Christine Jacobs-Wagner

All Publications

• Borrelia burgdorferi loses essential genetic elements and cell proliferative potential during stationary phase in culture but not in the tick vector. bioRxiv : the preprint server for biology Zhang, J., Takacs, C. N., McCausland, J. W., Mueller, E. A., Buron, J., Thappeta, Y., Wachter, J., Rosa, P. A., Jacobs-Wagner, C. 2024

  Abstract

  The Lyme disease agent Borrelia burgdorferi is a polyploid bacterium with a segmented genome in which both the chromosome and over 20 distinct plasmids are present in multiple copies per cell. This pathogen can survive at least nine months in its tick vector in an apparent dormant state between blood meals, without losing cell proliferative capability when re-exposed to nutrients. Cultivated B. burgdorferi cells grown to stationary phase or resuspended in nutrient-limited media are often used to study the effects of nutrient deprivation. However, a thorough assessment of the spirochete's ability to recover from nutrient depletion has been lacking. Our study shows that starved B. burgdorferi cultures rapidly lose cell proliferative. Loss of genetic elements essential for cell proliferation contributes to the observed proliferative defect in stationary phase. The gradual decline in copies of genetic elements is not perfectly synchronized between chromosomes and plasmids, generating cells that harbor one or more copies of the essential chromosome but lack all copies of one or more non-essential plasmids. This phenomenon likely contributes to the well-documented issue of plasmid loss during in vitro cultivation of B. burgdorferi. In contrast, B. burgdorferi cells from ticks starved for 14 months showed no evidence of reduced cell proliferative ability or plasmid loss. Beyond their practical implications for studying B. burgdorferi, these findings suggest that the midgut of the tick vector offers a unique environment that supports the maintenance of B. burgdorferi's segmented genome and cell proliferative potential during periods of tick fasting.

  View details for DOI 10.1101/2024.10.28.620338

  View details for PubMedID 39554112

  View details for PubMedCentralID PMC11565743

• The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria. bioRxiv : the preprint server for biology Schubert, K., Braly, M., Zhang, J., Muscolo, M. E., Lam, H. N., Hug, K., Moore, H., McCausland, J. W., Terciano, D., Lowe, T., Lesser, C. F., Jacobs-Wagner, C., Wang, H., Auerbuch, V. 2024

  Abstract

  Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37°C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence.

  View details for DOI 10.1101/2024.10.11.617751

  View details for PubMedID 39416138

  View details for PubMedCentralID PMC11482874

• Coupling of cell growth modulation to asymmetric division and cell cycle regulation inCaulobacter crescentus. Proceedings of the National Academy of Sciences of the United States of America Glenn, S., Fragasso, A., Lin, W., Papagiannakis, A., Kato, S., Jacobs-Wagner, C. 2024; 121 (41): e2406397121

  Abstract

  In proliferating bacteria, growth rate is often assumed to be similar between daughter cells. However, most of our knowledge of cell growth derives from studies on symmetrically dividing bacteria. In many alpha-proteobacteria, asymmetric division is a normal part of the life cycle, with each division producing daughter cells with different sizes and fates. Here, we demonstrate that the functionally distinct swarmer and stalked daughter cells produced by the model alpha-proteobacterium Caulobacter crescentus can have different average growth rates under nutrient-replete conditions despite sharing an identical genome and environment. The discrepancy in growth rate is due to a growth slowdown associated with the cell cycle stage preceding DNA replication (the G1 phase), which initiates in the late predivisional mother cell before daughter cell separation. Both progenies experience a G1-associated growth slowdown, but the effect is more severe in swarmer cells because they have a longer G1 phase. Activity of SpoT, which produces the (p)ppGpp alarmone and extends the G1 phase, accentuates the cell cycle-dependent growth slowdown. Collectively, our data identify a coupling between cell growth, the G1 phase, and asymmetric division that C. crescentus may exploit for environmental adaptation through SpoT activity. This coupling differentially modulates the growth rate of functionally distinct daughter cells, thereby altering the relative abundance of ecologically important G1-specific traits within the population.

  View details for DOI 10.1073/pnas.2406397121

  View details for PubMedID 39361646

• Synthesis of a Borrelia burgdorferi-Derived Muropeptide Standard Fragment Library. Molecules (Basel, Switzerland) Putnik, R., Zhou, J., Irnov, I., Garner, E., Liu, M., Bersch, K. L., Jacobs-Wagner, C., Grimes, C. L. 2024; 29 (14)

  Abstract

  The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn's disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi, is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.

  View details for DOI 10.3390/molecules29143297

  View details for PubMedID 39064876

  View details for PubMedCentralID PMC11279244

• Glycogen phase separation drives macromolecular rearrangement and asymmetric division in E. coli. bioRxiv : the preprint server for biology Thappeta, Y., Cañas-Duarte, S. J., Kallem, T., Fragasso, A., Xiang, Y., Gray, W., Lee, C., Cegelski, L., Jacobs-Wagner, C. 2024

  Abstract

  Bacteria often experience nutrient limitation in nature and the laboratory. While exponential and stationary growth phases are well characterized in the model bacterium Escherichia coli, little is known about what transpires inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the position of nucleoids and cell division sites becomes increasingly asymmetric during transition phase. These asymmetries were coupled with spatial reorganization of proteins, ribosomes, and RNAs to nucleoid-centric localizations. Results from live-cell imaging experiments, complemented with genetic and 13C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. In vitro experiments suggest that these phenotypes are likely due to the propensity of glycogen to phase separate in crowded environments, as glycogen condensates exclude fluorescent proteins under physiological crowding conditions. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase separation allows cells to store large glucose reserves without counting them as cytoplasmic space.

  View details for DOI 10.1101/2024.04.19.590186

  View details for PubMedID 38659787

  View details for PubMedCentralID PMC11042326

• Through the looking glass: An adventure into the metastable world of the bacterial cytoplasm. Cell Jacobs-Wagner, C. 2024; 187 (2): 228-234

  Abstract

  This personal story recounts the accidental observation, the struggles, the breakthroughs, and the collaborative spirit of a few individuals that led to the discovery that bacterial cells expend energy to effectively fluidize their otherwise "glass-like" cytoplasm and promote the dispersal of large cytoplasmic components. This adventure, which led us into an uncharted world at the intersection of cell biology and condensed matter physics about ten years ago, forever transformed the way I view cells and conduct research.

  View details for DOI 10.1016/j.cell.2023.11.034

  View details for PubMedID 38242080

• Apparent simplicity and emergent robustness in the control of the Escherichia coli cell cycle. Cell systems Govers, S. K., Campos, M., Tyagi, B., Laloux, G., Jacobs-Wagner, C. 2023

  Abstract

  To examine how bacteria achieve robust cell proliferation across diverse conditions, we developed a method that quantifies 77 cell morphological, cell cycle, and growth phenotypes of a fluorescently labeled Escherichia coli strain and >800 gene deletion derivatives under multiple nutrient conditions. This approach revealed extensive phenotypic plasticity and deviating mutant phenotypes were often nutrient dependent. From this broad phenotypic landscape emerged simple and robust unifying rules (laws) that connect DNA replication initiation, nucleoid segregation, FtsZ ring formation, and cell constriction to specific aspects of cell size (volume, length, or added length) at the population level. Furthermore, completion of cell division followed the initiation of cell constriction after a constant time delay across strains and nutrient conditions, identifying cell constriction as a key control point for cell size determination. Our work provides a population-level description of the governing principles by which E.coli integrates cell cycle processes and growth rate with cell size to achieve its robust proliferative capability. A record of this paper's transparent peer review process is included in the supplemental information.

  View details for DOI 10.1016/j.cels.2023.12.001

  View details for PubMedID 38157847

• Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development. Cell chemical biology Carlson, D. L., Kowalewski, M., Bodoor, K., Lietzan, A. D., Hughes, P. F., Gooden, D., Loiselle, D. L., Alcorta, D., Dingman, Z., Mueller, E. A., Irnov, I., Modla, S., Chaya, T., Caplan, J., Embers, M., Miller, J. C., Jacobs-Wagner, C., Redinbo, M. R., Spector, N., Haystead, T. A. 2023

  Abstract

  Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

  View details for DOI 10.1016/j.chembiol.2023.10.004

  View details for PubMedID 37918401

• Author Correction: Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete. Nature communications Takacs, C. N., Wachter, J., Xiang, Y., Ren, Z., Karaboja, X., Scott, M., Stoner, M. R., Irnov, I., Jannetty, N., Rosa, P. A., Wang, X., Jacobs-Wagner, C. 2023; 14 (1): 6298

  View details for DOI 10.1038/s41467-023-42035-6

  View details for PubMedID 37813840

• Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi. PLoS genetics Ren, Z., Takacs, C. N., Brandao, H. B., Jacobs-Wagner, C., Wang, X. 2023; 19 (7): e1010857

  Abstract

  Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/Smc. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that Smc and the Smc-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

  View details for DOI 10.1371/journal.pgen.1010857

  View details for PubMedID 37494383

• Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi. bioRxiv : the preprint server for biology Ren, Z., Takacs, C. N., Brandão, H. B., Jacobs-Wagner, C., Wang, X. 2023

  Abstract

  Borrelia burgdorferi , a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/SMC. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that SMC and the SMC-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC . Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

  View details for DOI 10.1101/2023.03.19.532819

  View details for PubMedID 37066390

  View details for PubMedCentralID PMC10103936

• Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete. Nature communications Takacs, C. N., Wachter, J., Xiang, Y., Ren, Z., Karaboja, X., Scott, M., Stoner, M. R., Irnov, I., Jannetty, N., Rosa, P. A., Wang, X., Jacobs-Wagner, C. 2022; 13 (1): 7173

  Abstract

  Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochete's length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

  View details for DOI 10.1038/s41467-022-34876-4

  View details for PubMedID 36450725

  View details for PubMedCentralID 7853543

• Connecting single-cell ATP dynamics to overflow metabolism, cell growth, and the cell cycle in Escherichia coli. Current biology : CB Lin, W. H., Jacobs-Wagner, C. 2022

  Abstract

  Adenosine triphosphate (ATP) is an abundant and essential metabolite that cells consume and regenerate in large amounts to support growth. Although numerous studies have inferred the intracellular concentration of ATP in bacterial cultures, what happens in individual bacterial cells under stable growth conditions is less clear. Here, we use the QUEEN-2m biosensor to quantify ATP dynamics in single Escherichia coli cells in relation to their growth rate, metabolism, cell cycle, and cell lineage. We find that ATP dynamics are more complex than expected from population studies and are associated with growth-rate variability. Under stable nutrient-rich condition, cells can display large fluctuations in ATP level that are partially coordinated with the cell cycle. Abrogation of aerobic acetate fermentation (overflow metabolism) through genetic deletion considerably reduces both the amplitude of ATP level fluctuations and the cell-cycle trend. Similarly, growth in media in which acetate fermentation is lower or absent results in the reduction of ATP level fluctuation and cell-cycle trend. This suggests that overflow metabolism exhibits temporal dynamics, which contributes to fluctuating ATP levels during growth. Remarkably, at the single-cell level, growth rate negatively correlates with the amplitude of ATP fluctuation for each tested condition, linking ATP dynamics to growth-rate heterogeneity in clonal populations. Our work highlights the importance of single-cell analysis in studying metabolism and its implication to phenotypic diversity and cell growth.

