Bio


Arthur Robert Grossman (born 1950): Arthur Grossman has been a Staff Scientist at The Carnegie Institution for Science, Department of Plant Biology since 1982, and holds a courtesy appointment as Professor in the Department of Biology at Stanford University. He has performed research across fields ranging from plant biology, microbiology, marine biology, ecology, genomics, engineering and photosynthesis and initiated large scale algal genomics by leading the Chlamydomonas genome project (sequencing of the genome coupled to transcriptomics). During his tenure at Carnegie, he mentored more than fifteen PhD students and approximately 40 post-doctoral fellows (many of whom have become very successful independent scientists at both major universities and in industry). In 2002 he received the Darbaker Prize (Botanical Society of America) for work on microalgae and in 2009 received the Gilbert Morgan Smith Medal (National Academy of Sciences) for the quality of his publications on marine and freshwater algae. In 2015 he was Vice Chair of the Gordon Research Conference on Photosynthesis and in 2017 was Chair of that same conference (Photosynthetic plasticity: From the environment to synthetic systems). He also gave the Arnon endowed lecture on photosynthesis in Berkeley in March of 2017, has given numerous plenary lectures and received a number of fellowships throughout his career, including the Visiting Scientist Fellowship - Department of Life and Environmental Sciences (DiSVA), Università Politecnica delle Marche (UNIVPM) (Italy, 2014), the Lady Davis Fellowship (Israel, 2011) and most recently the Chaire Edmond de Rothschild (to work IBPC in Paris in 2017-2018). He has been Co-Editor in Chief of Journal of Phycology and has served on the editorial boards of many well-respected biological journals including the Annual Review of Genetics, Plant Physiology, Eukaryotic Cell, Journal of Biological Chemistry, Molecular Plant, and Current Genetics. He has also reviewed innumerable papers and grants, served on many scientific panels that has evaluated various programs for granting agencies [NSF, CNRS, Marden program (New Zealand)] and private companies. He has also served in a scientific advisory capacity for both nonprofit and for profit companies including Phoenix Bioinformatics, Excelixis, Martex, Solazyme/TerraVia, Checkerspot, Phycoil and CarbonDrop. Recently he has worked with Francis-Andre Wollman, Susan Dutcher and Ursula Goodenough to complete a highly expanded third edition of the Chlamydomonas Sourcebook (Elsevier, 2023).

Academic Appointments


  • Visiting Professor (By courtesy), Biology

Current Research and Scholarly Interests


How photosynthetic organisms perceive and respond to their environment

Stanford Advisees


Graduate and Fellowship Programs


  • Biology (School of Humanities and Sciences) (Phd Program)

All Publications


  • One step further toward a crop CO2-concentrating mechanism. Journal of experimental botany Findinier, J., Grossman, A. R. 2023; 74 (12): 3402-3405

    Abstract

    This article comments on: Förster B, Rourke LM, Weerasooriya HN, Pabuayon ICM, Rolland V, Au EK, Bala S, Bajsa-Hirschel J, Kaines S, Kasili RW, LaPlace LM, Machingura MC, Massey B, Rosati VC, Stuart-Williams H, Badger MR, Price GD, Moroney JV. 2023. The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta. Journal of Experimental Botany 74, 3651–3666

    View details for DOI 10.1093/jxb/erad200

    View details for PubMedID 37369104

    View details for PubMedCentralID PMC10299783

  • Light-independent regulation of algal photoprotection by CO2 availability. Nature communications Aguila Ruiz-Sola, M., Flori, S., Yuan, Y., Villain, G., Sanz-Luque, E., Redekop, P., Tokutsu, R., Kuken, A., Tsichla, A., Kepesidis, G., Allorent, G., Arend, M., Iacono, F., Finazzi, G., Hippler, M., Nikoloski, Z., Minagawa, J., Grossman, A. R., Petroutsos, D. 2023; 14 (1): 1977

    Abstract

    Photosynthetic algae have evolved mechanisms to cope with suboptimal light and CO2 conditions. When light energy exceeds CO2 fixation capacity, Chlamydomonas reinhardtii activates photoprotection, mediated by LHCSR1/3 and PSBS, and the CO2 Concentrating Mechanism (CCM). How light and CO2 signals converge to regulate these processes remains unclear. Here, we show that excess light activates photoprotection- and CCM-related genes by altering intracellular CO2 concentrations and that depletion of CO2 drives these responses, even in total darkness. High CO2 levels, derived from respiration or impaired photosynthetic fixation, repress LHCSR3/CCM genes while stabilizing the LHCSR1 protein. Finally, we show that the CCM regulator CIA5 also regulates photoprotection, controlling LHCSR3 and PSBS transcript accumulation while inhibiting LHCSR1 protein accumulation. This work has allowed us to dissect the effect of CO2 and light on CCM and photoprotection, demonstrating that light often indirectly affects these processes by impacting intracellular CO2 levels.

    View details for DOI 10.1038/s41467-023-37800-6

    View details for PubMedID 37031262

  • Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light-sensitive. The Plant cell Huang, W., Krishnan, A., Plett, A., Meagher, M., Linka, N., Wang, Y., Ren, B., Findinier, J., Redekop, P., Fakhimi, N., Kim, R. G., Karns, D. A., Boyle, N., Posewitz, M. C., Grossman, A. R. 2023

    Abstract

    Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the CreTPT transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

    View details for DOI 10.1093/plcell/koad095

    View details for PubMedID 36970811

  • Restricting electron flow at cytochrome b6f when downstream electron acceptors are severely limited. Plant physiology Saroussi, S., Redekop, P., Karns, D. A., Thomas, D. C., Wittkopp, T. M., Posewitz, M. C., Grossman, A. R. 2023

    Abstract

    Photosynthetic organisms frequently experience abiotic stress that restricts their growth and development. Under such circumstances, most absorbed solar energy cannot be used for CO2 fixation and can cause the photoproduction of reactive oxygen species (ROS) that can damage the photosynthetic reaction centers of photosystem I and II (PSI and PSII), resulting in a decline in primary productivity. This work describes a biological 'switch' in the green alga Chlamydomonas reinhardtii that reversibly restricts photosynthetic electron transport (PET) at the cytochrome b6f (Cyt b6f) complex when the capacity for accepting electrons downstream of PSI is severely limited. We specifically show this restriction in STARCHLESS6 (sta6) mutant cells, which cannot synthesize starch when they are limited for nitrogen (growth inhibition) and subjected to a dark-to-light transition. This restriction represents a form of photosynthetic control that causes diminished electron flow to PSI and thereby prevents PSI photodamage but does not appear to rely on a ΔpH. Furthermore, when electron flow is restricted, the plastid alternative oxidase (PTOX) becomes active, functioning as an electron valve that dissipates some excitation energy absorbed by PSII and allows the formation of a proton motive force (PMF) that would drive some ATP production [potentially sustaining PSII repair and non-photochemical quenching (NPQ)]. The restriction at the Cyt b6f complex can be gradually relieved with continued illumination. This study provides insights into how photosynthetic electron transport responds to a marked reduction in availability of downstream electron acceptors and the protective mechanisms involved.

    View details for DOI 10.1093/plphys/kiad185

    View details for PubMedID 36960590

  • The Influence of Symbiosis on the Proteome of the Exaiptasia Endosymbiont Breviolum minutum. Microorganisms Mashini, A. G., Oakley, C. A., Beepat, S. S., Peng, L., Grossman, A. R., Weis, V. M., Davy, S. K. 2023; 11 (2)

    Abstract

    The cellular mechanisms responsible for the regulation of nutrient exchange, immune response, and symbiont population growth in the cnidarian-dinoflagellate symbiosis are poorly resolved. Here, we employed liquid chromatography-mass spectrometry to elucidate proteomic changes associated with symbiosis in Breviolum minutum, a native symbiont of the sea anemone Exaiptasia diaphana ('Aiptasia'). We manipulated nutrients available to the algae in culture and to the holobiont in hospite (i.e., in symbiosis) and then monitored the impacts of our treatments on host-endosymbiont interactions. Both the symbiotic and nutritional states had significant impacts on the B. minutum proteome. B. minutum in hospite showed an increased abundance of proteins involved in phosphoinositol metabolism (e.g., glycerophosphoinositol permease 1 and phosphatidylinositol phosphatase) relative to the free-living alga, potentially reflecting inter-partner signalling that promotes the stability of the symbiosis. Proteins potentially involved in concentrating and fixing inorganic carbon (e.g., carbonic anhydrase, V-type ATPase) and in the assimilation of nitrogen (e.g., glutamine synthase) were more abundant in free-living B. minutum than in hospite, possibly due to host-facilitated access to inorganic carbon and nitrogen limitation by the host when in hospite. Photosystem proteins increased in abundance at high nutrient levels irrespective of the symbiotic state, as did proteins involved in antioxidant defences (e.g., superoxide dismutase, glutathione s-transferase). Proteins involved in iron metabolism were also affected by the nutritional state, with an increased iron demand and uptake under low nutrient treatments. These results detail the changes in symbiont physiology in response to the host microenvironment and nutrient availability and indicate potential symbiont-driven mechanisms that regulate the cnidarian-dinoflagellate symbiosis.

