PubMedID 4688664

• DEOXYRIBONUCLEIC ACID-DEPENDENT RIBONUCLEIC-ACID POLYMERASE OF CAULOBACTER-CRESCENTUS JOURNAL OF BACTERIOLOGY BENDIS, I. K., Shapiro, L. 1973; 115 (3): 848-857

  Abstract

  Deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) was purified from the dimorphic bacterium Caulobacter crescentus at three stages in development. Enzyme from pure populations of stalked cells, as well as populations enriched in swarmer and predivisional cells, appeared identical in subunit structure and template requirements. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. By analogy with RNA polymerase from other bacterial sources, they are considered to be components of the C. crescentus holoenzyme, beta', beta, sigma, and alpha, respectively. The C. crescentus enzyme appeared similar to the Pseudomonas aeruginosa enzyme and unlike the Escherichia coli enzyme with respect to subunit molecular weights and failure to separate into core and sigma components upon phosphocellulose chromatography. In addition, the effects of ionic strength on the time course of polymerization varied both with the sources of bacterial polymerase and bacteriophage DNA.

  View details for Web of Science ID A1973Q490900017

  View details for PubMedID 4734061

• Structural studies on the capsid of Caulobacter crescentus bacteriophage phiCbK. Journal of molecular biology Leonard, K. R., Kleinschmidt, A. K., Agabian-Keshishian, N., Shapiro, L., MAIZEL, J. V. 1972; 71 (2): 201-216

  View details for PubMedID 4635986

• CHARACTERIZATION OF A PROTEIN ACYL KINASE FROM CAULOBACTER-CRESCENTUS BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Agabian, N., Rosen, O. M., Shapiro, L. 1972; 49 (6): 1690-?

  View details for Web of Science ID A1972O259100047

  View details for PubMedID 4674284

• STRUCTURAL STUDIES ON CAPSID OF CAULOBACTER-CRESCENTUS BACTERIOPHAGE PHICBK JOURNAL OF MOLECULAR BIOLOGY Leonard, K. R., Maizel, J. V., AGABIANK, N., Shapiro, L., KLEINSCH, A. K. 1972; 71 (2): 201-?

  View details for Web of Science ID A1972O049700006

• EFFECT OF DIBUTYRYLADENOSINE 3'-5'-CYCLIC MONOPHOSPHATE ON GROWTH AND DIFFERENTIATION IN CAULOBACTER-CRESCENTUS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shapiro, L., Rosen, O. M., AGABIANK, N., Hirsch, A. 1972; 69 (5): 1225-?

  Abstract

  Caulobacter crescentus goes through a series of morphological changes during its life cycle, including the coincident expression of synthesis of flagella, pili, and receptor sites for DNA bacteriophage. Upon transfer of a mixed population of cells to medium containing lactose as the sole carbon source, these changes were blocked for about 20 hr until beta-galactosidase activity became apparent. The addition of dibutyryl cyclic AMP to the blocked cultures brought about the resumption of cell differentiation, growth, and the appearance of beta-galactosidase activity within 1 hr. Unlike Escherichia coli, the intracellular and extracellular concentrations of cyclic AMP in C. crescentus did not vary under several growth conditions, including catabolite repression. It would appear, therefore, that although there is an effect of cyclic AMP on the induction of beta-galactosidase and differentiation in C. crescentus, regulation of these processes occurs without consistent changes in the cellular level of this nucleotide.

  View details for Web of Science ID A1972M472200040

  View details for PubMedID 4338585

• BACTERIAL DIFFERENTIATION SCIENCE Shapiro, L., AGABIANK, N., BENDIS, I. 1971; 173 (4000): 884-?