  View details for DOI 10.1016/j.cub.2022.07.035

  View details for PubMedID 35961315

• Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi. PloS one Takacs, C. N., Nakajima, Y., Haber, J. E., Jacobs-Wagner, C. 2022; 17 (11): e0278151

  Abstract

  The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

  View details for DOI 10.1371/journal.pone.0278151

  View details for PubMedID 36441794

• Christine Jacobs-Wagner. Current biology : CB Jacobs-Wagner, C. 2021; 31 (14): R882-R883

  Abstract

  Interview with Christine Jacobs-Wagner, who studies the replication of bacterial cells at Stanford University.

  View details for DOI 10.1016/j.cub.2021.05.060

  View details for PubMedID 34314707

• Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization. Current biology : CB Sanchez, A. D., Branon, T. C., Cote, L. E., Papagiannakis, A., Liang, X., Pickett, M. A., Shen, K., Jacobs-Wagner, C., Ting, A. Y., Feldman, J. L. 2021

  Abstract

  Microtubules are polarized intracellular polymers that play key roles in the cell, including in transport, polarity, and cell division. Across eukaryotic cell types, microtubules adopt diverse intracellular organization to accommodate these distinct functions coordinated by specific cellular sites called microtubule-organizing centers (MTOCs). Over 50 years of research on MTOC biology has focused mainly on the centrosome; however, most differentiated cells employ non-centrosomal MTOCs (ncMTOCs) to organize their microtubules into diverse arrays, which are critical to cell function. To identify essential ncMTOC components, we developed the biotin ligase-based, proximity-labeling approach TurboID for use in C.elegans. We identified proteins proximal to the microtubule minus end protein PTRN-1/Patronin at the apical ncMTOC of intestinal epithelial cells, focusing on two conserved proteins: spectraplakin protein VAB-10B/MACF1 and WDR-62, a protein we identify as homologous to vertebrate primary microcephaly disease protein WDR62. VAB-10B and WDR-62 do not associate with the centrosome and instead specifically regulate non-centrosomal microtubules and the apical targeting of microtubule minus-end proteins. Depletion of VAB-10B resulted in microtubule mislocalization and delayed localization of a microtubule nucleation complex ɣ-tubulin ring complex (gamma-TuRC), while loss of WDR-62 decreased the number of dynamic microtubules and abolished gamma-TuRC localization. This regulation occurs downstream of cell polarity and in conjunction with actin. As this is the first report for non-centrosomal roles of WDR62 family proteins, we expand the basic cell biological roles of this important disease protein. Our studies identify essential ncMTOC components and suggest a division of labor where microtubule growth and localization are distinctly regulated.

  View details for DOI 10.1016/j.cub.2021.06.021

  View details for PubMedID 34242576

• Interconnecting solvent quality, transcription, and chromosome folding in Escherichia coli. Cell Xiang, Y., Surovtsev, I. V., Chang, Y., Govers, S. K., Parry, B. R., Liu, J., Jacobs-Wagner, C. 2021

  Abstract

  All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.

  View details for DOI 10.1016/j.cell.2021.05.037

  View details for PubMedID 34186018

• A human secretome library screen reveals a role for Peptidoglycan Recognition Protein 1 in Lyme borreliosis. PLoS pathogens Gupta, A., Arora, G., Rosen, C. E., Kloos, Z., Cao, Y., Cerny, J., Sajid, A., Hoornstra, D., Golovchenko, M., Rudenko, N., Munderloh, U., Hovius, J. W., Booth, C. J., Jacobs-Wagner, C., Palm, N. W., Ring, A. M., Fikrig, E. 2020; 16 (11): e1009030

  Abstract

  Lyme disease, the most common vector-borne illness in North America, is caused by the spirochete Borrelia burgdorferi. Infection begins in the skin following a tick bite and can spread to the hearts, joints, nervous system, and other organs. Diverse host responses influence the level of B. burgdorferi infection in mice and humans. Using a systems biology approach, we examined potential molecular interactions between human extracellular and secreted proteins and B. burgdorferi. A yeast display library expressing 1031 human extracellular proteins was probed against 36 isolates of B. burgdorferi sensu lato. We found that human Peptidoglycan Recognition Protein 1 (PGLYRP1) interacted with the vast majority of B. burgdorferi isolates. In subsequent experiments, we demonstrated that recombinant PGLYRP1 interacts with purified B. burgdorferi peptidoglycan and exhibits borreliacidal activity, suggesting that vertebrate hosts may use PGLYRP1 to identify B. burgdorferi. We examined B. burgdorferi infection in mice lacking PGLYRP1 and observed an increased spirochete burden in the heart and joints, along with splenomegaly. Mice lacking PGLYRP1 also showed signs of immune dysregulation, including lower serum IgG levels and higher levels of IFNgamma, CXCL9, and CXCL10.Taken together, our findings suggest that PGLYRP1 plays a role in the host's response to B. burgdorferi and further demonstrate the utility of expansive yeast display screening in capturing biologically relevant interactions between spirochetes and their hosts.

  View details for DOI 10.1371/journal.ppat.1009030

  View details for PubMedID 33175909

• Caulobacter crescentus: model system extraordinaire CURRENT BIOLOGY Govers, S. K., Jacobs-Wagner, C. 2020; 30 (19): R1151–R1158

  View details for Web of Science ID 000579845200031

• Caulobacter crescentus: model system extraordinaire. Current biology : CB Govers, S. K., Jacobs-Wagner, C. n. 2020; 30 (19): R1151–R1158

  Abstract

  In scientific research, we often rely on well-established model systems to tackle important questions. In this context, extensive characterization of specific bacterial species such as Escherichia coli and Bacillus subtilis has provided a vast amount of knowledge that extends well beyond the biology of these two organisms. However, the bacterial world is large and extremely diverse, necessitating the development of additional models that complement the classical rod-shaped and symmetrically dividing systems. Caulobacter crescentus is a species that has met this need effectively, as its dimorphic lifestyle showcases distinctive features, including cellular asymmetry and differentiation during the cell cycle. Studying C. crescentus has reformed our understanding of bacterial intracellular organization, cellular development, and cell-cycle regulation. These findings have, in turn, stimulated studies in other bacteria, shedding light on how protein function and cell morphology can evolve and diversify. Studies in C. crescentus have also deepened our knowledge of other topics (e.g. cell mechanosensing, motility, and bacterial aging), while opening the door to biotechnological innovations. In this Primer, we provide some general background to this peculiar bacterium and highlight specific features that have contributed to its rise as a versatile bacterial model. This Primer is not meant to be exhaustive on any topic and is instead intended to provide a taste of the power of C. crescentus as a model system to explore a diverse range of topics.

  View details for DOI 10.1016/j.cub.2020.07.033

  View details for PubMedID 33022259

• A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi. Applied and environmental microbiology Takacs, C. N., Scott, M. n., Chang, Y. n., Kloos, Z. A., Irnov, I. n., Rosa, P. A., Liu, J. n., Jacobs-Wagner, C. n. 2020

  Abstract

  The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen.IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

  View details for DOI 10.1128/AEM.02519-20

  View details for PubMedID 33257311

• Origin of exponential growth in nonlinear reaction networks. Proceedings of the National Academy of Sciences of the United States of America Lin, W. H., Kussell, E. n., Young, L. S., Jacobs-Wagner, C. n. 2020

  Abstract

  Exponentially growing systems are prevalent in nature, spanning all scales from biochemical reaction networks in single cells to food webs of ecosystems. How exponential growth emerges in nonlinear systems is mathematically unclear. Here, we describe a general theoretical framework that reveals underlying principles of long-term growth: scalability of flux functions and ergodicity of the rescaled systems. Our theory shows that nonlinear fluxes can generate not only balanced growth but also oscillatory or chaotic growth modalities, explaining nonequilibrium dynamics observed in cell cycles and ecosystems. Our mathematical framework is broadly useful in predicting long-term growth rates from natural and synthetic networks, analyzing the effects of system noise and perturbations, validating empirical and phenomenological laws on growth rate, and studying autocatalysis and network evolution.

  View details for DOI 10.1073/pnas.2013061117

  View details for PubMedID 33093194

• Long-Distance Cooperative and Antagonistic RNA Polymerase Dynamics via DNA Supercoiling CELL Kim, S., Beltran, B., Irnov, I., Jacobs-Wagner, C. 2019; 179 (1): 106-+

  Abstract

  Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.

  View details for DOI 10.1016/j.cell.2019.08.033

  View details for Web of Science ID 000486618500017

  View details for PubMedID 31539491

• Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jutras, B. L., Lochhead, R. B., Kloos, Z. A., Biboy, J., Strle, K., Booth, C. J., Govers, S. K., Gray, J., Schumann, P., Vollmer, W., Bockenstedt, L. K., Steere, A. C., Jacobs-Wagner, C. 2019; 116 (27): 13498–507

  Abstract

  Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

  View details for DOI 10.1073/pnas.1904170116

  View details for Web of Science ID 000473427900054

  View details for PubMedID 31209025

  View details for PubMedCentralID PMC6613144

• Nucleoid Size Scaling and Intracellular Organization of Translation across Bacteria CELL Gray, W. T., Govers, S. K., Xiang, Y., Parry, B. R., Campos, M., Kim, S., Jacobs-Wagner, C. 2019; 177 (6): 1632-+

  Abstract

  The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.

  View details for DOI 10.1016/j.cell.2019.05.017

  View details for Web of Science ID 000469415100024

  View details for PubMedID 31150626

  View details for PubMedCentralID PMC6629263

• Fluorescent Proteins, Promoters, and Selectable Markers for Applications in the Lyme Disease Spirochete Borrelia burgdorferi APPLIED AND ENVIRONMENTAL MICROBIOLOGY Takacs, C. N., Kloos, Z. A., Scott, M., Rosa, P. A., Jacobs-Wagner, C. 2018; 84 (24)

  Abstract

  Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging in B. burgdorferi We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.IMPORTANCE Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium's transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

  View details for DOI 10.1128/AEM.01824-18

  View details for Web of Science ID 000451988000010

  View details for PubMedID 30315081

  View details for PubMedCentralID PMC6275353

• De novo design of self-assembling helical protein filaments SCIENCE Shen, H., Fallas, J. A., Lynch, E., Sheffler, W., Parry, B., Jannetty, N., Decarreau, J., Wagenbach, M., Vicente, J., Chen, J., Wang, L., Dowling, Q., Oberdorfer, G., Stewart, L., Wordeman, L., De Yoreo, J., Jacobs-Wagner, C., KolInan, J., Baker, D. 2018; 362 (6415): 705-+

  Abstract

  We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo-electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

  View details for DOI 10.1126/science.aau3775

  View details for Web of Science ID 000450474500045

  View details for PubMedID 30409885

  View details for PubMedCentralID PMC6637945

• mTORC1 Controls Phase Separation and the Biophysical Properties of the Cytoplasm by Tuning Crowding CELL Delarue, M., Brittingham, G. P., Pfeffer, S., Surovtsev, I. V., Pinglay, S., Kennedy, K. J., Schaffer, M., Gutierrez, J. I., Sang, D., Poterewicz, G., Chung, J. K., Plitzko, J. M., Groves, J. T., Jacobs-Wagner, C., Engel, B. D., Holt, L. J. 2018; 174 (2): 338-+

  Abstract

  Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed genetically encoded multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein that self-assemble into bright, stable particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can modulate the effective diffusion coefficient of particles ≥20 nm in diameter more than 2-fold by tuning ribosome concentration, without any discernable effect on the motion of molecules ≤5 nm. This change in ribosome concentration affected phase separation both in vitro and in vivo. Together, these results establish a role for mTORC1 in controlling both the mesoscale biophysical properties of the cytoplasm and biomolecular condensation.