    View details for DOI 10.3390/microorganisms11020292

    View details for PubMedID 36838257

  • Symbiosis induces unique volatile profiles in the model cnidarian Aiptasia. The Journal of experimental biology Wuerz, M., Lawson, C. A., Ueland, M., Oakley, C. A., Grossman, A. R., Weis, V. M., Suggett, D. J., Davy, S. K. 2022

    Abstract

    The establishment and maintenance of the symbiosis between a cnidarian host and its dinoflagellate symbionts is central to the success of coral reefs. To explore the metabolite production underlying this symbiosis, we focused on a group of low weight secondary metabolites, biogenic volatile organic compounds (BVOCs). BVOCs are released from an organism or environment, and can be collected in the gas phase, allowing non-invasive analysis of an organism's metabolism (i.e. 'volatilomics'). We characterised volatile profiles of the sea anemone Exaiptasia diaphana ('Aiptasia'), a model system for cnidarian-dinoflagellate symbiosis, using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry. We compared volatile profiles between: 1) symbiotic anemones containing their native symbiont, Breviolum minutum; 2) aposymbiotic anemones; and 3) cultured isolates of B. minutum. Overall, 152 BVOCs were detected, and classified into 14 groups based on their chemical structure, the most numerous groups being alkanes and aromatic compounds. A total of 53 BVOCs were differentially abundant between aposymbiotic anemones and B. minutum cultures; 13 between aposymbiotic and symbiotic anemones; and 60 between symbiotic anemones and cultures of B. minutum. More BVOCs were differentially abundant between cultured and symbiotic dinoflagellates than between aposymbiotic and symbiotic anemones, suggesting that symbiosis may modify symbiont physiology more than host physiology. This is the first volatilome analysis of the Aiptasia model system and provides a foundation from which to explore how BVOC production is perturbed under environmental stress, and ultimately the role they play in this important symbiosis.

    View details for DOI 10.1242/jeb.244600

    View details for PubMedID 36156083

  • Lifestyle differences and carbon acquisition in Paragymnodinium dinoflagellates. Journal of phycology Grossman, A. R. 2022; 58 (4): 487-489

    View details for DOI 10.1111/jpy.13274

    View details for PubMedID 35946485

  • Immunolocalization of Metabolite Transporter Proteins in a Model Cnidarian-Dinoflagellate Symbiosis. Applied and environmental microbiology Mashini, A. G., Oakley, C. A., Grossman, A. R., Weis, V. M., Davy, S. K. 2022: e0041222

    Abstract

    Bidirectional nutrient flow between partners is integral to the cnidarian-dinoflagellate endosymbiosis. However, our current knowledge of the transporter proteins that regulate nutrient and metabolite trafficking is nascent. Four transmembrane transporters that likely play an important role in interpartner nitrogen and carbon exchange were investigated with immunocytochemistry in the model sea anemone Exaiptasia diaphana ("Aiptasia"; strain NZ1): ammonium transporter 1 (AMT1), V-type proton ATPase (VHA), facilitated glucose transporter member 8 (GLUT8), and aquaporin-3 (AQP3). Anemones lacking symbionts were compared with those in symbiosis with either their typical, homologous dinoflagellate symbiont, Breviolum minutum, or the heterologous species, Durusdinium trenchii and Symbiodinium microadriaticum. AMT1 and VHA were only detected in symbiotic Aiptasia, irrespective of symbiont type. However, GLUT8 and AQP3 were detected in both symbiotic and aposymbiotic states. All transporters were localized to both the epidermis and gastrodermis, though localization patterns in host tissues were heavily influenced by symbiont identity, with S. microadriaticum-colonized anemones showing the most distinct patterns. These patterns suggested disruption of fixed carbon and inorganic nitrogen fluxes when in symbiosis with heterologous versus homologous symbionts. This study enhances our understanding of nutrient transport and host-symbiont integration, while providing a platform for further investigation of nutrient transporters and the host-symbiont interface in the cnidarian-dinoflagellate symbiosis. IMPORTANCE Coral reefs are in serious decline, in particular due to the thermally induced dysfunction of the cnidarian-dinoflagellate symbiosis that underlies their success. Yet our ability to react to this crisis is hindered by limited knowledge of how this symbiosis functions. Indeed, we still have much to learn about the cellular integration that determines whether a particular host-symbiont combination can persist, and hence whether corals might be able to adapt by acquiring new, more thermally resistant symbionts. Here, we employed immunocytochemistry to localize and quantify key nutrient transporters in tissues of the sea anemone Aiptasia, a globally adopted model system for this symbiosis, and compared the expression of these transporters when the host is colonized by native versus nonnative symbionts. We showed a clear link between transporter expression and symbiont identity, elucidating the cellular events that dictate symbiosis success, and we provide a methodological platform for further examination of cellular integration in this ecologically important symbiosis.

    View details for DOI 10.1128/aem.00412-22

    View details for PubMedID 35678605

  • Retrotransposition facilitated the establishment of a primary plastid in the thecate amoeba Paulinella. Proceedings of the National Academy of Sciences of the United States of America Calatrava, V., Stephens, T. G., Gabr, A., Bhaya, D., Bhattacharya, D., Grossman, A. R. 2022; 119 (23): e2121241119

    Abstract

    SignificancePrimary endosymbiosis allowed the evolution of complex life on Earth. In this process, a prokaryote was engulfed and retained in the cytoplasm of another microbe, where it developed into a new organelle (mitochondria and plastids). During organelle evolution, genes from the endosymbiont are transferred to the host nuclear genome, where they must become active despite differences in the genetic nature of the "partner" organisms. Here, we show that in the amoeba Paulinella micropora, which harbors a nascent photosynthetic organelle, the "copy-paste" mechanism of retrotransposition allowed domestication of endosymbiont-derived genes in the host nuclear genome. This duplication mechanism is widespread in eukaryotes and may be a major facilitator for host-endosymbiont integration and the evolution of organelles.

    View details for DOI 10.1073/pnas.2121241119

    View details for PubMedID 35639693

  • Transcriptional regulation of photoprotection in dark-to-light transition-More than just a matter of excess light energy. Science advances Redekop, P., Sanz-Luque, E., Yuan, Y., Villain, G., Petroutsos, D., Grossman, A. R. 2022; 8 (22): eabn1832

    Abstract

    In nature, photosynthetic organisms are exposed to different light spectra and intensities depending on the time of day and atmospheric and environmental conditions. When photosynthetic cells absorb excess light, they induce nonphotochemical quenching to avoid photodamage and trigger expression of "photoprotective" genes. In this work, we used the green alga Chlamydomonas reinhardtii to assess the impact of light intensity, light quality, photosynthetic electron transport, and carbon dioxide on induction of the photoprotective genes (LHCSR1, LHCSR3, and PSBS) during dark-to-light transitions. Induction (mRNA accumulation) occurred at very low light intensity and was independently modulated by blue and ultraviolet B radiation through specific photoreceptors; only LHCSR3 was strongly controlled by carbon dioxide levels through a putative enhancer function of CIA5, a transcription factor that controls genes of the carbon concentrating mechanism. We propose a model that integrates inputs of independent signaling pathways and how they may help the cells anticipate diel conditions and survive in a dynamic light environment.

    View details for DOI 10.1126/sciadv.abn1832

    View details for PubMedID 35658034

  • Intelligent image-activated sorting of Chlamydomonas reinhardtii by mitochondrial localization. Cytometry. Part A : the journal of the International Society for Analytical Cytology Harmon, J., Findinier, J., Ishii, N. T., Herbig, M., Isozaki, A., Grossman, A., Goda, K. 2022

    Abstract

    Organelle positioning in cells is associated with various metabolic functions and signaling in unicellular organisms. Specifically, the microalga Chlamydomonas reinhardtii repositions its mitochondria, depending on the levels of inorganic carbon. Mitochondria are typically randomly distributed in the Chlamydomonas cytoplasm, but relocate towards the cell periphery at low inorganic carbon levels. This mitochondrial relocation is linked with the carbon-concentrating mechanism, but its significance is not yet thoroughly understood. A genotypic understanding of this relocation would require a high-throughput method to isolate rare mutant cells not exhibiting this relocation. However, this task is technically challenging due to the complex intracellular morphological difference between mutant and wild-type cells, rendering conventional non-image-based high-event-rate methods unsuitable. Here, we report our demonstration of intelligent image-activated cell sorting by mitochondrial localization. Specifically, we applied an intelligent image-activated cell sorting system to sort for C. reinhardtii cells displaying no mitochondrial relocation. We trained a convolutional neural network (CNN) to distinguish the cell types based on the complex morphology of their mitochondria. The CNN was employed to perform image-activated sorting for the mutant cell type at 180 events per second, which is 1-2 orders of magnitude faster than automated microscopy with robotic pipetting, resulting in an enhancement of the concentration from 5% to 56.5% corresponding to an enrichment factor of 11.3. These results show the potential of image-activated cell sorting for connecting genotype-phenotype relations for rare-cell populations, which require a high throughput and could lead to a better understanding of metabolic functions in cells.