  Abstract

  The foregoing studies are intended to define a differentiation process and to permit genetic access to the mechanisms that control this process. In order to elucidate the basic mechanisms whereby a cell dictates its own defined morphogenic changes, we have found it helpful to study an organism that can be manipulated both biochemically and genetically. We have attempted to develop the studies initiated by Poindexter,Stove and Stanier, and Schmidt and Stanier (16, 17, 20) with the Caulobacter genus so that these bacteria can serve as a model system for prokaryotic differentiation. The Caulobacter life cycle, defined in synchronously growing cultures, includes a sequential series of morphological changes that occur at specific times in the cycle and at specific locations in the cell. Six distinct cellular characteristics, which are peculiar to these bacteria, have been defined and include (i) the synthesis of a polar organelle which may be membranous (21-23), (ii) a satellite DNA in the stalked cell (26), (iii) pili to which RNA bacteriophage specifically adsorb (16, 33), (iv) a single polar flagellum(17), (v) a lipopolysaccharide phage receptor site (27), and (vi) new cell wall material at the flagellated pole of the cell giving rise to a stalk (19, 20). Cell division, essential for the viability of the organism, is dependent on the irreversible differentiation of a flagellated swarmer cell to a mature stalked cell. The specific features of the Caulobacter system which make it a system of choice for studies of the control of sequential events resulting in cellular differentiation can be summarized as follows. 1) Cell populations can be synchronized, and homogeneous populations at each stage in the differentiation cycle can thus be obtained. 2) A specific technique has been developed whereby the progress of the differentiation cycle can be accurately measured by adsorption of labeled RNA phage or penetration of labeled phage DNA into specific cell forms. This technique can be used to select for mutants blocked in the various stages of morphogenesis. 3) Temperature-sensitive mutants of Caulobacter that are restricted in macromolecular synthesis and development at elevated temperatures have been isolated. 4) Genetic exchange in the Calflobacter genus has been demonstrated and is now being defined. Two questions related to control processes can now readily be approached experimentally. (i) Is the temporal progression of events occurring during bacterial differentiation controlled by regulator gene products? (ii) Is the differentiation cycle like a biosynthetic pathway where one event must follow another? The availability of temperature-sensitive mutants blocked at various stages of development permits access to both questions. An interesting feature of the differentiation cycle is that the polar organelle may represent a special segregated unit which is operative in the control of the differentiation process. Perhaps the sequential morphogenic changes exhibited by Caulobacter are dependent on the initial synthesis of this organelle. Because the ultimate expression of cell changes are dependent on selective protein synthesis, specific messenger RNA production-either from DNA present in an organelle or from the chromosome-may prove to be a controlling factor in cell differentiation. We have begun studies with RNA polymerase purified from Caulobacter crescentus to determine whether cell factors or alterations in the enzyme structure serve to change the specificity of transcription during the cell cycle. Control of sequential cell changes at the level of transcription has long been postulated and has recently been substantiated in the case of Bacillus sporulation (6). The Caulobacter bacteria now present another system in which direct analysis of these control mechanisms is feasible.

  View details for Web of Science ID A1971K176100008

  View details for PubMedID 5572165

• BACTERIAL DIFFERENTIATION AND PHAGE INFECTION VIROLOGY AGABIANK, N., Shapiro, L. 1971; 44 (1): 46-?

  View details for Web of Science ID A1971J193000005

  View details for PubMedID 4105994

• Specific Assay for Differentiation in the Stalked Bacterium Caulobacter crescentus. Proceedings of the National Academy of Sciences of the United States of America Shapiro, L., Agabian-Keshishian, N. 1970; 67 (1): 200-203

  Abstract

  The bacterium Caulobacter crescentus carries out a distinct differentiation process during its life cycle. Cultures of the bacterium have been synchronized and an assay has been developed for monitoring the course of morphogenesis by the selective adsorption of radioactive RNA bacteriophage.

  View details for PubMedID 16591854

• STALKED BACTERIA - PROPERTIES OF DEOXRIYBONUCLEIC ACID BACTERIOPHAGE PHICBK JOURNAL OF VIROLOGY AGABIANK, N., Shapiro, L. 1970; 5 (6): 795-?

  Abstract

  The Caulobacter crescentus bacteriophage phiCbK was studied with respect to the physical and chemical properties of both the phage and its deoxyribonucleic acid (DNA). Electron micrographs reveal the phage to be among the largest DNA bacteriophages reported, with head dimensions of 64 by 195 nm and a flexible tail 275 nm in length. The phage is composed of 57% DNA. This DNA is double-stranded as indicated by (i) the sharp increase in extinction coefficient over a narrow range of temperature increase, (ii) an increase in density in CsCl upon thermal denaturation, and (iii) the equivalence of guanine and cytosine as well as adenine and thymine, determined by chemical analysis. phiCbK DNA cosediments with Escherichia coli phage T2 DNA and has therefore been assigned an S(20,w) value of 63.5S. The size of the phage and its DNA and the percentage of DNA indicate that the phiCbK phage head is relatively loosely packed. The properties of the DNA from bacteriophage phiCbK are similar to those of host C. crescentus DNA with respect to buoyant density, thermal transition point, and guanine plus cytosine content.

  View details for Web of Science ID A1970G593000016

  View details for PubMedID 4193836

• SPECIFIC ASSAY FOR DIFFERENTIATION IN STALKED BACTERIUM CAULOBACTER-CRESCENTUS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shapiro, L., AGABIANK, N. 1970; 67 (1): 200-?