  View details for DOI 10.1016/j.cell.2018.05.042

  View details for Web of Science ID 000438482800011

  View details for PubMedID 29937223

• Genomewide phenotypic analysis of growth, cell morphogenesis, and cell cycle events in Escherichia coli MOLECULAR SYSTEMS BIOLOGY Campos, M., Govers, S. K., Irnov, I., Dobihal, G. S., Cornet, F., Jacobs-Wagner, C. 2018; 14 (6): e7573

  Abstract

  Cell size, cell growth, and cell cycle events are necessarily intertwined to achieve robust bacterial replication. Yet, a comprehensive and integrated view of these fundamental processes is lacking. Here, we describe an image-based quantitative screen of the single-gene knockout collection of Escherichia coli and identify many new genes involved in cell morphogenesis, population growth, nucleoid (bulk chromosome) dynamics, and cell division. Functional analyses, together with high-dimensional classification, unveil new associations of morphological and cell cycle phenotypes with specific functions and pathways. Additionally, correlation analysis across ~4,000 genetic perturbations shows that growth rate is surprisingly not predictive of cell size. Growth rate was also uncorrelated with the relative timings of nucleoid separation and cell constriction. Rather, our analysis identifies scaling relationships between cell size and nucleoid size and between nucleoid size and the relative timings of nucleoid separation and cell division. These connections suggest that the nucleoid links cell morphogenesis to the cell cycle.

  View details for DOI 10.15252/msb.20177573

  View details for Web of Science ID 000436804300007

  View details for PubMedID 29941428

  View details for PubMedCentralID PMC6018989

• Effects of mRNA Degradation and Site-Specific Transcriptional Pausing on Protein Expression Noise BIOPHYSICAL JOURNAL Kim, S., Jacobs-Wagner, C. 2018; 114 (7): 1718–29

  Abstract

  Genetically identical cells exhibit diverse phenotypes even when experiencing the same environment. This phenomenon in part originates from cell-to-cell variability (noise) in protein expression. Although various kinetic schemes of stochastic transcription initiation are known to affect gene expression noise, how posttranscription initiation events contribute to noise at the protein level remains incompletely understood. To address this question, we developed a stochastic simulation-based model of bacterial gene expression that integrates well-known dependencies between transcription initiation, transcription elongation dynamics, mRNA degradation, and translation. We identified realistic conditions under which mRNA lifetime and transcriptional pauses modulate the protein expression noise initially introduced by the promoter architecture. For instance, we found that the short lifetime of bacterial mRNAs facilitates the production of protein bursts. Conversely, RNA polymerase (RNAP) pausing at specific sites during transcription elongation can attenuate protein bursts by fluidizing the RNAP traffic to the point of erasing the effect of a bursty promoter. Pause-prone sites, if located close to the promoter, can also affect noise indirectly by reducing both transcription and translation initiation due to RNAP and ribosome congestion. Our findings highlight how the interplay between transcription initiation, transcription elongation, translation, and mRNA degradation shapes the distribution in protein numbers. They also have implications for our understanding of gene evolution and suggest combinatorial strategies for modulating phenotypic variability by genetic engineering.

  View details for DOI 10.1016/j.bpj.2018.02.010

  View details for Web of Science ID 000430214500020

  View details for PubMedID 29642040

  View details for PubMedCentralID PMC5954620

• Subcellular Organization: A Critical Feature of Bacterial Cell Replication CELL Surovtsev, I. V., Jacobs-Wagner, C. 2018; 172 (6): 1271–93

  Abstract

  Spatial organization is a hallmark of all living systems. Even bacteria, the smallest forms of cellular life, display defined shapes and complex internal organization, showcasing a highly structured genome, cytoskeletal filaments, localized scaffolding structures, dynamic spatial patterns, active transport, and occasionally, intracellular organelles. Spatial order is required for faithful and efficient cellular replication and offers a powerful means for the development of unique biological properties. Here, we discuss organizational features of bacterial cells and highlight how bacteria have evolved diverse spatial mechanisms to overcome challenges cells face as self-replicating entities.

  View details for DOI 10.1016/j.cell.2018.01.014

  View details for Web of Science ID 000427482100028

  View details for PubMedID 29522747

  View details for PubMedCentralID PMC5870143

• Combinatorial Origin of Protein Expression Noise Kim, S., Jacobs-Wagner, C. CELL PRESS. 2018: 395A

  View details for DOI 10.1016/j.bpj.2017.11.2184

  View details for Web of Science ID 000430450000458

• Crosstalk between the tricarboxylic acid cycle and peptidoglycan synthesis in Caulobacter crescentus through the homeostatic control of alpha-ketoglutarate PLOS GENETICS Irnov, I., Wang, Z., Jannetty, N. D., Bustamante, J. A., Rhee, K. Y., Jacobs-Wagner, C. 2017; 13 (8): e1006978

  Abstract

  To achieve robust replication, bacteria must integrate cellular metabolism and cell wall growth. While these two processes have been well characterized, the nature and extent of cross-regulation between them is not well understood. Here, using classical genetics, CRISPRi, metabolomics, transcriptomics and chemical complementation approaches, we show that a loss of the master regulator Hfq in Caulobacter crescentus alters central metabolism and results in cell shape defects in a nutrient-dependent manner. We demonstrate that the cell morphology phenotype in the hfq deletion mutant is attributable to a disruption of α-ketoglutarate (KG) homeostasis. In addition to serving as a key intermediate of the tricarboxylic acid (TCA) cycle, KG is a by-product of an enzymatic reaction required for the synthesis of peptidoglycan, a major component of the bacterial cell wall. Accumulation of KG in the hfq deletion mutant interferes with peptidoglycan synthesis, resulting in cell morphology defects and increased susceptibility to peptidoglycan-targeting antibiotics. This work thus reveals a direct crosstalk between the TCA cycle and cell wall morphogenesis. This crosstalk highlights the importance of metabolic homeostasis in not only ensuring adequate availability of biosynthetic precursors, but also in preventing interference with cellular processes in which these intermediates arise as by-products.

  View details for DOI 10.1371/journal.pgen.1006978

  View details for Web of Science ID 000408763800043

  View details for PubMedID 28827812

  View details for PubMedCentralID PMC5578688

• A Tick Antivirulence Protein Potentiates Antibiotics against Staphylococcus aureus ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Abraham, N. M., Liu, L., Jutras, B. L., Murfin, K., Acar, A., Yarovinsky, T. O., Sutton, E., Heisig, M., Jacobs-Wagner, C., Fikriga, E. 2017; 61 (7)

  Abstract

  New strategies are needed to combat antibiotic resistance, especially against pathogens such as methicillin-resistant Staphylococcus aureus A tick antifreeze glycoprotein, IAFGP, possesses potent antibiofilm properties against a variety of clinical pathogens, including S. aureus Synergy between IAFGP, or a peptide (P1) representative of a repeat region of the protein, with different antibiotics was assessed in vitro Antibiotics that synergized with either IAFPG or P1 were further evaluated in vivo using vertebrate and invertebrate infection models. IAFGP readily enhanced the efficacy of antibiotics against S. aureus Synergy with daptomycin, an antibiotic used to treat methicillin-resistant S. aureus, was observed in vitro and in vivo using iafgp-transgenic mice and flies. Furthermore, synergy with ciprofloxacin or gentamicin, antibiotics not generally used to treat S. aureus, was also perceived. The combined effect of the antibiotic and IAFGP was associated with improved permeation of the antibiotic into the cell. Our results highlight that synergy of IAFGP with antibiotics traditionally used to treat this pathogen, and enhancement of the potency of antibiotics not commonly used against this microbe, can provide novel alternative therapeutic strategies to combat bacterial infections.

  View details for DOI 10.1128/AAC.00113-17

  View details for Web of Science ID 000406257600012

  View details for PubMedID 28438938

  View details for PubMedCentralID PMC5487661

• Replication fork passage drives asymmetric dynamics of a critical nucleoid-associated protein in Caulobacter EMBO JOURNAL Arias-Cartin, R., Dobihal, G. S., Campos, M., Surovtsev, I. V., Parry, B., Jacobs-Wagner, C. 2017; 36 (3): 301–18

  Abstract

  In bacteria, chromosome dynamics and gene expression are modulated by nucleoid-associated proteins (NAPs), but little is known about how NAP activity is coupled to cell cycle progression. Using genomic techniques, quantitative cell imaging, and mathematical modeling, our study in Caulobacter crescentus identifies a novel NAP (GapR) whose activity over the cell cycle is shaped by DNA replication. GapR activity is critical for cellular function, as loss of GapR causes severe, pleiotropic defects in growth, cell division, DNA replication, and chromosome segregation. GapR also affects global gene expression with a chromosomal bias from origin to terminus, which is associated with a similar general bias in GapR binding activity along the chromosome. Strikingly, this asymmetric localization cannot be explained by the distribution of GapR binding sites on the chromosome. Instead, we present a mechanistic model in which the spatiotemporal dynamics of GapR are primarily driven by the progression of the replication forks. This model represents a simple mechanism of cell cycle regulation, in which DNA-binding activity is intimately linked to the action of DNA replication.

  View details for DOI 10.15252/embj.201695513

  View details for Web of Science ID 000394444900008

  View details for PubMedID 28011580

  View details for PubMedCentralID PMC5286365

• Pathogen-mediated manipulation of arthropod microbiota to promote infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Abraham, N. M., Liu, L., Jutras, B., Yadav, A. K., Narasimhan, S., Gopalakrishnan, V., Ansari, J. M., Jefferson, K. K., Cava, F., Jacobs-Wagner, C., Fikrig, E. 2017; 114 (5): E781–E790

  Abstract

  Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood. Ixodes scapularis ticks harbor numerous human pathogens, including Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We now demonstrate that A. phagocytophilum modifies the I. scapularis microbiota to more efficiently infect the tick. A. phagocytophilum induces ticks to express Ixodes scapularis antifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier-critical obstacles for Anaplasma colonization. Mechanistically, IAFGP binds the terminal d-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

  View details for DOI 10.1073/pnas.1613422114

  View details for Web of Science ID 000393196300016

  View details for PubMedID 28096373

  View details for PubMedCentralID PMC5293115

• DNA-relay mechanism is sufficient to explain ParA-dependent intracellular transport and patterning of single and multiple cargos PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Surovtsev, I. V., Campos, M., Jacobs-Wagner, C. 2016; 113 (46): E7268–E7276

  Abstract

  Spatial ordering of macromolecular components inside cells is important for cellular physiology and replication. In bacteria, ParA/B systems are known to generate various intracellular patterns that underlie the transport and partitioning of low-copy-number cargos such as plasmids. ParA/B systems consist of ParA, an ATPase that dimerizes and binds DNA upon ATP binding, and ParB, a protein that binds the cargo and stimulates ParA ATPase activity. Inside cells, ParA is asymmetrically distributed, forming a propagating wave that is followed by the ParB-rich cargo. These correlated dynamics lead to cargo oscillation or equidistant spacing over the nucleoid depending on whether the cargo is in single or multiple copies. Currently, there is no model that explains how these different spatial patterns arise and relate to each other. Here, we test a simple DNA-relay model that has no imposed asymmetry and that only considers the ParA/ParB biochemistry and the known fluctuating and elastic dynamics of chromosomal loci. Stochastic simulations with experimentally derived parameters demonstrate that this model is sufficient to reproduce the signature patterns of ParA/B systems: the propagating ParA gradient correlated with the cargo dynamics, the single-cargo oscillatory motion, and the multicargo equidistant patterning. Stochasticity of ATP hydrolysis breaks the initial symmetry in ParA distribution, resulting in imbalance of elastic force acting on the cargo. Our results may apply beyond ParA/B systems as they reveal how a minimal system of two players, one binding to DNA and the other modulating this binding, can transform directionally random DNA fluctuations into directed motion and intracellular patterning.