    View details for DOI 10.1002/cyto.a.24661

    View details for PubMedID 35643943

  • Symbiosis with Dinoflagellates Alters Cnidarian Cell-Cycle Gene Expression CELLULAR MICROBIOLOGY Gorman, L. M., Konciute, M. K., Cui, G., Oakley, C. A., Grossman, A. R., Weis, V. M., Aranda, M., Davy, S. K. 2022; 2022
  • Systematic characterization of gene function in the photosynthetic alga Chlamydomonas reinhardtii. Nature genetics Fauser, F., Vilarrasa-Blasi, J., Onishi, M., Ramundo, S., Patena, W., Millican, M., Osaki, J., Philp, C., Nemeth, M., Salome, P. A., Li, X., Wakao, S., Kim, R. G., Kaye, Y., Grossman, A. R., Niyogi, K. K., Merchant, S. S., Cutler, S. R., Walter, P., Dinneny, J. R., Jonikas, M. C., Jinkerson, R. E. 2022

    Abstract

    Most genes in photosynthetic organisms remain functionally uncharacterized. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. A total of 59% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles place uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism and ciliogenesis. We illustrate the value of this resource by validating phenotypes and gene functions, including three new components of an actin cytoskeleton defense pathway. The data also inform phenotype discovery in land plants; mutants in Arabidopsis thaliana genes exhibit phenotypes similar to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

    View details for DOI 10.1038/s41588-022-01052-9

    View details for PubMedID 35513725

  • Differential Phototactic Behavior of Closely Related Cyanobacterial Isolates from Yellowstone Hot Spring Biofilms. Applied and environmental microbiology Bunbury, F., Rivas, C., Calatrava, V., Shelton, A. N., Grossman, A., Bhaya, D. 2022: e0019622

    Abstract

    Phototrophic biofilms in most environments experience major changes in light levels throughout a diel cycle. Phototaxis can be a useful strategy for optimizing light exposure under these conditions, but little is known about its role in cyanobacteria from thermal springs. We examined two closely related Synechococcus isolates (Synechococcus OS-A dominates at 60 to 65°C and OS-B' at 50 to 55°C) from outflows of Octopus Spring in Yellowstone National Park. Both isolates exhibited phototaxis and photokinesis in white light, but with differences in speed and motility bias. OS-B' exhibited phototaxis toward UVA, blue, green, and red wavelengths, while OS-A primarily exhibited phototaxis toward red and green. OS-A also exhibited negative phototaxis under certain conditions. The repertoires of photoreceptors and signal transduction elements in both isolates were quite different from those characterized in other unicellular cyanobacteria. These differences in the photoresponses between OS-A and OS-B' in conjunction with in situ observations indicate that phototactic strategies may be quite versatile and finely tuned to the light and local environment. IMPORTANCE Optimizing light absorption is of paramount importance to photosynthetic organisms. Some photosynthetic microbes have evolved a sophisticated process called phototaxis to move toward or away from a light source. In many hot springs in Yellowstone National Park, cyanobacteria thrive in thick, laminated biofilms or microbial mats, where small movements can result in large changes in light exposure. We quantified the light-dependent motility behaviors in isolates representing two of the most abundant and closely related cyanobacterial species from these springs. We found that they exhibited unexpected differences in their speed, directionality, and responses to different intensities or qualities of light. An examination of their genomes revealed several variations from well-studied phototaxis-related genes. Studying these recently isolated cyanobacteria reveals that diverse phototactic strategies can exist even among close relatives in the same environment. It also provides insights into the importance of phototaxis for growth and survival in microbial biofilm communities.

    View details for DOI 10.1128/aem.00196-22

    View details for PubMedID 35499327

  • Cnidarian-Symbiodiniaceae symbiosis establishment is independent of photosynthesis. Current biology : CB Jinkerson, R. E., Russo, J. A., Newkirk, C. R., Kirk, A. L., Chi, R. J., Martindale, M. Q., Grossman, A. R., Hatta, M., Xiang, T. 2022

    Abstract

    Photosynthesis shapes the symbiotic relationships between cnidarians and Symbiodiniaceae algae-with many cnidarian hosts requiring symbiont photosynthate for survival-but little is known about how photosynthesis impacts symbiosis establishment. Here, we show that during symbiosis establishment, infection, proliferation, and maintenance can proceed without photosynthesis, but the ability to do so is dependent on specific cnidarian-Symbiodiniaceae relationships. The evaluation of 31 pairs of symbiotic relationships (five species of Symbiodiniaceae in sea anemone, coral, and jellyfish hosts) revealed that infection can occur without photosynthesis. A UV mutagenesis method for Symbiodiniaceae was established and used to generate six photosynthetic mutants that can infect these hosts. Without photosynthesis, Symbiodiniaceae cannot proliferate in the sea anemone Aiptasia or jellyfish Cassiopea but can proliferate in the juvenile polyps of the coral Acropora. After 6months of darkness, Breviolum minutum is maintained within Aiptasia, indicating that Symbiodiniaceae maintenance can be independent of photosynthesis. Manipulating photosynthesis provides insights into cnidarian-Symbiodiniaceae symbiosis.

    View details for DOI 10.1016/j.cub.2022.04.021

    View details for PubMedID 35504283

  • The chromatin organization of a chlorarachniophyte nucleomorph genome. Genome biology Marinov, G. K., Chen, X., Wu, T., He, C., Grossman, A. R., Kundaje, A., Greenleaf, W. J. 2022; 23 (1): 65

    Abstract

    BACKGROUND: Nucleomorphs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have suggested that nucleomorph chromatin likely exhibits a number of divergent features.RESULTS: In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organization for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleomorph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions and show the relative contact frequencies between the multiple genomic compartments of distinct origin that B. natans cells contain.CONCLUSIONS: We provide the first study of a nucleomorph genome using modern functional genomic tools, and derive numerous novel insights into the physical and functional organization of these unique genomes.

    View details for DOI 10.1186/s13059-022-02639-5

    View details for PubMedID 35232465

  • Deep imaging flow cytometry. Lab on a chip Huang, K., Matsumura, H., Zhao, Y., Herbig, M., Yuan, D., Mineharu, Y., Harmon, J., Findinier, J., Yamagishi, M., Ohnuki, S., Nitta, N., Grossman, A. R., Ohya, Y., Mikami, H., Isozaki, A., Goda, K. 2022

    Abstract

    Imaging flow cytometry (IFC) has become a powerful tool for diverse biomedical applications by virtue of its ability to image single cells in a high-throughput manner. However, there remains a challenge posed by the fundamental trade-off between throughput, sensitivity, and spatial resolution. Here we present deep-learning-enhanced imaging flow cytometry (dIFC) that circumvents this trade-off by implementing an image restoration algorithm on a virtual-freezing fluorescence imaging (VIFFI) flow cytometry platform, enabling higher throughput without sacrificing sensitivity and spatial resolution. A key component of dIFC is a high-resolution (HR) image generator that synthesizes "virtual" HR images from the corresponding low-resolution (LR) images acquired with a low-magnification lens (10*/0.4-NA). For IFC, a low-magnification lens is favorable because of reduced image blur of cells flowing at a higher speed, which allows higher throughput. We trained and developed the HR image generator with an architecture containing two generative adversarial networks (GANs). Furthermore, we developed dIFC as a method by combining the trained generator and IFC. We characterized dIFC using Chlamydomonas reinhardtii cell images, fluorescence in situ hybridization (FISH) images of Jurkat cells, and Saccharomyces cerevisiae (budding yeast) cell images, showing high similarities of dIFC images to images obtained with a high-magnification lens (40*/0.95-NA), at a high flow speed of 2 m s-1. We lastly employed dIFC to show enhancements in the accuracy of FISH-spot counting and neck-width measurement of budding yeast cells. These results pave the way for statistical analysis of cells with high-dimensional spatial information.

    View details for DOI 10.1039/d1lc01043c

    View details for PubMedID 35142325

  • Genomic conservation and putative downstream functionality of the phosphatidylinositol signalling pathway in the cnidarian-dinoflagellate symbiosis. Frontiers in microbiology Ashley, I. A., Kitchen, S. A., Gorman, L. M., Grossman, A. R., Oakley, C. A., Suggett, D. J., Weis, V. M., Rosset, S. L., Davy, S. K. 2022; 13: 1094255

    Abstract

    The mutualistic cnidarian-dinoflagellate symbiosis underpins the evolutionary success of stony corals and the persistence of coral reefs. However, a molecular understanding of the signalling events that lead to the successful establishment and maintenance of this symbiosis remains unresolved. For example, the phosphatidylinositol (PI) signalling pathway has been implicated during the establishment of multiple mutualistic and parasitic interactions across the kingdoms of life, yet its role within the cnidarian-dinoflagellate symbiosis remains unexplored. Here, we aimed to confirm the presence and assess the specific enzymatic composition of the PI signalling pathway across cnidaria and dinoflagellates by compiling 21 symbiotic anthozoan (corals and sea anemones) and 28 symbiotic dinoflagellate (Symbiodiniaceae) transcriptomic and genomic datasets and querying genes related to this pathway. Presence or absence of PI-kinase and PI-phosphatase orthologs were also compared between a broad sampling of taxonomically related symbiotic and non-symbiotic species. Across the symbiotic anthozoans analysed, there was a complete and highly conserved PI pathway, analogous to the pathway found in model eukaryotes. The Symbiodiniaceae pathway showed similarities to its sister taxon, the Apicomplexa, with the absence of PI 4-phosphatases. However, conversely to Apicomplexa, there was also an expansion of homologs present in the PI5-phosphatase and PI5-kinase groups, with unique Symbiodiniaceae proteins identified that are unknown from non-symbiotic unicellular organisms. Additionally, we aimed to unravel the putative functionalities of the PI signalling pathway in this symbiosis by analysing phosphoinositide (PIP)-binding proteins. Analysis of phosphoinositide (PIP)-binding proteins showed that, on average, 2.23 and 1.29% of the total assemblies of anthozoan and Symbiodiniaceae, respectively, have the potential to bind to PIPs. Enrichment of Gene Ontology (GO) terms associated with predicted PIP-binding proteins within each taxon revealed a broad range of functions, including compelling links to processes putatively involved in symbiosis regulation. This analysis establishes a baseline for current understanding of the PI pathway across anthozoans and Symbiodiniaceae, and thus a framework to target future research.