  View details for Web of Science ID A1970H419900033

• PHAGE-SPECIFIC AND HOST PROTEINS IN REPLICATION OF BACTERIOPHAGE RNA FEDERATION PROCEEDINGS August, J. T., EOYANG, L., DEFERNAN, M. T., Hasegawa, S., Kuo, C. H., RENSING, U., Shapiro, L. 1970; 29 (3): 1170-?

  View details for Web of Science ID A1970G466200017

  View details for PubMedID 4315363

• PROPERTIES OF CAULOBACTER RIBONUCLEIC ACID BACTERIOPHAGE PHICB5 JOURNAL OF VIROLOGY BENDIS, I., Shapiro, L. 1970; 6 (6): 847-?

  Abstract

  The ribonucleic acid (RNA) bacteriophage phiCb5, which specifically infects only one form of the dimorphic stalked bacterium Caulobacter crescentus, has been obtained in high yield. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. Phage phiCb5 was studied with respect to the physical and chemical properties of both the phage and its RNA. In an electron microscope, the phage particles appear as small polyhedra, 23 nm in diameter. The phage is similar to the Escherichia coli RNA phages in that it (i) sediments at an S(20, w) of 70.6S, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. Phage phiCb5 differs from the E. coli RNA phages in (i) host specificity, (ii) salt sensitivity, and (iii) the presence of histidine, but not methionine, in the coat protein.

  View details for Web of Science ID A1970I035400018

  View details for PubMedID 5495512

• Replication of the RNA genome. Journal of cellular physiology August, J. T., EOYANG, L., FRANZE DE FERNANDEZ, M. T., Hasegawa, S., Kuo, C. H., RENSING, U., Shapiro, L. 1969; 74 (2): 187-?

  View details for PubMedID 5363333

• Resolution of two factors required in the Q-beta-RNA polymerase reaction. Nature Shapiro, L., FRANZE DE FERNANDEZ, M. T., August, J. T. 1968; 220 (5166): 478-480

  View details for PubMedID 4879600

• Ribonucleic acid virus replication. Research publications - Association for Research in Nervous and Mental Disease August, J. T., Shapiro, L. 1968; 44: 71-86

  View details for PubMedID 4894473

• SYNTHESIS OF BACTERIOPHAGE QBETA RNA COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY August, J. T., Banerjee, A. K., EOYANG, L., DEFERNAN, M. T., Hori, K., Kuo, C. H., RENSING, U., Shapiro, L. 1968; 33: 73-?

  View details for Web of Science ID A1968D122300009

  View details for PubMedID 5254583

• PHYSICAL STUDIES ON STRUCTURE OF YEAST MITOCHONDRIAL DNA JOURNAL OF MOLECULAR BIOLOGY Shapiro, L., Grossman, L. I., Marmur, J., KLEISCHM, A. K. 1968; 33 (3): 907-?

  View details for Web of Science ID A1968B197000024

  View details for PubMedID 5700425

• SYMPOSIUM ON REPLICATION OF VIRAL NUCLEIC ACIDS .2. RIBONUCLEIC ACID VIRUS REPLICATION BACTERIOLOGICAL REVIEWS Shapiro, L., August, J. T. 1966; 30 (2): 279-?

  View details for Web of Science ID A19667876500003

  View details for PubMedID 5327758

• REPLICATION OF RNA VIRUSES .3. UTILIZATION OF RIBONUCLEOTIDE ANALOGS IN REACTION CATALYZED BY A RNA VIRUS RNA POLYMERASE JOURNAL OF MOLECULAR BIOLOGY Shapiro, L., August, J. T. 1965; 14 (1): 214-?

  View details for Web of Science ID A19657086600018

  View details for PubMedID 5883914

• REPLICATION OF RNA VIRUSES .2. RNA PRODUCT OF A REACTION CATALYZED BY A VIRAL RNA-DEPENDENT RNA POLYMERASE JOURNAL OF MOLECULAR BIOLOGY Shapiro, L., August, J. T. 1965; 11 (2): 272-?

  View details for Web of Science ID A19656243300011

  View details for PubMedID 14290345

• REPLICATION OF RNA VIRUSES .I. CHARACTERIZATION OF A VIRAL RNA-DEPENDENT RNA POLYMERASE JOURNAL OF MOLECULAR BIOLOGY August, J. T., Shapiro, L., EOYANG, L. 1965; 11 (2): 257-?

  View details for Web of Science ID A19656243300010

  View details for PubMedID 14290344