  View details for DOI 10.1073/pnas.1616118113

  View details for Web of Science ID 000388970100017

  View details for PubMedID 27799522

  View details for PubMedCentralID PMC5135302

• Lyme disease and relapsing fever Borrelia elongate through zones of peptidoglycan synthesis that mark division sites of daughter cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jutras, B., Scott, M., Parry, B., Biboy, J., Gray, J., Vollmer, W., Jacobs-Wagner, C. 2016; 113 (33): 9162–70

  Abstract

  Agents that cause Lyme disease, relapsing fever, leptospirosis, and syphilis belong to the phylum Spirochaetae-a unique lineage of bacteria most known for their long, spiral morphology. Despite the relevance to human health, little is known about the most fundamental aspects of spirochete growth. Here, using quantitative microscopy to track peptidoglycan cell-wall synthesis, we found that the Lyme disease spirochete Borrelia burgdorferi displays a complex pattern of growth. B. burgdorferi elongates from discrete zones that are both spatially and temporally regulated. In addition, some peptidoglycan incorporation occurs along the cell body, with the notable exception of a large region at the poles. Newborn cells inherit a highly active zone of peptidoglycan synthesis at midcell that contributes to elongation for most of the cell cycle. Concomitant with the initiation of nucleoid separation and cell constriction, second and third zones of elongation are established at the 1/4 and 3/4 cellular positions, marking future sites of division for the subsequent generation. Positioning of elongation zones along the cell is robust to cell length variations and is relatively precise over long distances (>30 µm), suggesting that cells ‟sense" relative, as opposed to absolute, cell length to establish zones of peptidoglycan synthesis. The transition from one to three zones of peptidoglycan growth during the cell cycle is also observed in relapsing fever Borrelia. However, this mode of growth does not extend to representative species from other spirochetal genera, suggesting that this distinctive growth mode represents an evolutionary divide in the spirochete phylum.

  View details for DOI 10.1073/pnas.1610805113

  View details for Web of Science ID 000381399200035

  View details for PubMedID 27506799

  View details for PubMedCentralID PMC4995948

• Ultra-High Resolution 3D Imaging of Whole Cells CELL Huang, F., Sirinakis, G., Allgeyer, E. S., Schroeder, L. K., Duim, W. C., Kromann, E. B., Phan, T., Rivera-Molina, F. E., Myers, J. R., Irnov, I., Lessard, M., Zhang, Y., Handel, M., Jacobs-Wagner, C., Lusk, C., Rothman, J. E., Toomre, D., Booth, M. J., Bewersdorf, J. 2016; 166 (4): 1028–40

  Abstract

  Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50-80 nm) and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.

  View details for DOI 10.1016/j.cell.2016.06.016

  View details for Web of Science ID 000382258700023

  View details for PubMedID 27397506

  View details for PubMedCentralID PMC5005454

• The Slow Mobility of the ParA Partitioning Protein Underlies Its Steady-State Patterning in Caulobacter BIOPHYSICAL JOURNAL Surovtsev, I. V., Lim, H., Jacobs-Wagner, C. 2016; 110 (12): 2790–99

  Abstract

  In bacteria, ParABS systems mediate intracellular transport of various cargos, including chromosomal regions in Caulobacter crescentus. Transport of the ParB/parS partition complex requires the DNA-binding activity of ParA, which transiently tethers the partition complex during translocation. In C. crescentus, the directionality of the transport is set up by a gradient of ParA whose concentration gradually increases from one end of the cell (old pole) to the other (new pole). Importantly, this ParA gradient is already observed before DNA replication and segregation are initiated when the partition complex is anchored at the old pole. How such micron-scale ParA pattern is established and maintained before the initiation of chromosome segregation has not been experimentally established. Although the stimulation of ParA ATPase activity by the localized ParB/parS partition complex is thought to be involved, this activity alone cannot quantitatively describe the ParA pattern observed inside cells. Instead, our experimental and theoretical study shows that the missing key component for achieving the experimentally observed steady-state ParA patterning is the slow mobility of ParA dimers (D ∼10(-3)μm(2)/s) due to intermittent DNA binding. Our model recapitulates the entire steady-state ParA distribution observed experimentally, including the shape of the gradient as well as ParA accumulation at the location of the partition complex. Stochastic simulations suggest that cell-to-cell variability in ParA pattern is due to the low ParA copy number in C. crescentus cells. The model also accounts for an apparent exclusion of ParA from regions with small spacing between partition complexes observed in filamentous cells. Collectively, our work demonstrates that in addition to its function in mediating transport, the conserved DNA-binding property of ParA has a critical function before DNA segregation by setting up a ParA pattern required for transport directionality.

  View details for DOI 10.1016/j.bpj.2016.05.014

  View details for Web of Science ID 000378383300026

  View details for PubMedID 27332137

  View details for PubMedCentralID PMC4919595

• Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis MOLECULAR MICROBIOLOGY Paintdakhi, A., Parry, B., Campos, M., Irnov, I., Elf, J., Surovtsev, I., Jacobs-Wagner, C. 2016; 99 (4): 767–77

  Abstract

  With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills.

  View details for DOI 10.1111/mmi.13264

  View details for Web of Science ID 000370338900011

  View details for PubMedID 26538279

  View details for PubMedCentralID PMC4752901

• Bacterial Evolution: What Goes Around Comes Around CURRENT BIOLOGY Jutras, B. L., Jacobs-Wagner, C. 2015; 25 (12): R496–R498

  Abstract

  Over 3 billion years ago, cellular life began anaerobically. A new study now establishes a key link between oxidative stress and proliferation of wall-less bacteria known as L-forms. The finding provides insights into both the origin of life and the potential threat posed by pathogenic L-forms.

  View details for DOI 10.1016/j.cub.2015.05.002

  View details for Web of Science ID 000356562500007

  View details for PubMedID 26079079

• Transferred interbacterial antagonism genes augment eukaryotic innate immune function NATURE Chou, S., Daugherty, M. D., Peterson, S., Biboy, J., Yang, Y., Jutras, B. L., Fritz-Laylin, L. K., Ferrin, M. A., Harding, B. N., Jacobs-Wagner, C., Yang, X., Vollmer, W., Malik, H. S., Mougous, J. D. 2015; 518 (7537): 98-+

  Abstract

  Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

  View details for DOI 10.1038/nature13965

  View details for Web of Science ID 000349098000039

  View details for PubMedID 25470067

  View details for PubMedCentralID PMC4713192

• Mycofumigation by the Volatile Organic Compound-Producing Fungus Muscodor albus Induces Bacterial Cell Death through DNA Damage APPLIED AND ENVIRONMENTAL MICROBIOLOGY Alpha, C. J., Campos, M., Jacobs-Wagner, C., Strobela, S. A. 2015; 81 (3): 1147–56

  Abstract

  Muscodor albus belongs to a genus of endophytic fungi that inhibit and kill other fungi, bacteria, and insects through production of a complex mixture of volatile organic compounds (VOCs). This process of mycofumigation has found commercial application for control of human and plant pathogens, but the mechanism of the VOC toxicity is unknown. Here, the mode of action of these volatiles was investigated through a series of genetic screens and biochemical assays. A single-gene knockout screen revealed high sensitivity for Escherichia coli lacking enzymes in the pathways of DNA repair, DNA metabolic process, and response to stress when exposed to the VOCs of M. albus. Furthermore, the sensitivity of knockouts involved in the repair of specific DNA alkyl adducts suggests that the VOCs may induce alkylation. Evidence of DNA damage suggests that these adducts lead to breaks during DNA replication or transcription if not properly repaired. Additional cytotoxicity profiling indicated that during VOC exposure, E. coli became filamentous and demonstrated an increase in cellular membrane fluidity. The volatile nature of the toxic compounds produced by M. albus and their broad range of inhibition make this fungus an attractive biological agent. Understanding the antimicrobial effects and the VOC mode of action will inform the utility and safety of potential mycofumigation applications for M. albus.

  View details for DOI 10.1128/AEM.03294-14

  View details for Web of Science ID 000347914800037

  View details for PubMedID 25452287

  View details for PubMedCentralID PMC4292491

• A Constant Size Extension Drives Bacterial Cell Size Homeostasis CELL Campos, M., Surovtsev, I. V., Kato, S., Paintdakhi, A., Beltran, B., Ebmeier, S. E., Jacobs-Wagner, C. 2014; 159 (6): 1433-1446

  Abstract

  Cell size control is an intrinsic feature of the cell cycle. In bacteria, cell growth and division are thought to be coupled through a cell size threshold. Here, we provide direct experimental evidence disproving the critical size paradigm. Instead, we show through single-cell microscopy and modeling that the evolutionarily distant bacteria Escherichia coli and Caulobacter crescentus achieve cell size homeostasis by growing, on average, the same amount between divisions, irrespective of cell length at birth. This simple mechanism provides a remarkably robust cell size control without the need of being precise, abating size deviations exponentially within a few generations. This size homeostasis mechanism is broadly applicable for symmetric and asymmetric divisions, as well as for different growth rates. Furthermore, our data suggest that constant size extension is implemented at or close to division. Altogether, our findings provide fundamentally distinct governing principles for cell size and cell-cycle control in bacteria.

  View details for DOI 10.1016/j.cell.2014.11.022

  View details for Web of Science ID 000346652900020

  View details for PubMedID 25480302

  View details for PubMedCentralID PMC4258233

• G1-arrested newborn cells are the predominant infectious form of the pathogen Brucella abortus NATURE COMMUNICATIONS Deghelt, M., Mullier, C., Sternon, J., Francis, N., Laloux, G., Dotreppe, D., Van der Henst, C., Jacobs-Wagner, C., Letesson, J., De Bolle, X. 2014; 5: 4366

  Abstract

  Several intracellular pathogens, such as Brucella abortus, display a biphasic infection process starting with a non-proliferative stage of unclear nature. Here, we study the cell cycle of B. abortus at the single-cell level, in culture and during infection of HeLa cells and macrophages. The localization of segregation and replication loci of the two bacterial chromosomes indicates that, immediately after being engulfed by host-cell endocytic vacuoles, most bacterial cells are newborn. These bacterial cells do not initiate DNA replication for the next 4 to 6 h, indicating a G1 arrest. Moreover, growth is completely stopped during that time, reflecting a global cell cycle block. Growth and DNA replication resume later, although bacteria still reside within endosomal-like compartments. We hypothesize that the predominance of G1-arrested bacteria in the infectious population, and the bacterial cell cycle arrest following internalization, may constitute a widespread strategy among intracellular pathogens to colonize new proliferation niches.