    View details for DOI 10.3389/fmicb.2022.1094255

    View details for PubMedID 36777026

  • INSIGHTS INTO THE EVOLUTION OF A PRIMARY ENDOSYMBIOSIS THROUGH ANALYSIS OF THE PAULINELLA GENOME Stephens, T. G., Calatrava, V., Gabr, A., Grossman, A., Bhattacharya, D. TAYLOR & FRANCIS LTD. 2021: 54
  • WHY IS PRIMARY ENDOSYMBIOSIS SO RARE? Bhattacharya, D., Stephens, T. G., Gabr, A., Calatreva, V., Grossman, A. R. TAYLOR & FRANCIS LTD. 2021: 41-42
  • Why is primary endosymbiosis so rare? The New phytologist Stephens, T. G., Gabr, A., Calatrava, V., Grossman, A. R., Bhattacharya, D. 2021

    Abstract

    Endosymbiosis is a relationship between two organisms wherein one cell resides inside the other. This affiliation, when stable and beneficial for the "host" cell, can result in massive genetic innovation with the foremost examples being the evolution of eukaryotic organelles, the mitochondria and plastids. Despite its critical evolutionary role, there is limited knowledge about how endosymbiosis is initially established and how host - endosymbiont biology is integrated. Here, we explore this issue using, as our model, the rhizarian amoeba Paulinella, which represents an independent case of primary plastid origin that occurred ~120 Mya. We propose the "chassis and engine" model that provides a theoretical framework for understanding primary plastid endosymbiosis, potentially explaining why it is so rare.

    View details for DOI 10.1111/nph.17478

    View details for PubMedID 34018613

  • Transcription-dependent domain-scale three-dimensional genome organization in the dinoflagellate Breviolum minutum. Nature genetics Marinov, G. K., Trevino, A. E., Xiang, T., Kundaje, A., Grossman, A. R., Greenleaf, W. J. 2021

    Abstract

    Dinoflagellate chromosomes represent a unique evolutionary experiment, as they exist in a permanently condensed, liquid crystalline state; are not packaged by histones; and contain genes organized into tandem gene arrays, with minimal transcriptional regulation. We analyze the three-dimensional genome of Breviolum minutum, and find large topological domains (dinoflagellate topologically associating domains, which we term 'dinoTADs') without chromatin loops, which are demarcated by convergent gene array boundaries. Transcriptional inhibition disrupts dinoTADs, implicating transcription-induced supercoiling as the primary topological force in dinoflagellates.

    View details for DOI 10.1038/s41588-021-00848-5

    View details for PubMedID 33927397

  • Responses of Chlamydomonas reinhardtii during the transition from P-deficient to P-sufficient growth (the P-overplus response): The roles of the vacuolar transport chaperones and polyphosphate synthesis. Journal of phycology Plouviez, M., Fernandez, E., Grossman, A. R., Sanz-Luque, E., Sells, M., Wheeler, D., Guieysse, B. 2021

    Abstract

    Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO4 3- ; 10mg P·L-1 ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C.reinhardtii assimilated PO4 3- and stored polyP within minutes of adding PO4 3- to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO4 3- . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO4 3- assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C.reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO4 3- assimilation in C.reinhardtii. RNA-sequencing was carried out on C.reinhardtii cells that were either P-depleted (control) or supplied with PO4 3- following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24h after the addition of PO4 3- to the cells, although polyP granules were continuously synthesized during this 24h period. These results suggest that the VTC complex remains active for at least 24h after supplying the cells with PO4 3- . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.

    View details for DOI 10.1111/jpy.13145

    View details for PubMedID 33778959

  • Interplay of four auxiliary factors is required for the assembly of photosystem I reaction center subcomplex. The Plant journal : for cell and molecular biology Nellaepalli, S., Kim, R. G., Grossman, A. R., Takahashi, Y. 2021

    Abstract

    The photosystem I (PSI) complex consisting of reaction center (RC) subunits, several peripheral subunits, and many cofactors, is present in the thylakoid membranes of chloroplasts and cyanobacteria. The assembly of RC subunits (PsaA/B) which bind electron transfer cofactors and antenna pigments is an intricate process and is mediated by several auxiliary factors such as Ycf3, Y3IP1/CGL59, Ycf4, and Ycf37/PYG7/CGL71. However, their precise molecular mechanisms in RC assembly remain to be addressed. Here we purified four PSI auxiliary factors by affinity chromatography and characterized copurified PSI assembly intermediates. We suggest that Ycf3 assists the initial assembly of newly synthesized PsaA/B subunits into an RC subcomplex while Y3IP1 may be involved in transferring the RC subcomplex from Ycf3 to the Ycf4 module which stabilizes it. CGL71 may form an oligomer that transiently interacts with the PSI RC subcomplex, physically protecting it under oxic conditions until association with the peripheral PSI subunits occurs. Together, our results reveal the interplay among four auxiliary factors required for the stepwise assembly of the PSI RC.

    View details for DOI 10.1111/tpj.15220

    View details for PubMedID 33655619

  • moving toward more model algae. Journal of phycology Grossman, A. 2021; 57 (1): 51–53

    View details for DOI 10.1111/jpy.13094

    View details for PubMedID 33570196

  • A phytophotonic approach to enhanced photosynthesis ENERGY & ENVIRONMENTAL SCIENCE Kunz, L. Y., Redekop, P., Ort, D. R., Grossman, A. R., Cargnello, M., Majumdar, A. 2020; 13 (12): 4794–4807

    View details for DOI 10.1039/d0ee02960b

    View details for Web of Science ID 000599751100013

  • Phylogenetic analysis of cell-cycle regulatory proteins within the Symbiodiniaceae. Scientific reports Gorman, L. M., Wilkinson, S. P., Kitchen, S. A., Oakley, C. A., Grossman, A. R., Weis, V. M., Davy, S. K. 2020; 10 (1): 20473

    Abstract

    In oligotrophic waters, cnidarian hosts rely on symbiosis with their photosynthetic dinoflagellate partners (family Symbiodiniaceae) to obtain the nutrients they need to grow, reproduce and survive. For this symbiosis to persist, the host must regulate the growth and proliferation of its symbionts. One of the proposed regulatory mechanisms is arrest of the symbiont cell cycle in the G1 phase, though the cellular mechanisms involved remain unknown. Cell-cycle progression in eukaryotes is controlled by the conserved family of cyclin-dependent kinases (CDKs) and their partner cyclins. We identified CDKs and cyclins in different Symbiodiniaceae species and examined their relationship to homologs in other eukaryotes. Cyclin proteins related to eumetazoan cell-cycle-related cyclins A, B, D, G/I and Y, and transcriptional cyclin L, were identified in theSymbiodiniaceae, alongside several alveolate-specific cyclin A/B proteins, and proteins related to protist P/U-type cyclins and apicomplexan cyclins. The largest expansion of Symbiodiniaceaecyclins was in the P/U-type cyclin groups. Proteins related to eumetazoan cell-cycle-related CDKs (CDK1) were identified as well as transcription-related CDKs. The largest expansion of CDK groups was, however, in alveolate-specific groups which comprised 11 distinct CDK groups (CDKA-J) with CDKB being the most widely distributed CDK protein. As a result of its phylogenetic position, conservation across Symbiodiniaceae species, and the presence of the canonical CDK motif, CDKB emerged as a likely candidate for a Saccharomyces cerevisiae Cdc28/Pho85-like homolog in Symbiodiniaceae. Similar to cyclins, two CDK-groups found in Symbiodiniaceae species were solely associated with apicomplexan taxa. A comparison of Breviolum minutum CDK and cyclin gene expression between free-living and symbiotic states showed that several alveolate-specific CDKs and two P/U-type cyclins exhibited altered expression in hospite, suggesting that symbiosis influences the cell cycle of symbionts on a molecular level. These results highlight the divergence of Symbiodiniaceae cell-cycle proteins across species. These results have important implications for host control of the symbiont cell cycle in novel cnidarian-dinoflagellate symbioses.

    View details for DOI 10.1038/s41598-020-76621-1

    View details for PubMedID 33235281

  • Transcriptome Reprogramming of Symbiodiniaceae Breviolum minutum in Response to Casein Amino Acids Supplementation FRONTIERS IN PHYSIOLOGY Kirk, A. L., Clowez, S., Lin, F., Grossman, A. R., Xiang, T. 2020; 11
  • Impact of menthol on growth and photosynthetic function of Breviolum minutum (Dinoflagellata, Dinophyceae, Symbiodiniaceae) and interactions with its Aiptasia host. Journal of phycology Clowez, S., Renicke, C., Pringle, J. R., Grossman, A. R. 2020

    Abstract

    Environmental change, including global warming and chemical pollution, can compromise cnidarian (e.g., coral) -dinoflagellate symbioses and cause coral bleaching. Understanding the mechanisms that regulate these symbioses will inform strategies for sustaining healthy coral-reef communities. A model system for corals is the symbiosis between the sea anemone Exaiptasia pallida (common name Aiptasia) and its dinoflagellate partners (family Symbiodiniaceae). To complement existing studies of the interactions between these organisms, we examined the impact of menthol, a reagent often used to render cnidarians aposymbiotic, on the dinoflagellate Breviolum minutum, both in culture and in hospite. In both environments, the growth and photosynthesis of this alga were compromised at either 100 or 300M menthol. We observed reduction of PSII and PSI functions, the abundances of reaction-center proteins, and, at 300M menthol, of total cellular proteins. Interestingly, for free-living algae exposed to 100M menthol, an initial decline in growth, photosynthetic activities, pigmentation, and protein abundances reversed after 5-15 d, eventually approaching control levels. This behavior was observed in cells maintained in continuous light, but not in cells experiencing a light-dark regimen, suggesting that B. minutum can detoxify menthol or acclimate and repair damaged photosynthetic complexes in a light- and/or energy-dependent manner. Extended exposures of cultured algae to 300M menthol ultimately resulted in algal death. Most symbiotic anemones were also unable to survive this menthol concentration for 30 d. Additionally, cells impaired for photosynthesis by pre-treatment with 300M menthol exhibited reduced efficiency in re-populating the anemone host.

    View details for DOI 10.1111/jpy.13081

    View details for PubMedID 33025575

  • Metabolite pools of the reef building coralMontipora capitataare unaffected by Symbiodiniaceae community composition CORAL REEFS Matthews, J. L., Cunning, R., Ritson-Williams, R., Oakley, C. A., Lutz, A., Roessner, U., Grossman, A. R., Weis, V. M., Gates, R. D., Davy, S. K. 2020
  • Sub-cellular imaging shows reduced photosynthetic carbon and increased nitrogen assimilation by the non-native endosymbiont Durusdinium trenchii in the model cnidarian Aiptasia. Environmental microbiology Sproles, A. E., Oakley, C. A., Krueger, T., Grossman, A. R., Weis, V. M., Meibom, A., Davy, S. K. 2020

    Abstract

    Hosting different symbiont species can affect inter-partner nutritional fluxes within the cnidarian-dinoflagellate symbiosis. Using nanoscale secondary ion mass spectrometry (NanoSIMS), we measured the spatial incorporation of photosynthetically-fixed 13 C and heterotrophically-derived 15 N into host and symbiont cells of the model symbiotic cnidarian Aiptasia (Exaiptasia pallida) when colonised with its native symbiont Breviolum minutum or the non-native Durusdinium trenchii. B. minutum exhibited high photosynthetic carbon assimilation per cell and translocation to host tissue throughout symbiosis establishment, while D. trenchii assimilated significantly less carbon, but obtained more host nitrogen. These findings suggest that D. trenchii has less potential to provide photosynthetically-fixed carbon to the host despite obtaining considerable amounts of heterotrophically-derived nitrogen. These sub-cellular events help explain previous observations that demonstrate differential effects of D. trenchii compared to B. minutum on the host transcriptome, proteome, metabolome, and host growth and asexual reproduction. Together, these differential effects suggest that the non-native host-symbiont pairing is sub-optimal with respect to the host's nutritional benefits under normal environmental conditions. This contributes to our understanding of the ways in which metabolic integration impacts the benefits of a symbiotic association, and the potential evolution of novel host-symbiont pairings. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1111/1462-2920.15142

    View details for PubMedID 32592285

  • Paulinella, a model for understanding plastid primary endosymbiosis. Journal of phycology Gabr, A., Grossman, A. R., Bhattacharya, D. 2020

    Abstract

    The uptake and conversion of a free-living cyanobacterium into a photosynthetic organelle by the single-celled Archaeplastida ancestor helped transform the biosphere from low to high oxygen. There are two documented, independent cases of plastid primary endosymbiosis. The first is the well-studied instance in Archaeplastida that occurred ca. 1.6 billion years ago, whereas the second occurred 90-140 million years ago establishing a permanent photosynthetic compartment (the chromatophore) in amoebae in the genus Paulinella. Here, we briefly summarize knowledge about plastid origin in the Archaeplastida and then focus on the Paulinella model. In particular, we describe features of the Paulinella chromatophore that make it a model for examining earlier events in the evolution of photosynthetic organelles. Our review stresses recently gained insights into the evolution of chromatophore and nuclear encoded DNA sequences in Paulinella, metabolic connectivity between the endosymbiont and cytoplasm, and systems that target proteins into the chromatophore. We also describe future work with Paulinella, and the potential rewards and challenges associated with developing further this model system.

    View details for DOI 10.1111/jpy.13003

    View details for PubMedID 32289879

  • Symbiont population control by host-symbiont metabolic interaction in Symbiodiniaceae-cnidarian associations. Nature communications Xiang, T. n., Lehnert, E. n., Jinkerson, R. E., Clowez, S. n., Kim, R. G., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. 2020; 11 (1): 108

    Abstract

    In cnidarian-Symbiodiniaceae symbioses, algal endosymbiont population control within the host is needed to sustain a symbiotic relationship. However, the molecular mechanisms that underlie such population control are unclear. Here we show that a cnidarian host uses nitrogen limitation as a primary mechanism to control endosymbiont populations. Nitrogen acquisition and assimilation transcripts become elevated in symbiotic Breviolum minutum algae as they reach high-densities within the sea anemone host Exaiptasia pallida. These same transcripts increase in free-living algae deprived of nitrogen. Symbiotic algae also have an elevated carbon-to-nitrogen ratio and shift metabolism towards scavenging nitrogen from purines relative to free-living algae. Exaiptasia glutamine synthetase and glutamate synthase transcripts concomitantly increase with the algal endosymbiont population, suggesting an increased ability of the host to assimilate ammonium. These results suggest algal growth and replication in hospite is controlled by access to nitrogen, which becomes limiting for the algae as their population within the host increases.

    View details for DOI 10.1038/s41467-019-13963-z

    View details for PubMedID 31913264

  • Polyphosphate: A Multifunctional Metabolite in Cyanobacteria and Algae. Frontiers in plant science Sanz-Luque, E., Bhaya, D., Grossman, A. R. 2020; 11: 938

    Abstract

    Polyphosphate (polyP), a polymer of orthophosphate (PO4 3-) of varying lengths, has been identified in all kingdoms of life. It can serve as a source of chemical bond energy (phosphoanhydride bond) that may have been used by biological systems prior to the evolution of ATP. Intracellular polyP is mainly stored as granules in specific vacuoles called acidocalcisomes, and its synthesis and accumulation appear to impact a myriad of cellular functions. It serves as a reservoir for inorganic PO4 3- and an energy source for fueling cellular metabolism, participates in maintaining adenylate and metal cation homeostasis, functions as a scaffold for sequestering cations, exhibits chaperone function, covalently binds to proteins to modify their activity, and enables normal acclimation of cells to stress conditions. PolyP also appears to have a role in symbiotic and parasitic associations, and in higher eukaryotes, low polyP levels seem to impact cancerous proliferation, apoptosis, procoagulant and proinflammatory responses and cause defects in TOR signaling. In this review, we discuss the metabolism, storage, and function of polyP in photosynthetic microbes, which mostly includes research on green algae and cyanobacteria. We focus on factors that impact polyP synthesis, specific enzymes required for its synthesis and degradation, sequestration of polyP in acidocalcisomes, its role in cellular energetics, acclimation processes, and metal homeostasis, and then transition to its potential applications for bioremediation and medical purposes.