  View details for DOI 10.1038/ncomms5366

  View details for Web of Science ID 000340615500057

  View details for PubMedID 25006695

  View details for PubMedCentralID PMC4104442

• Evidence for a DNA-relay mechanism in ParABS-mediated chromosome segregation ELIFE Lim, H. C., Surovtsev, I. V., Beltran, B. G., Huang, F., Bewersdorf, J., Jacobs-Wagner, C. 2014; 3

  Abstract

  The widely conserved ParABS system plays a major role in bacterial chromosome segregation. How the components of this system work together to generate translocation force and directional motion remains uncertain. Here, we combine biochemical approaches, quantitative imaging and mathematical modeling to examine the mechanism by which ParA drives the translocation of the ParB/parS partition complex in Caulobacter crescentus. Our experiments, together with simulations grounded on experimentally-determined biochemical and cellular parameters, suggest a novel 'DNA-relay' mechanism in which the chromosome plays a mechanical function. In this model, DNA-bound ParA-ATP dimers serve as transient tethers that harness the elastic dynamics of the chromosome to relay the partition complex from one DNA region to another across a ParA-ATP dimer gradient. Since ParA-like proteins are implicated in the partitioning of various cytoplasmic cargos, the conservation of their DNA-binding activity suggests that the DNA-relay mechanism may be a general form of intracellular transport in bacteria.DOI: http://dx.doi.org/10.7554/eLife.02758.001.

  View details for DOI 10.7554/eLife.02758

  View details for Web of Science ID 000336253600001

  View details for PubMedID 24859756

  View details for PubMedCentralID PMC4067530

• The Bacterial Cytoplasm Has Glass-like Properties and Is Fluidized by Metabolic Activity CELL Parry, B. R., Surovtsev, I. V., Cabeen, M. T., O'Hem, C. S., Dufresne, E. R., Jacobs-Wagner, C. 2014; 156 (1-2): 183–94

  Abstract

  The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.

  View details for DOI 10.1016/j.cell.2013.11.028

  View details for Web of Science ID 000329912200020

  View details for PubMedID 24361104

  View details for PubMedCentralID PMC3956598

• How do bacteria localize proteins to the cell pole? JOURNAL OF CELL SCIENCE Laloux, G., Jacobs-Wagner, C. 2014; 127 (1): 11–19

  Abstract

  It is now well appreciated that bacterial cells are highly organized, which is far from the initial concept that they are merely bags of randomly distributed macromolecules and chemicals. Central to their spatial organization is the precise positioning of certain proteins in subcellular domains of the cell. In particular, the cell poles - the ends of rod-shaped cells - constitute important platforms for cellular regulation that underlie processes as essential as cell cycle progression, cellular differentiation, virulence, chemotaxis and growth of appendages. Thus, understanding how the polar localization of specific proteins is achieved and regulated is a crucial question in bacterial cell biology. Often, polarly localized proteins are recruited to the poles through their interaction with other proteins or protein complexes that were already located there, in a so-called diffusion-and-capture mechanism. Bacteria are also starting to reveal their secrets on how the initial pole 'recognition' can occur and how this event can be regulated to generate dynamic, reproducible patterns in time (for example, during the cell cycle) and space (for example, at a specific cell pole). Here, we review the major mechanisms that have been described in the literature, with an emphasis on the self-organizing principles. We also present regulation strategies adopted by bacterial cells to obtain complex spatiotemporal patterns of protein localization.

  View details for DOI 10.1242/jcs.138628

  View details for Web of Science ID 000329122500002

  View details for PubMedID 24345373

  View details for PubMedCentralID PMC3874780

• Suppression of Amber Codons in Caulobacter crescentus by the Orthogonal Escherichia coli Histidyl-tRNA Synthetase/tRNA(His) Pair PLOS ONE Ko, J., Llopis, P., Heinritz, J., Jacobs-Wagner, C., Soell, D. 2013; 8 (12): e83630

  Abstract

  While translational read-through of stop codons by suppressor tRNAs is common in many bacteria, archaea and eukaryotes, this phenomenon has not yet been observed in the α-proteobacterium Caulobacter crescentus. Based on a previous report that C. crescentus and Escherichia coli tRNA(His) have distinctive identity elements, we constructed E. coli tRNA(His) CUA, a UAG suppressor tRNA for C. crescentus. By examining the expression of three UAG codon- containing reporter genes (encoding a β-lactamase, the fluorescent mCherry protein, or the C. crescentus xylonate dehydratase), we demonstrated that the E. coli histidyl-tRNA synthetase/tRNA(His) CUA pair enables in vivo UAG suppression in C. crescentus. E. coli histidyl-tRNA synthetase (HisRS) or tRNA(His) CUA alone did not achieve suppression; this indicates that the E. coli HisRS/tRNA(His) CUA pair is orthogonal in C. crescentus. These results illustrate that UAG suppression can be achieved in C. crescentus with an orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair.

  View details for DOI 10.1371/journal.pone.0083630

  View details for Web of Science ID 000329194700053

  View details for PubMedID 24386240

  View details for PubMedCentralID PMC3875453

• Transcriptomic and phylogenetic analysis of a bacterial cell cycle reveals strong associations between gene co-expression and evolution BMC GENOMICS Fang, G., Passalacqua, K. D., Hocking, J., Llopis, P., Gerstein, M., Bergman, N. H., Jacobs-Wagner, C. 2013; 14: 450

  Abstract

  The genetic network involved in the bacterial cell cycle is poorly understood even though it underpins the remarkable ability of bacteria to proliferate. How such network evolves is even less clear. The major aims of this work were to identify and examine the genes and pathways that are differentially expressed during the Caulobacter crescentus cell cycle, and to analyze the evolutionary features of the cell cycle network.We used deep RNA sequencing to obtain high coverage RNA-Seq data of five C. crescentus cell cycle stages, each with three biological replicates. We found that 1,586 genes (over a third of the genome) display significant differential expression between stages. This gene list, which contains many genes previously unknown for their cell cycle regulation, includes almost half of the genes involved in primary metabolism, suggesting that these "house-keeping" genes are not constitutively transcribed during the cell cycle, as often assumed. Gene and module co-expression clustering reveal co-regulated pathways and suggest functionally coupled genes. In addition, an evolutionary analysis of the cell cycle network shows a high correlation between co-expression and co-evolution. Most co-expression modules have strong phylogenetic signals, with broadly conserved genes and clade-specific genes predominating different substructures of the cell cycle co-expression network. We also found that conserved genes tend to determine the expression profile of their module.We describe the first phylogenetic and single-nucleotide-resolution transcriptomic analysis of a bacterial cell cycle network. In addition, the study suggests how evolution has shaped this network and provides direct biological network support that selective pressure is not on individual genes but rather on the relationship between genes, which highlights the importance of integrating phylogenetic analysis into biological network studies.

  View details for DOI 10.1186/1471-2164-14-450

  View details for Web of Science ID 000328624600001

  View details for PubMedID 23829427

  View details for PubMedCentralID PMC3829707

• Spatiotemporal control of PopZ localization through cell cycle-coupled multimerization JOURNAL OF CELL BIOLOGY Laloux, G., Jacobs-Wagner, C. 2013; 201 (6): 827–41

  Abstract

  Bacterial cell poles constitute defined subcellular domains where numerous proteins localize, often at specific times, to affect various physiological processes. How pole recognition occurs and what governs the timing of protein localization are often unknown. In this paper, we investigate the mechanisms governing the localization of PopZ, a chromosome-anchoring protein whose unipolar to bipolar localization pattern is critical for cell cycle progression in Caulobacter crescentus. We provide evidence that polar localization of PopZ relied on its self-assembly into a higher-order structure (matrix) and that the unipolar to bipolar transition was coupled to the asymmetric distribution of ParA during the translocation of the origin-proximal ParB-parS partition complex. Collectively, our data suggest a model in which a local increase of ParA concentration promotes the assembly of a PopZ matrix precisely when and where this matrix is needed. Such coupling of protein assembly with a cell cycle-associated molecular asymmetry may represent a principle of cellular organization for controlling protein localization in both time and space.

  View details for DOI 10.1083/jcb.201303036

  View details for Web of Science ID 000320121900006

  View details for PubMedID 23751494

  View details for PubMedCentralID PMC3678156

• Cellular organization of the transfer of genetic information CURRENT OPINION IN MICROBIOLOGY Campos, M., Jacobs-Wagner, C. 2013; 16 (2): 171–76

  Abstract

  Each step involved in the transfer of genetic information is spatially regulated in eukaryotic cells, as transcription, translation and mRNA degradation mostly occur in distinct functional compartments (e.g., nucleus, cytoplasm and P-bodies). At first glance in bacteria, these processes seem to take place in the same compartment - the cytoplasm - because of the conspicuous absence of membrane-enclosed organelles. However, it is becoming increasingly evident that mRNA-related processes are also spatially organized inside bacterial cells, and that this organization affects cellular function. The aims of this review are to summarize the current knowledge about this organization and to consider the mechanisms and forces shaping the cell interior. The field stands at an exciting point where new technologies are making long-standing questions amenable to experimentation.

  View details for DOI 10.1016/j.mib.2013.01.007

  View details for Web of Science ID 000319180100010

  View details for PubMedID 23395479

  View details for PubMedCentralID PMC3646911

• Growth Medium-Dependent Glycine Incorporation into the Peptidoglycan of Caulobacter crescentus PLOS ONE Takacs, C. N., Hocking, J., Cabeen, M. T., Bui, N., Poggio, S., Vollmer, W., Jacobs-Wagner, C. 2013; 8 (2): e57579

  Abstract

  The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity.

  View details for DOI 10.1371/journal.pone.0057579

  View details for Web of Science ID 000315524900129

  View details for PubMedID 23469030

  View details for PubMedCentralID PMC3585186

• In Vivo Biochemistry in Bacterial Cells Using FRAP: Insight into the Translation Cycle BIOPHYSICAL JOURNAL Llopis, P., Sliusarenko, O., Heinritz, J., Jacobs-Wagner, C. 2012; 103 (9): 1848–59

  Abstract

  In vivo measurements of the mobility and binding kinetics of cellular components are essential to fully understand the biochemical processes occurring inside cells. Here, we describe a fluorescence recovery after photobleaching-based method that can be easily implemented to the study of reaction-diffusion processes in live bacteria despite their small size. We apply this method to provide new, to our knowledge, quantitative insight into multiple aspects of the bacterial translation cycle by measuring the binding kinetics and the micrometer-scale diffusive properties of the 50S ribosomal subunit in live Caulobacter cells. From our measurements, we infer that 70% of 50S subunits are engaged in translation and display, on average, limited motion on the micrometer scale, consistent with little mixing of transcripts undergoing translation. We also extract the average rate constants for the binding of 50S subunits to 30S initiation complexes during initiation and for their release from mRNAs when translation is completed. From this, we estimate the average time of protein synthesis and the average search time of 50S subunits before they engage in the next initiation event. Additionally, our experiments suggest that so-called free 50S subunits do not diffuse freely; instead their mobility is significantly slowed down, possibly through transient associations with mRNA.