    View details for DOI 10.3389/fpls.2020.00938

    View details for PubMedID 32670331

  • Photo-movement in the sea anemone Aiptasia influenced by light quality and symbiotic association CORAL REEFS Foo, S. A., Liddell, L., Grossman, A., Caldeira, K. 2019
  • Towards sustainable microalgal biomass processing: anaerobic induction of autolytic cell-wall self-ingestion in lipid-rich Nannochloropsis slurries GREEN CHEMISTRY Halim, R., Hill, D. A., Hanssen, E., Webley, P. A., Blackburn, S., Grossman, A. R., Posten, C., Martin, G. O. 2019; 21 (11): 2967–82

    View details for DOI 10.1039/c8gc03186j

    View details for Web of Science ID 000470709000035

  • Proteomics quantifies protein expression changes in a model cnidarian colonised by a thermally tolerant but suboptimal symbiont. The ISME journal Sproles, A. E., Oakley, C. A., Matthews, J. L., Peng, L., Owen, J. G., Grossman, A. R., Weis, V. M., Davy, S. K. 2019

    Abstract

    The acquisition of thermally tolerant algal symbionts by corals has been proposed as a natural or assisted mechanism of increasing coral reef resilience to anthropogenic climate change, but the cell-level processes determining the performance of new symbiotic associations are poorly understood. We used liquid chromatography-mass spectrometry to investigate the effects of an experimentally induced symbiosis on the host proteome of the model sea anemone Exaiptasia pallida. Aposymbiotic specimens were colonised by either the homologous dinoflagellate symbiont (Breviolum minutum) or a thermally tolerant, ecologically invasive heterologous symbiont (Durusdinium trenchii). Anemones containing D. trenchii exhibited minimal expression of Niemann-Pick C2 proteins, which have predicted biochemical roles in sterol transport and cell recognition, and glutamine synthetases, which are thought to be involved in nitrogen assimilation and recycling between partners. D. trenchii-colonised anemones had higher expression of methionine-synthesising betaine-homocysteine S-methyltransferases and proteins with predicted oxidative stress response functions. Multiple lysosome-associated proteins were less abundant in both symbiotic treatments compared with the aposymbiotic treatment. The differentially abundant proteins are predicted to represent pathways that may be involved in nutrient transport or resource allocation between partners. These results provide targets for specific experiments to elucidate the mechanisms underpinning compensatory physiology in the coral-dinoflagellate symbiosis.

    View details for DOI 10.1038/s41396-019-0437-5

    View details for PubMedID 31118473

  • Alternative outlets for sustaining photosynthetic electron transport during dark-to-light transitions. Proceedings of the National Academy of Sciences of the United States of America Saroussi, S., Karns, D. A., Thomas, D. C., Bloszies, C., Fiehn, O., Posewitz, M. C., Grossman, A. R. 2019

    Abstract

    Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O2 to H2O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O2 exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O2 reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O2 reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H2O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.

    View details for DOI 10.1073/pnas.1903185116

    View details for PubMedID 31101712

  • Building the GreenCut2 suite of proteins to unmask photosynthetic function and regulation. Microbiology (Reading, England) Grossman, A., Sanz-Luque, E., Yi, H., Yang, W. 2019

    Abstract

    The suite of GreenCut proteins, initially assembled in 2007 and updated in 2011 (GreenCut2), comprises 597 Chlamydomonas reinhardtii proteins; these proteins, identified as putative orthologues in all green lineage organisms examined, but not (or poorly conserved) in non-photosynthetic organisms, are potentially enriched for proteins affiliated with photosynthesis. The annotation of GreenCut2 proteins and the characterization of mutants with lesions in genes encoding those proteins identified catalytic components of the photosynthetic apparatus that were previously uncharacterized, as well as polypeptides likely associated with chloroplast biogenesis and potential regulatory factors and activities that link environmental conditions to dynamic control of photosynthetic activities. Analyses of strains devoid of specific GreenCut2 proteins are being aided by a genome-wide library of mutants for which the lesions are mapped, indexed and readily available to the community (https://www.chlamylibrary.org/). In this review we briefly include some milestones in the history of photosynthesis, explain the way in which the GreenCut protein assemblage was generated and describe potential functions of individual member proteins, especially those linked to photosynthesis.

    View details for DOI 10.1099/mic.0.000788

    View details for PubMedID 31063126

  • A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis NATURE GENETICS Li, X., Patena, W., Fauser, F., Jinkerson, R. E., Saroussi, S., Meyer, M. T., Ivanova, N., Robertson, J. M., Yue, R., Zhang, R., Vilarrasa-Blasi, J., Wittkopp, T. M., Ramundo, S., Blum, S. R., Goh, A., Laudon, M., Srikumar, T., Lefebvre, P. A., Grossman, A. R., Jonikas, M. C. 2019; 51 (4): 627-+
  • A genome-wide algal mutant library and functional screen identifies genes required for eukaryotic photosynthesis. Nature genetics Li, X., Patena, W., Fauser, F., Jinkerson, R. E., Saroussi, S., Meyer, M. T., Ivanova, N., Robertson, J. M., Yue, R., Zhang, R., Vilarrasa-Blasi, J., Wittkopp, T. M., Ramundo, S., Blum, S. R., Goh, A., Laudon, M., Srikumar, T., Lefebvre, P. A., Grossman, A. R., Jonikas, M. C. 2019

    Abstract

    Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.

    View details for PubMedID 30886426

  • The mitochondrial alternative oxidase from Chlamydomonas reinhardtii enables survival in high light JOURNAL OF BIOLOGICAL CHEMISTRY Kaye, Y., Huang, W., Clowez, S., Saroussi, S., Idoine, A., Sanz-Luque, E., Grossman, A. R. 2019; 294 (4): 1380–95

    Abstract

    Photosynthetic organisms often experience extreme light conditions that can cause hyper-reduction of the chloroplast electron transport chain, resulting in oxidative damage. Accumulating evidence suggests that mitochondrial respiration and chloroplast photosynthesis are coupled when cells are absorbing high levels of excitation energy.  This coupling helps protect the cells from hyper-reduction of photosynthetic electron carriers and diminishes the production of reactive oxygen species (ROS). To examine this cooperative protection, here we characterized Chlamydomonas reinhardtii mutants lacking the mitochondrial alternative terminal respiratory oxidases, CrAOX1 and CrAOX2. Using fluorescent fusion proteins, we experimentally demonstrated that both enzymes localize to mitochondria. We also observed that the mutant strains were more sensitive than wildtype cells to high light under mixotrophic and photoautotrophic conditions, with the aox1 strain being more sensitive than aox2 Additionally, the lack of CrAOX1 increased ROS accumulation, especially in very high light, and damaged the photosynthetic machinery ultimately resulting in cell death. These findings indicate that the Chlamydomonas AOX proteins can participate in acclimation of C. reinhardtii cells to excess absorbed light energy.  They suggest that when photosynthetic electron carriers are highly reduced, a chloroplast-mitochondria coupling allows safe dissipation of photosynthetically derived electrons via the reduction of O2 through AOX (especially AOX1)-dependent mitochondrial respiration.

    View details for DOI 10.1074/jbc.RA118.004667

    View details for Web of Science ID 000457879500027

    View details for PubMedID 30510139

    View details for PubMedCentralID PMC6349123

  • Partner switching and metabolic flux in a model cnidarian-dinoflagellate symbiosis. Proceedings. Biological sciences Matthews, J. L., Oakley, C. A., Lutz, A., Hillyer, K. E., Roessner, U., Grossman, A. R., Weis, V. M., Davy, S. K. 2018; 285 (1892)

    Abstract

    Metabolite exchange is fundamental to the viability of the cnidarian-Symbiodiniaceae symbiosis and survival of coral reefs. Coral holobiont tolerance to environmental change might be achieved through changes in Symbiodiniaceae species composition, but differences in the metabolites supplied by different Symbiodiniaceae species could influence holobiont fitness. Using 13C stable-isotope labelling coupled to gas chromatography-mass spectrometry, we characterized newly fixed carbon fate in the model cnidarian Exaiptasia pallida (Aiptasia) when experimentally colonized with either native Breviolum minutum or non-native Durusdinium trenchii Relative to anemones containing B. minutum, D. trenchii-colonized hosts exhibited a 4.5-fold reduction in 13C-labelled glucose and reduced abundance and diversity of 13C-labelled carbohydrates and lipogenesis precursors, indicating symbiont species-specific modifications to carbohydrate availability and lipid storage. Mapping carbon fate also revealed significant alterations to host molecular signalling pathways. In particular, D. trenchii-colonized hosts exhibited a 40-fold reduction in 13C-labelled scyllo-inositol, a potential interpartner signalling molecule in symbiosis specificity. 13C-labelling also highlighted differential antioxidant- and ammonium-producing pathway activities, suggesting physiological responses to different symbiont species. Such differences in symbiont metabolite contribution and host utilization may limit the proliferation of stress-driven symbioses; this contributes valuable information towards future scenarios that select in favour of less-competent symbionts in response to environmental change.

    View details for DOI 10.1098/rspb.2018.2336

    View details for PubMedID 30487315

  • From molecular manipulation of domesticated Chlamydomonas reinhardtii to survival in nature. eLife Sasso, S., Stibor, H., Mittag, M., Grossman, A. R. 2018; 7

    Abstract

    In the mid-20th century, the unicellular and genetically tractable green alga Chlamydomonas reinhardtii was first developed as a model organism to elucidate fundamental cellular processes such as photosynthesis, light perception and the structure, function and biogenesis of cilia. Various studies of C. reinhardtii have profoundly advanced plant and cell biology, and have also impacted algal biotechnology and our understanding of human disease. However, the 'real' life of C. reinhardtii in the natural environment has largely been neglected. To extend our understanding of the biology of C. reinhardtii, it will be rewarding to explore its behavior in its natural habitats, learning more about its abundance and life cycle, its genetic and physiological diversity, and its biotic and abiotic interactions.

    View details for DOI 10.7554/eLife.39233

    View details for PubMedID 30382941

  • A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii PLANT JOURNAL Heimerl, N., Hommel, E., Westermann, M., Meichsner, D., Lohr, M., Hertweck, C., Grossman, A. R., Mittag, M., Sasso, S. 2018; 95 (2): 268–81

    Abstract

    Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non-toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2-day-old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild-type zygospores contain knob-like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild-type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild-type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.

    View details for DOI 10.1111/tpj.13948

    View details for Web of Science ID 000437297100007

    View details for PubMedID 29729034

  • Phylogenetic characterization of transporter proteins in the cnidarian-dinoflagellate symbiosis MOLECULAR PHYLOGENETICS AND EVOLUTION Sproles, A. E., Kirk, N. L., Kitchen, S. A., Oakley, C. A., Grossman, A. R., Weis, V. M., Davy, S. K. 2018; 120: 307–20

    Abstract

    Metabolic exchange between cnidarians and their symbiotic dinoflagellates is central to maintaining their mutualistic relationship. Sugars are translocated to the host, while ammonium and nitrate are utilized by the dinoflagellates (Symbiodinium spp.). We investigated membrane protein sequences of each partner to identify potential transporter proteins that move sugars into cnidarian cells and nitrogen products into Symbiodinium cells. We examined the facilitated glucose transporters (GLUT), sodium/glucose cotransporters (SGLT), and aquaporin (AQP) channels in the cnidarian host as mechanisms for sugar uptake, and the ammonium and high-affinity nitrate transporters (AMT and NRT2, respectively) in the algal symbiont as mechanisms for nitrogen uptake. Homologous protein sequences were used for phylogenetic analysis and tertiary structure deductions. In cnidarians, we identified putative glucose transporters of the GLUT family and glycerol transporting AQP proteins, as well as sodium monocarboxylate transporters and sodium myo-inositol cotransporters homologous to SGLT proteins. We hypothesize that cnidarians use GLUT proteins as the primary mechanism for glucose uptake, while glycerol moves into cells by passive diffusion. We also identified putative AMT proteins in several Symbiodinium clades and putative NRT2 proteins only in a single clade. We further observed an upregulation of expressed putative AMT proteins in Symbiodinium, which may have emerged as an adaptation to conditions experienced inside the host cell. This study is the first to identify transporter sequences from a diversity of cnidarian species and Symbiodinium clades, which will be useful for future experimental analyses of the host-symbiont proteome and the nutritional exchange of Symbiodinium cells in hospite.

    View details for DOI 10.1016/j.ympev.2017.12.007

    View details for Web of Science ID 000426199700028

    View details for PubMedID 29233707

  • Glucose-Induced Trophic Shift in an Endosymbiont Dinoflagellate with Physiological and Molecular Consequences PLANT PHYSIOLOGY Xiang, T., Jinkerson, R. E., Clowez, S., Tran, C., Krediet, C. J., Onishi, M., Cleves, P. A., Pringle, J. R., Grossman, A. R. 2018; 176 (2): 1793–1807

    Abstract

    Interactions between the dinoflagellate endosymbiont Symbiodinium and its cnidarian hosts (e.g. corals, sea anemones) are the foundation of coral-reef ecosystems. Carbon flow between the partners is a hallmark of this mutualism, but the mechanisms governing this flow and its impact on symbiosis remain poorly understood. We showed previously that although Symbiodinium strain SSB01 can grow photoautotrophically, it can grow mixotrophically or heterotrophically when supplied with Glc, a metabolite normally transferred from the alga to its host. Here we show that Glc supplementation of SSB01 cultures causes a loss of pigmentation and photosynthetic activity, disorganization of thylakoid membranes, accumulation of lipid bodies, and alterations of cell-surface morphology. We used global transcriptome analyses to determine if these physiological changes were correlated with changes in gene expression. Glc-supplemented cells exhibited a marked reduction in levels of plastid transcripts encoding photosynthetic proteins, although most nuclear-encoded transcripts (including those for proteins involved in lipid synthesis and formation of the extracellular matrix) exhibited little change in their abundances. However, the altered carbon metabolism in Glc-supplemented cells was correlated with modest alterations (approximately 2x) in the levels of some nuclear-encoded transcripts for sugar transporters. Finally, Glc-bleached SSB01 cells appeared unable to efficiently populate anemone larvae. Together, these results suggest links between energy metabolism and cellular physiology, morphology, and symbiotic interactions. However, the results also show that in contrast to many other organisms, Symbiodinium can undergo dramatic physiological changes that are not reflected by major changes in the abundances of nuclear-encoded transcripts and thus presumably reflect posttranscriptional regulatory processes.

    View details for PubMedID 29217594

    View details for PubMedCentralID PMC5813547

  • GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6f complex accumulation. The Plant journal : for cell and molecular biology Wittkopp, T. M., Saroussi, S. n., Yang, W. n., Johnson, X. n., Kim, R. G., Heinnickel, M. L., Russell, J. J., Phuthong, W. n., Dent, R. M., Broeckling, C. D., Peers, G. n., Lohr, M. n., Wollman, F. A., Niyogi, K. K., Grossman, A. R. 2018

    Abstract

    The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b6f complex (Cytb6f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb6f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule that influences the assembly and activity of Cytb6f. This article is protected by copyright. All rights reserved.

    View details for PubMedID 29602195

  • Prolonged and highly efficient intracellular extraction of photosynthetic electrons from single algal cells by optimized nanoelectrode insertion NANO RESEARCH Hong, H., Kim, Y., Han, M., Yoo, G., Song, H., Chae, Y., Pyun, J., Grossman, A. R., Ryu, W. 2018; 11 (1): 397–409
  • Optimal nutrient exchange and immune responses operate in partner specificity in the cnidarian-dinoflagellate symbiosis (vol 114, pg 13194, 2017) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Matthews, J. L., Crowder, C., Oakley, C. A., Lutz, A., Roessner, U., Meyer, E., Grossman, A. R., Weis, V. M., Davy, S. K. 2017; 114 (51): E11058
  • Optimal nutrient exchange and immune responses operate in partner specificity in the cnidarian-dinoflagellate symbiosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Matthews, J. L., Crowder, C. M., Oakley, C. A., Lutz, A., Roessner, U., Meyer, E., Grossman, A. R., Weis, V. M., Davy, S. K. 2017; 114 (50): 13194–99

    Abstract

    The relationship between corals and dinoflagellates of the genus Symbiodinium is fundamental to the functioning of coral ecosystems. It has been suggested that reef corals may adapt to climate change by changing their dominant symbiont type to a more thermally tolerant one, although the capacity for such a shift is potentially hindered by the compatibility of different host-symbiont pairings. Here we combined transcriptomic and metabolomic analyses to characterize the molecular, cellular, and physiological processes that underlie this compatibility, with a particular focus on Symbiodinium trenchii, an opportunistic, thermally tolerant symbiont that flourishes in coral tissues after bleaching events. Symbiont-free individuals of the sea anemone Exaiptasia pallida (commonly referred to as Aiptasia), an established model system for the study of the cnidarian-dinoflagellate symbiosis, were colonized with the "normal" (homologous) symbiont Symbiodinium minutum and the heterologous S. trenchii Analysis of the host gene and metabolite expression profiles revealed that heterologous symbionts induced an expression pattern intermediate between the typical symbiotic state and the aposymbiotic state. Furthermore, integrated pathway analysis revealed that increased catabolism of fixed carbon stores, metabolic signaling, and immune processes occurred in response to the heterologous symbiont type. Our data suggest that both nutritional provisioning and the immune response induced by the foreign "invader" are important factors in determining the capacity of corals to adapt to climate change through the establishment of novel symbioses.

    View details for DOI 10.1073/pnas.1710733114

    View details for Web of Science ID 000417806200060

    View details for PubMedID 29158383

    View details for PubMedCentralID PMC5740609

  • Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH FRONTIERS IN PLANT SCIENCE Fan, J., Zheng, L., Bai, Y., Saroussi, S., Grossman, A. R. 2017; 8
  • Biotic interactions as drivers of algal origin and evolution NEW PHYTOLOGIST Brodie, J., Ball, S. G., Bouget, F., Chan, C., De Clerck, O., Cock, J., Gachon, C., Grossman, A. R., Mock, T., Raven, J. A., Saha, M., Smith, A. G., Vardi, A., Yoon, H., Bhattacharya, D. 2017; 216 (3): 670–81

    Abstract

    Contents 670 I. 671 II. 671 III. 676 IV. 678 678 References 678 SUMMARY: Biotic interactions underlie life's diversity and are the lynchpin to understanding its complexity and resilience within an ecological niche. Algal biologists have embraced this paradigm, and studies building on the explosive growth in omics and cell biology methods have facilitated the in-depth analysis of nonmodel organisms and communities from a variety of ecosystems. In turn, these advances have enabled a major revision of our understanding of the origin and evolution of photosynthesis in eukaryotes, bacterial-algal interactions, control of massive algal blooms in the ocean, and the maintenance and degradation of coral reefs. Here, we review some of the most exciting developments in the field of algal biotic interactions and identify challenges for scientists in the coming years. We foresee the development of an algal knowledgebase that integrates ecosystem-wide omics data and the development of molecular tools/resources to perform functional analyses of individuals in isolation and in populations. These assets will allow us to move beyond mechanistic studies of a single species towards understanding the interactions amongst algae and other organisms in both the laboratory and the field.