  View details for DOI 10.1016/j.bpj.2012.09.035

  View details for Web of Science ID 000310785300012

  View details for PubMedID 23199913

  View details for PubMedCentralID PMC3491719

• The evolution of new lipoprotein subunits of the bacterial outer membrane BAM complex MOLECULAR MICROBIOLOGY Anwari, K., Webb, C. T., Poggio, S., Perry, A. J., Belousoff, M., Celik, N., Ramm, G., Lovering, A., Sockett, R., Smit, J., Jacobs-Wagner, C., Lithgow, T. 2012; 84 (5): 832–44

  Abstract

  The β-barrel assembly machine (BAM) complex is an essential feature of all bacteria with an outer membrane. The core subunit of the BAM complex is BamA and, in Escherichia coli, four lipoprotein subunits: BamB, BamC, BamD and BamE, also function in the BAM complex. Hidden Markov model analysis was used to comprehensively assess the distribution of subunits of the BAM lipoproteins across all subclasses of proteobacteria. A patchwork distribution was detected which is readily reconciled with the evolution of the α-, β-, γ-, δ- and ε-proteobacteria. Our findings lead to a proposal that the ancestral BAM complex was composed of two subunits: BamA and BamD, and that BamB, BamC and BamE evolved later in a distinct sequence of events. Furthermore, in some lineages novel lipoproteins have evolved instead of the lipoproteins found in E. coli. As an example of this concept, we show that no known species of α-proteobacteria has a homologue of BamC. However, purification of the BAM complex from the model α-proteobacterium Caulobacter crescentus identified a novel subunit we refer to as BamF, which has a conserved sequence motif related to sequences found in BamC. BamF and BamD can be eluted from the BAM complex under similar conditions, mirroring the BamC:D module seen in the BAM complex of γ-proteobacteria such as E. coli.

  View details for DOI 10.1111/j.1365-2958.2012.08059.x

  View details for Web of Science ID 000304301500004

  View details for PubMedID 22524202

  View details for PubMedCentralID PMC3359395

• 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

• Probing Spatial Organization of mRNA in Bacterial Cells using 3D Super-Resolution Microscopy Kim, S., Mlodzianoski, M., Bewersdorf, J., Jacobs-Wagner, C. CELL PRESS. 2012: 278A

  View details for DOI 10.1016/j.bpj.2011.11.1537

  View details for Web of Science ID 000321561201717

• Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Charbon, G., Wang, J., Brustad, E., Schultz, P. G., Horwich, A. L., Jacobs-Wagner, C., Chapman, E. 2011; 21 (20): 6067–70

  Abstract

  The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.

  View details for DOI 10.1016/j.bmcl.2011.08.057

  View details for Web of Science ID 000295494500008

  View details for PubMedID 21890355

  View details for PubMedCentralID PMC3177974

• Subcellular Protein Localization by Using a Genetically Encoded Fluorescent Amino Acid CHEMBIOCHEM Charbon, G., Brustad, E., Scott, K. A., Wang, J., Lobner-Olesen, A., Schultz, P. G., Jacobs-Wagner, C., Chapman, E. 2011; 12 (12): 1818–21

  View details for DOI 10.1002/cbic.201100282

  View details for Web of Science ID 000294268600004

  View details for PubMedID 21681882

  View details for PubMedCentralID PMC3175735

• High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics MOLECULAR MICROBIOLOGY Sliusarenko, O., Heinritz, J., Emonet, T., Jacobs-Wagner, C. 2011; 80 (3): 612–27

  Abstract

  Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues. Quantitative, unbiased study of these spatio-temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high-throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini-colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old-pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position-dependent silencing is regulated by division.

  View details for DOI 10.1111/j.1365-2958.2011.07579.x

  View details for Web of Science ID 000289729800007

  View details for PubMedID 21414037

  View details for PubMedCentralID PMC3090749

• The Domain Organization of the Bacterial Intermediate Filament-Like Protein Crescentin is Important for Assembly and Function CYTOSKELETON Cabeen, M. T., Herrmann, H., Jacobs-Wagner, C. 2011; 68 (4): 205–19

  Abstract

  Crescentin is a bacterial filament-forming protein that exhibits domain organization features found in metazoan intermediate filament (IF) proteins. Structure-function studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantifiable phenotypes. Here we exploit the characteristic localization of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curvature associated with impaired crescentin function to analyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastructural analysis in vitro with cellular localization and functional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be functionally subdivided into two subdomains; the first (amino-terminal) is required for function but not assembly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assembly, and the linker L1 appears important to prevent runaway assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have critical functional roles in stabilizing crescentin structures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organization, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions throughout life from bacteria to humans.

  View details for DOI 10.1002/cm.20505

  View details for Web of Science ID 000288611100001

  View details for PubMedID 21360832

  View details for PubMedCentralID PMC3087291

• Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins EMBO JOURNAL Schofield, W. B., Lim, H., Jacobs-Wagner, C. 2010; 29 (18): 3068–81

  Abstract

  What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back-and-forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole-organizing protein PopZ, compensate for the defect in segregation regulation in ΔtipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self-organizing oscillator to create motion suitable for chromosome segregation.

  View details for DOI 10.1038/emboj.2010.207

  View details for Web of Science ID 000282991900005

  View details for PubMedID 20802464

  View details for PubMedCentralID PMC2944072

• A metabolic assembly line in bacteria NATURE CELL BIOLOGY Cabeen, M. T., Jacobs-Wagner, C. 2010; 12 (8): 731–33

  View details for DOI 10.1038/ncb0810-731

  View details for Web of Science ID 000280561600003

  View details for PubMedID 20680001

• A protein critical for cell constriction in the Gram-negative bacterium Caulobacter crescentus localizes at the division site through its peptidoglycan-binding LysM domains MOLECULAR MICROBIOLOGY Poggio, S., Takacs, C. N., Vollmer, W., Jacobs-Wagner, C. 2010; 77 (1): 74–89

  Abstract

  During division of Gram-negative bacteria, invagination of the cytoplasmic membrane and inward growth of the peptidoglycan (PG) are followed by the cleavage of connective septal PG to allow cell separation. This PG splitting process requires temporal and spatial regulation of cell wall hydrolases. In Escherichia coli, LytM factors play an important role in PG splitting. Here we identify and characterize a member of this family (DipM) in Caulobacter crescentus. Unlike its E. coli counterparts, DipM is essential for viability under fast-growth conditions. Under slow-growth conditions, the DeltadipM mutant displays severe defects in cell division and FtsZ constriction. Consistent with its function in division, DipM colocalizes with the FtsZ ring during the cell cycle. Mutagenesis suggests that the LytM domain of DipM is essential for protein function, despite being non-canonical. DipM also carries two tandems of the PG-binding LysM domain that are sufficient for FtsZ ring localization. Localization and fluorescence recovery after photobleaching microscopy experiments suggest that DipM localization is mediated, at least in part, by the ability of the LysM tandems to distinguish septal, multilayered PG from non-septal, monolayered PG.

  View details for DOI 10.1111/j.1365-2958.2010.07223.x

  View details for Web of Science ID 000279168200008

  View details for PubMedID 20497503

  View details for PubMedCentralID PMC2907422

• Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Cabeen, M. T., Murolo, M. A., Briegel, A., Bui, N., Vollmer, W., Ausmees, N., Jensen, G. J., Jacobs-Wagner, C. 2010; 192 (13): 3368–78

  Abstract

  Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant O-polysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis.

  View details for DOI 10.1128/JB.01371-09

  View details for Web of Science ID 000278806100016

  View details for PubMedID 20435724

  View details for PubMedCentralID PMC2897673

• Spatial organization of the flow of genetic information in bacteria NATURE Llopis, P., Jackson, A. F., Sliusarenko, O., Surovtsev, I., Heinritz, J., Emonet, T., Jacobs-Wagner, C. 2010; 466 (7302): 77–U90

  Abstract

  Eukaryotic cells spatially organize mRNA processes such as translation and mRNA decay. Much less is clear in bacterial cells where the spatial distribution of mature mRNA remains ambiguous. Using a sensitive method based on quantitative fluorescence in situ hybridization, we show here that in Caulobacter crescentus and Escherichia coli, chromosomally expressed mRNAs largely display limited dispersion from their site of transcription during their lifetime. We estimate apparent diffusion coefficients at least two orders of magnitude lower than expected for freely diffusing mRNA, and provide evidence in C. crescentus that this mRNA localization restricts ribosomal mobility. Furthermore, C. crescentus RNase E appears associated with the DNA independently of its mRNA substrates. Collectively, our findings show that bacteria can spatially organize translation and, potentially, mRNA decay by using the chromosome layout as a template. This chromosome-centric organization has important implications for cellular physiology and for our understanding of gene expression in bacteria.

  View details for DOI 10.1038/nature09152

  View details for Web of Science ID 000279343800037

  View details for PubMedID 20562858

  View details for PubMedCentralID PMC2896451

• Processivity of peptidoglycan synthesis provides a built-in mechanism for the robustness of straight-rod cell morphology PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sliusarenko, O., Cabeen, M. T., Wolgemuth, C. W., Jacobs-Wagner, C., Emonet, T. 2010; 107 (22): 10086–91

  Abstract

  The propagation of cell shape across generations is remarkably robust in most bacteria. Even when deformations are acquired, growing cells progressively recover their original shape once the deforming factors are eliminated. For instance, straight-rod-shaped bacteria grow curved when confined to circular microchambers, but straighten in a growth-dependent fashion when released. Bacterial cell shape is maintained by the peptidoglycan (PG) cell wall, a giant macromolecule of glycan strands that are synthesized by processive enzymes and cross-linked by peptide chains. Changes in cell geometry require modifying the PG and therefore depend directly on the molecular-scale properties of PG structure and synthesis. Using a mathematical model we quantify the straightening of curved Caulobacter crescentus cells after disruption of the cell-curving crescentin structure. We observe that cells straighten at a rate that is about half (57%) the cell growth rate. Next we show that in the absence of other effects there exists a mathematical relationship between the rate of cell straightening and the processivity of PG synthesis-the number of subunits incorporated before termination of synthesis. From the measured rate of cell straightening this relationship predicts processivity values that are in good agreement with our estimates from published data. Finally, we consider the possible role of three other mechanisms in cell straightening. We conclude that regardless of the involvement of other factors, intrinsic properties of PG processivity provide a robust mechanism for cell straightening that is hardwired to the cell wall synthesis machinery.

  View details for DOI 10.1073/pnas.1000737107

  View details for Web of Science ID 000278246000034

  View details for PubMedID 20479277

  View details for PubMedCentralID PMC2890421

• MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall JOURNAL OF BACTERIOLOGY Takacs, C. N., Poggio, S., Charbon, G., Pucheault, M., Vollmer, W., Jacobs-Wagner, C. 2010; 192 (6): 1671–84

  Abstract

  MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.

  View details for DOI 10.1128/JB.01311-09

  View details for Web of Science ID 000274891300022

  View details for PubMedID 20023035

  View details for PubMedCentralID PMC2832515

• A Modular BAM Complex in the Outer Membrane of the alpha-Proteobacterium Caulobacter crescentus PLOS ONE Anwari, K., Poggio, S., Perry, A., Gatsos, X., Ramarathinam, S., Williamson, N. A., Noinaj, N., Buchanan, S., Gabriel, K., Purcell, A. W., Jacobs-Wagner, C., Lithgow, T. 2010; 5 (1): e8619

  Abstract

  Mitochondria are organelles derived from an intracellular alpha-proteobacterium. The biogenesis of mitochondria relies on the assembly of beta-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an alpha-proteobacterium, and the BAM (beta-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A approximately 150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.

  View details for DOI 10.1371/journal.pone.0008619

  View details for Web of Science ID 000273414200010

  View details for PubMedID 20062535

  View details for PubMedCentralID PMC2797634

• Function and Regulation of the Bacterial Cytoskeleton Jacobs-Wagner, C. CELL PRESS. 2010: 3A

  View details for DOI 10.1016/j.bpj.2009.12.020

  View details for Web of Science ID 000208762000017

• Polar Localization of the CckA Histidine Kinase and Cell Cycle Periodicity of the Essential Master Regulator CtrA in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Angelastro, P. S., Sliusarenko, O., Jacobs-Wagner, C. 2010; 192 (2): 539–52

  Abstract

  The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two cis-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between beta-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression.

  View details for DOI 10.1128/JB.00985-09

  View details for Web of Science ID 000273097500017

  View details for PubMedID 19897656

  View details for PubMedCentralID PMC2805319

• The Bacterial Cytoskeleton ANNUAL REVIEW OF GENETICS, VOL 44 Cabeen, M. T., Jacobs-Wagner, C., Campbell, A., Lichten, M., Schupbach, G. 2010; 44: 365–92

  Abstract

  Bacteria, like eukaryotes, employ cytoskeletal elements to perform many functions, including cell morphogenesis, cell division, DNA partitioning, and cell motility. They not only possess counterparts of eukaryotic actin, tubulin, and intermediate filament proteins, but they also have cytoskeletal elements of their own. Unlike the rigid sequence and structural conservation often observed for eukaryotic cytoskeletal proteins, the bacterial counterparts can display considerable diversity in sequence and function across species. Their wide range of function highlights the flexibility of core cytoskeletal protein motifs, such that one type of cytoskeletal element can perform various functions, and one function can be performed by different types of cytoskeletal elements.

  View details for DOI 10.1146/annurev-genet-102108-134845

  View details for Web of Science ID 000286042600016

  View details for PubMedID 21047262

• The reducible complexity of a mitochondrial molecular machine PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Clements, A., Bursac, D., Gatsos, X., Perry, A. J., Civciristov, S., Celik, N., Likic, V. A., Poggio, S., Jacobs-Wagner, C., Strugnell, R. A., Lithgow, T. 2009; 106 (37): 15791–95

  Abstract

  Molecular machines drive essential biological processes, with the component parts of these machines each contributing a partial function or structural element. Mitochondria are organelles of eukaryotic cells, and depend for their biogenesis on a set of molecular machines for protein transport. How these molecular machines evolved is a fundamental question. Mitochondria were derived from an alpha-proteobacterial endosymbiont, and we identified in alpha-proteobacteria the component parts of a mitochondrial protein transport machine. In bacteria, the components are found in the inner membrane, topologically equivalent to the mitochondrial proteins. Although the bacterial proteins function in simple assemblies, relatively little mutation would be required to convert them to function as a protein transport machine. This analysis of protein transport provides a blueprint for the evolution of cellular machinery in general.

  View details for DOI 10.1073/pnas.0908264106

  View details for Web of Science ID 000269806600052

  View details for PubMedID 19717453

  View details for PubMedCentralID PMC2747197

• Bacterial cell curvature through mechanical control of cell growth EMBO JOURNAL Cabeen, M. T., Charbon, G., Vollmer, W., Born, P., Ausmees, N., Weibel, D. B., Jacobs-Wagner, C. 2009; 28 (9): 1208–19

  Abstract

  The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.

  View details for DOI 10.1038/emboj.2009.61

  View details for Web of Science ID 000265885000004

  View details for PubMedID 19279668

  View details for PubMedCentralID PMC2683044

• Bacterial intermediate filaments: in vivo assembly, organization, and dynamics of crescentin GENES & DEVELOPMENT Charbon, G., Cabeen, M. T., Jacobs-Wagner, C. 2009; 23 (9): 1131–44

  Abstract

  Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin also shares in vivo properties of assembly and dynamics with IF proteins by forming stable filamentous structures that continuously incorporate subunits along their length and that grow in a nonpolar fashion. De novo assembly of crescentin is biphasic and involves a cell size-dependent mechanism that controls the length of the structure by favoring lateral insertion of crescentin subunits over bipolar longitudinal extension when the structure ends reach the cell poles. The crescentin structure is stably anchored to the cell envelope, and this cellular organization requires MreB function, identifying a new function for MreB and providing a parallel to the role of actin in IF assembly and organization in metazoan cells. Additionally, analysis of an MreB localization mutant suggests that cell wall insertion during cell elongation normally occurs along two helices of opposite handedness, each counterbalancing the other's torque.

  View details for DOI 10.1101/gad.1795509

  View details for Web of Science ID 000265757900012

  View details for PubMedID 19417107

  View details for PubMedCentralID PMC2682956

• RodZ, a component of the bacterial core morphogenic apparatus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Alyahya, S., Alexander, R., Costa, T., Henriques, A. O., Emonet, T., Jacobs-Wagner, C. 2009; 106 (4): 1239–44

  Abstract

  The molecular basis of bacterial cell morphogenesis remains largely an open question. Here we discover a morphogenic protein, RodZ, which is widely conserved across the bacterial kingdom. In Caulobacter crescentus, RodZ is essential for viability and is involved in all aspects of this organism's complex morphology. Depletion or over-production of RodZ results in grossly misshapen cells with stalk defects. RodZ exhibits a localization pattern during the cell cycle corresponding to sites of active peptidoglycan synthesis. The temporal transition of RodZ between patchy/helical and mid-cell localization mimics and depends on the actin-like MreB cytoskeleton. In Escherichia coli, an organism with a distinct mode of growth and MreB localization dynamics, RodZ follows MreB and retains its crucial role in cell morphogenesis, demonstrating conservation of function. Genomic analysis shows that RodZ represents an ancient function unique to bacteria. Multiple sequence alignment of 143 RodZ sequences from species across bacterial phyla identifies an N-terminal cytoplasmic domain with a helix-turn-helix motif, a transmembrane sequence, and a previously unidentified, conserved periplasmic or extracellular C-terminal domain. Both the N- and C-terminal domains are important for function, with the N-terminal domain containing localization determinants. This study uncovers a key missing player in the cytoskeleton-based growth machinery enabling heritable and defined cellular forms in bacteria.

  View details for DOI 10.1073/pnas.0810794106

  View details for Web of Science ID 000262831600051

  View details for PubMedID 19164570

  View details for PubMedCentralID PMC2633561

• Localization of PBP3 in Caulobacter crescentus is highly dynamic and largely relies on its functional transpeptidase domain MOLECULAR MICROBIOLOGY Costa, T., Priyadarshini, R., Jacobs-Wagner, C. 2008; 70 (3): 634–51

  Abstract

  In rod-shaped bacteria, septal peptidoglycan synthesis involves the late recruitment of the ftsI gene product (PBP3 in Escherichia coli) to the FtsZ ring. We show that in Caulobacter crescentus, PBP3 accumulates at the new pole at the beginning of the cell cycle. Fluorescence recovery after photobleaching experiments reveal that polar PBP3 molecules are, constantly and independently of FtsZ, replaced by those present in the cellular pool, implying that polar PBP3 is not a remnant of the previous division. By the time cell constriction is initiated, all PBP3 polar accumulation has disappeared in favour of an FtsZ-dependent localization near midcell, consistent with PBP3 function in cell division. Kymograph analysis of time-lapse experiments shows that the recruitment of PBP3 to the FtsZ ring is progressive and initiated very early on, shortly after FtsZ ring formation and well before cell constriction starts. Accumulation of PBP3 near midcell is also highly dynamic with a rapid exchange of PBP3 molecules between midcell and cellular pools. Localization of PBP3 at both midcell and pole appears multifactorial, primarily requiring the catalytic site of PBP3. Collectively, our results suggest a role for PBP3 in pole morphogenesis and provide new insights into the process of peptidoglycan assembly during division.

  View details for DOI 10.1111/j.1365-2958.2008.06432.x

  View details for Web of Science ID 000262305000009

  View details for PubMedID 18786147

  View details for PubMedCentralID PMC2581642

• A self-associating protein critical for chromosome attachment, division, and polar organization in Caulobacter CELL Ebersbach, G., Briegel, A., Jensen, G. J., Jacobs-Wagner, C. 2008; 134 (6): 956–68

  Abstract

  Cell polarization is an integral part of many unrelated bacterial processes. How intrinsic cell polarization is achieved is poorly understood. Here, we provide evidence that Caulobacter crescentus uses a multimeric pole-organizing factor (PopZ) that serves as a hub to concurrently achieve several polarizing functions. During chromosome segregation, polar PopZ captures the ParB*ori complex and thereby anchors sister chromosomes at opposite poles. This step is essential for stabilizing bipolar gradients of a cell division inhibitor and setting up division near midcell. PopZ also affects polar stalk morphogenesis and mediates the polar localization of the morphogenetic and cell cycle signaling proteins CckA and DivJ. Polar accumulation of PopZ, which is central to its polarizing activity, can be achieved independently of division and does not appear to be dictated by the pole curvature. Instead, evidence suggests that localization of PopZ largely relies on PopZ multimerization in chromosome-free regions, consistent with a self-organizing mechanism.

  View details for DOI 10.1016/j.cell.2008.07.016

  View details for Web of Science ID 000259318100016

  View details for PubMedID 18805089

  View details for PubMedCentralID PMC2614312

• PflI, a protein involved in flagellar positioning in Caulobacter crescentus JOURNAL OF BACTERIOLOGY Obuchowski, P. L., Jacobs-Wagner, C. 2008; 190 (5): 1718–29

  Abstract

  The bacterial flagellum is important for motility and adaptation to environmental niches. The sequence of events required for the synthesis of the flagellar apparatus has been extensively studied, yet the events that dictate where the flagellum is placed at the onset of flagellar biosynthesis remain largely unknown. We addressed this question for alphaproteobacteria by using the polarly flagellated alphaproteobacterium Caulobacter crescentus as an experimental model system. To identify candidates for a role in flagellar placement, we searched all available alphaproteobacterial genomes for genes of unknown function that cluster with early flagellar genes and that are present in polarly flagellated alphaproteobacteria while being absent in alphaproteobacteria with other flagellation patterns. From this in silico screen, we identified pflI. Loss of PflI function in C. crescentus results in an abnormally high frequency of cells with a randomly placed flagellum, while other aspects of cell polarization remain normal. In a wild-type background, a fusion of green fluorescent protein (GFP) and PflI localizes to the pole where the flagellum develops. This polar localization is independent of the flagellar protein FliF, whose oligomerization into the MS ring is thought to define the site of flagellar synthesis, suggesting that PflI acts before or independently of this event. Overproduction of PflI-GFP often leads to ectopic localization at the wrong, stalked pole. This is accompanied by a high frequency of flagellum formation at this ectopic site, suggesting that the location of PflI is a sufficient marker for a site for flagellar assembly.

  View details for DOI 10.1128/JB.01706-07

  View details for Web of Science ID 000253554300027

  View details for PubMedID 18165296

  View details for PubMedCentralID PMC2258662

• Skin and bones: the bacterial cytoskeleton, cell wall, and cell morphogenesis JOURNAL OF CELL BIOLOGY Cabeen, M. T., Jacobs-Wagner, C. 2007; 179 (3): 381–87

  Abstract

  The bacterial world is full of varying cell shapes and sizes, and individual species perpetuate a defined morphology generation after generation. We review recent findings and ideas about how bacteria use the cytoskeleton and other strategies to regulate cell growth in time and space to produce different shapes and sizes.

  View details for DOI 10.1083/jcb.200708001

  View details for Web of Science ID 000250705200005

  View details for PubMedID 17967949

  View details for PubMedCentralID PMC2064785

• The tubulin homologue FtsZ contributes to cell elongation by guiding cell wall precursor synthesis in Caulobacter crescentus MOLECULAR MICROBIOLOGY Aaron, M., Charbon, G., Lam, H., Schwarz, H., Vollmer, W., Jacobs-Wagner, C. 2007; 64 (4): 938–52

  Abstract

  The tubulin homologue FtsZ is well known for its essential function in bacterial cell division. Here, we show that in Caulobacter crescentus, FtsZ also plays a major role in cell elongation by spatially regulating the location of MurG, which produces the essential lipid II peptidoglycan cell wall precursor. The early assembly of FtsZ into a highly mobile ring-like structure during cell elongation is quickly followed by the recruitment of MurG and a major redirection of peptidoglycan precursor synthesis to the midcell region. These FtsZ-dependent events occur well before cell constriction and contribute to cell elongation. In the absence of FtsZ, MurG fails to accumulate near midcell and cell elongation proceeds unperturbed in appearance by insertion of peptidoglycan material along the entire sidewalls. Evidence suggests that bacteria use both a FtsZ-independent and a FtsZ-dependent mode of peptidoglycan synthesis to elongate, the importance of each mode depending on the timing of FtsZ assembly during elongation.

  View details for DOI 10.1111/j.1365-2958.2007.05720.x

  View details for Web of Science ID 000246398900008

  View details for PubMedID 17501919

• The asymmetric distribution of the essential histidine kinase PdhS indicates a differentiation event in Brucella abortus EMBO JOURNAL Hallez, R., Mignolet, J., Van Mullem, V., Wery, M., Vandenhaute, J., Letesson, J., Jacobs-Wagner, C., De Bolle, X. 2007; 26 (5): 1444–55

  Abstract

  Many organisms use polar localization of signalling proteins to control developmental events in response to completion of asymmetric cell division. Asymmetric division was recently reported for Brucella abortus, a class III facultative intracellular pathogen generating two sibling cells of slightly different size. Here we characterize PdhS, a cytoplasmic histidine kinase essential for B. abortus viability and homologous to the asymmetrically distributed PleC and DivJ histidine kinases from Caulobacter crescentus. PdhS is localized at the old pole of the large cell, and after division and growth, the small cell acquires PdhS at its old pole. PdhS may therefore be considered as a differentiation marker as it labels the old pole of the large cell. Moreover, PdhS colocalizes with its paired response regulator DivK. Finally, PdhS is able to localize at one pole in other alpha-proteobacteria, suggesting that a polar structure associating PdhS with one pole is conserved in these bacteria. We propose that a differentiation event takes place after the completion of cytokinesis in asymmetrically dividing alpha-proteobacteria. Altogether, these data suggest that prokaryotic differentiation may be much more widespread than expected.

  View details for DOI 10.1038/sj.emboj.7601577

  View details for Web of Science ID 000244716000022

  View details for PubMedID 17304218

  View details for PubMedCentralID PMC1817626

• Exploration into the spatial and temporal mechanisms of bacterial polarity TRENDS IN MICROBIOLOGY Ebersbach, G., Jacobs-Wagner, C. 2007; 15 (3): 101–8

  Abstract

  The recognition of bacterial asymmetry is not new: the first high-resolution microscopy studies revealed that bacteria come in a multitude of shapes and sometimes carry asymmetrically localized external structures such as flagella on the cell surface. Even so, the idea that bacteria could have an inherent overall polarity, which affects not only their outer appearance but also many of their vital processes, has only recently been appreciated. In this review, we focus on recent advances in our understanding of the molecular mechanisms underlying the establishment of polarized functions and cell polarity in bacteria.

  View details for DOI 10.1016/j.tim.2007.01.004

  View details for Web of Science ID 000245465700003

  View details for PubMedID 17275310

• A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell CELL Lam, H., Schofield, W. B., Jacobs-Wagner, C. 2006; 124 (5): 1011–23

  Abstract

  Polarity is often an intrinsic property of the cell, yet little is known about its origin or its maintenance over generations. Here we identify a landmark protein, TipN, which acts as a spatial and temporal cue for setting up the correct polarity in the bacterium Caulobacter crescentus. TipN marks the new pole throughout most of the cell cycle, and its relocation to the nascent poles at the end of division provides a preexisting reference point for orienting the polarity axis in the progeny. Deletion of tipN causes pleiotropic polarity defects, including frequently reversed asymmetry in progeny size and mislocalization of proteins and organelles. Ectopic localization of TipN along the lateral side of the cell creates new axes of polarity leading to cell branching and formation of competent cell poles. Localization defects of the actin-like protein MreB in the DeltatipN mutant suggest that TipN is upstream of MreB in regulating cell polarity.

  View details for DOI 10.1016/j.cell.2005.12.040

  View details for Web of Science ID 000237241200022

  View details for PubMedID 16530047

• Bacterial cell shape NATURE REVIEWS MICROBIOLOGY Cabeen, M. T., Jacobs-Wagner, C. 2005; 3 (8): 601–10

  Abstract

  Bacterial species have long been classified on the basis of their characteristic cell shapes. Despite intensive research, the molecular mechanisms underlying the generation and maintenance of bacterial cell shape remain largely unresolved. The field has recently taken an important step forward with the discovery that eukaryotic cytoskeletal proteins have homologues in bacteria that affect cell shape. Here, we discuss how a bacterium gains and maintains its shape, the challenges still confronting us and emerging strategies for answering difficult questions in this rapidly evolving field.

  View details for DOI 10.1038/nrmicro1205

  View details for Web of Science ID 000230879700011

  View details for PubMedID 16012516

• Cytokinesis monitoring during development: Rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter CELL Matroule, J. Y., Lam, H., Burnette, D. T., Jacobs-Wagner, C. 2004; 118 (5): 579–90

  Abstract

  For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development.

  View details for DOI 10.1016/j.cell.2004.08.019

  View details for Web of Science ID 000223730300009

  View details for PubMedID 15339663

• Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter MOLECULAR MICROBIOLOGY Jacobs-Wagner, C. 2004; 51 (1): 7–13

  Abstract

  Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single-celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two-component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle-dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.

  View details for DOI 10.1046/j.1365-2958.2003.03828.x

  View details for Web of Science ID 000186974100003

  View details for PubMedID 14651607

• The bacterial cytoskeleton: An intermediate filament-like function CELL Ausmees, N., Kuhn, Jacobs-Wagner, C. 2003; 115 (6): 705–13

  Abstract

  Various cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly understood. Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape in higher organisms. No such filaments have been found in prokaryotes. Here, we describe a bacterial equivalent to IF proteins, named crescentin, whose cytoskeletal function is required for the vibrioid and helical shapes of Caulobacter crescentus. Without crescentin, the cells adopt a straight-rod morphology. Crescentin has characteristic features of IF proteins including the ability to assemble into filaments in vitro without energy or cofactor requirements. In vivo, crescentin forms a helical structure that colocalizes with the inner cell curvatures beneath the cytoplasmic membrane. We propose that IF-like filaments of crescentin assemble into a helical structure, which by applying its geometry to the cell, generates a vibrioid or helical cell shape depending on the length of the cell.

  View details for DOI 10.1016/S0092-8674(03)00935-8

  View details for Web of Science ID 000187366100010

  View details for PubMedID 14675535

• The asymmetric spatial distribution of bacterial signal transduction proteins coordinates cell cycle events DEVELOPMENTAL CELL Lam, H., Matroule, J. Y., Jacobs-Wagner, C. 2003; 5 (1): 149–59

  Abstract

  The polar localization of signaling proteins that are essential for Caulobacter cell cycle control is temporally regulated. Here we provide evidence that phosphorylation of the essential response regulator, DivK, is required for both its function and its cell cycle-regulated localization. The asymmetric location of the DivJ and PleC histidine kinases and their antagonistic activities on the cellular concentration of phosphorylated DivK provide positional and temporal information for the ordered sequence of DivK localization during the cell cycle. DivJ activity on DivK affects its correct localization, which, in turn, is required for PleC function. Since DivJ and PleC regulate different cell cycle events, the interconnected function of these two histidine kinases through localization of a common response regulator provides a mechanism for coordinating cell cycle progression. Study of a DivK homolog in the morphologically symmetric bacterium Sinorhizobium meliloti suggests that this type of cell cycle mechanism is widespread in prokaryotes.

  View details for DOI 10.1016/S1534-5807(03)00191-6

  View details for Web of Science ID 000184114800017

  View details for PubMedID 12852859

• Spatial and temporal control of differentiation and cell cycle progression in Caulobacter crescentus ANNUAL REVIEW OF MICROBIOLOGY Ausmees, N., Jacobs-Wagner, C. 2003; 57: 225–47

  Abstract

  The dimorphic and intrinsically asymmetric bacterium Caulobacter crescentus has become an important model organism to study the bacterial cell cycle, cell polarity, and polar differentiation. A multifaceted regulatory network orchestrates the precise coordination between the development of polar organelles and the cell cycle. One master response regulator, CtrA, directly controls the initiation of chromosome replication as well as several aspects of polar morphogenesis and cell division. CtrA activity is temporally and spatially regulated by multiple partially redundant control mechanisms, such as transcription, phosphorylation, and targeted proteolysis. A multicomponent signal transduction network upstream CtrA, containing histidine kinases CckA, PleC, DivJ, and DivL and the essential response regulator DivK, contributes to the control of CtrA activity in response to cell cycle and developmental cues. An intriguing feature of this signaling network is the dynamic cell cycle-dependent polar localization of its components, which is believed to have a novel regulatory function.

  View details for DOI 10.1146/annurev.micro.57.030502.091006

  View details for Web of Science ID 000186493700011

  View details for PubMedID 14527278