    View details for DOI 10.1111/nph.14760

    View details for Web of Science ID 000417215200009

    View details for PubMedID 28857164

  • Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii PLANT CELL Wittkopp, T. M., Schmollinger, S., Saroussi, S., Hu, W., Zhang, W., Fan, Q., Gallaher, S. D., Leonard, M. T., Soubeyrand, E., Basset, G. J., Merchant, S. S., Grossman, A. R., Duanmu, D., Lagarias, J. 2017; 29 (11): 2711–26

    Abstract

    In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant (hmox1) of the green alga Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. hmox1 cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 5'-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The hmox1 mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IXα, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of hmox1, genetically complemented hmox1, and chemically rescued hmox1 reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the hmox1 mutant at the transcript level. We propose that a bilin-based, blue-light-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.

    View details for PubMedID 29084873

    View details for PubMedCentralID PMC5728120

  • Nutrient scavenging and energy management: acclimation responses in nitrogen and sulfur deprived Chlamydomonas. Current opinion in plant biology Saroussi, S., Sanz-Luque, E., Kim, R. G., Grossman, A. R. 2017; 39: 114-122

    Abstract

    Photosynthetic organisms have evolved to modulate their metabolism to accommodate the highly dynamic light and nutrient conditions in nature. In this review we discuss ways in which the green alga Chlamydomonas reinhardtii acclimates to nitrogen and sulfur deprivation, conditions that would limit the anabolic use of excitation energy because of a markedly reduced capacity for cell growth and division. Major aspects of this acclimation process are stringently regulated and involve scavenging the limited nutrient from internal and external sources, and the redirection of fixed carbon toward energy storage (e.g. starch, oil). However, photosynthetic organisms have also evolved mechanisms to dissipate excess absorbed light energy, and to eliminate potentially dangerous energetic electrons through the reduction of O2 and H+ to H2O; this reduction can occur both through photosynthetic electron transport (e.g. Mehler reaction, chlororespiration) and mitochondrial respiration. Furthermore, algal cells likely exploit other energy management pathways that are currently not linked to nutrient limitation responses or that remain to be identified.

    View details for DOI 10.1016/j.pbi.2017.06.002

    View details for PubMedID 28692856

  • Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brawley, S. H., Blouin, N. A., Ficko-Blean, E., Wheeler, G. L., Lohr, M., Goodson, H. V., Jenkins, J. W., Blaby-Haas, C. E., Helliwell, K. E., Chan, C., Marriage, T. N., Bhattacharya, D., Klein, A. S., Badis, Y., Brodie, J., Cao, Y., Collen, J., Dittami, S. M., Gachon, C. M., Green, B. R., Karpowicz, S. J., Kim, J. W., Kudahl, U., Lin, S., Michel, G., Mittag, M., Olson, B. C., Pangilinan, J. L., Peng, Y., Qiu, H., Shu, S., Singer, J. T., Smith, A. G., Sprecher, B. N., Wagner, V., Wang, W., Wang, Z., Yan, J., Yarish, C., Zauner-Riek, S., Zhuang, Y., Zou, Y., Lindquist, E. A., Grimwood, J., Barry, K. W., Rokhsar, D. S., Schmutz, J., Stiller, J. W., Grossman, A. R., Prochnik, S. E. 2017; 114 (31): E6361–E6370

    Abstract

    Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

    View details for DOI 10.1073/pnas.1703088114

    View details for Web of Science ID 000406653300014

    View details for PubMedID 28716924

    View details for PubMedCentralID PMC5547612

  • Thermal Shock Induces Host Proteostasis Disruption and Endoplasmic Reticulum Stress in the Model Symbiotic Cnidarian Aiptasia JOURNAL OF PROTEOME RESEARCH Oakley, C. A., Durand, E., Wilkinson, S. P., Peng, L., Weis, V. M., Grossman, A. R., Davy, S. K. 2017; 16 (6): 2121–34

    Abstract

    Coral bleaching has devastating effects on coral survival and reef ecosystem function, but many of the fundamental cellular effects of thermal stress on cnidarian physiology are unclear. We used label-free liquid chromatography-tandem mass spectrometry to compare the effects of rapidly (33.5 °C, 24 h) and gradually (30 and 33.5 °C, 12 days) elevated temperatures on the proteome of the model symbiotic anemone Aiptasia. We identified 2133 proteins in Aiptasia, 136 of which were differentially abundant between treatments. Thermal shock, but not acclimation, resulted in significant abundance changes in 104 proteins, including those involved in protein folding and synthesis, redox homeostasis, and central metabolism. Nineteen abundant structural proteins showed particularly reduced abundance, demonstrating proteostasis disruption and potential protein synthesis inhibition. Heat shock induced antioxidant mechanisms and proteins involved in stabilizing nascent proteins, preventing protein aggregation and degrading damaged proteins, which is indicative of endoplasmic reticulum stress. Host proteostasis disruption occurred before either bleaching or symbiont photoinhibition was detected, suggesting host-derived reactive oxygen species production as the proximate cause of thermal damage. The pronounced abundance changes in endoplasmic reticulum proteins associated with proteostasis and protein turnover indicate that these processes are essential in the cellular response of symbiotic cnidarians to severe thermal stress.

    View details for DOI 10.1021/acs.jproteome.6b00797

    View details for Web of Science ID 000402850800002

    View details for PubMedID 28474894

  • Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency. Journal of experimental botany Caspari, O. D., Meyer, M. T., Tolleter, D., Wittkopp, T. M., Cunniffe, N. J., Lawson, T., Grossman, A. R., Griffiths, H. 2017; 68 (14): 3903-3913

    Abstract

    The pyrenoid of the unicellular green alga Chlamydomonas reinhardtii is a microcompartment situated in the centre of the cup-shaped chloroplast, containing up to 90% of cellular Rubisco. Traversed by a network of dense, knotted thylakoid tubules, the pyrenoid has been proposed to influence thylakoid biogenesis and ultrastructure. Mutants that are unable to assemble a pyrenoid matrix, due to expressing a vascular plant version of the Rubisco small subunit, exhibit severe growth and photosynthetic defects and have an ineffective carbon-concentrating mechanism (CCM). The present study set out to determine the cause of photosynthetic limitation in these pyrenoid-less lines. We tested whether electron transport and light use were compromised as a direct structural consequence of pyrenoid loss or as a metabolic effect downstream of lower CCM activity and resulting CO2 limitation. Thylakoid organization was unchanged in the mutants, including the retention of intrapyrenoid-type thylakoid tubules, and photosynthetic limitations associated with the absence of the pyrenoid were rescued by exposing cells to elevated CO2 levels. These results demonstrate that Rubisco aggregation in the pyrenoid functions as an essential element for CO2 delivery as part of the CCM, and does not play other roles in maintenance of photosynthetic membrane energetics.

    View details for DOI 10.1093/jxb/erx197

    View details for PubMedID 28911055

    View details for PubMedCentralID PMC5853600

  • A robust protocol for efficient generation, and genomic characterization of insertional mutants of Chlamydomonas reinhardtii PLANT METHODS Pollock, S. V., Mukherjee, B., Bajsa-Hirschel, J., Machingura, M. C., Mukherjee, A., Grossman, A. R., Moroney, J. V. 2017; 13
  • Impact of light intensity and quality on chromatophore and nuclear gene expression in Paulinella chromatophora, an amoeba with nascent photosynthetic organelles PLANT JOURNAL Zhang, R., Nowack, E. C., Price, D. C., Bhattacharya, D., Grossman, A. R. 2017; 90 (2): 221-234

    Abstract

    Plastid evolution has been attributed to a single primary endosymbiotic event that occurred about 1.6 billion years ago (BYA) in which a cyanobacterium was engulfed and retained by a eukaryotic cell, although early steps in plastid integration are poorly understood. The photosynthetic amoeba Paulinella chromatophora represents a unique model for the study of plastid evolution because it contains cyanobacterium-derived photosynthetic organelles termed 'chromatophores' that originated relatively recently (0.09-0.14 BYA). The chromatophore genome is about a third the size of the genome of closely related cyanobacteria, but 10-fold larger than most plastid genomes. Several genes have been transferred from the chromatophore genome to the host nuclear genome through endosymbiotic gene transfer (EGT). Some EGT-derived proteins could be imported into chromatophores for function. Two photosynthesis-related genes (psaI and csos4A) are encoded by both the nuclear and chromatophore genomes, suggesting that EGT in Paulinella chromatophora is ongoing. Many EGT-derived genes encode proteins that function in photosynthesis and photoprotection, including an expanded family of high-light-inducible (ncHLI) proteins. Cyanobacterial hli genes are high-light induced and required for cell viability under excess light. We examined the impact of light on Paulinella chromatophora and found that this organism is light sensitive and lacks light-induced transcriptional regulation of chromatophore genes and most EGT-derived nuclear genes. However, several ncHLI genes have reestablished light-dependent regulation, which appears analogous to what is observed in cyanobacteria. We postulate that expansion of the ncHLI gene family and its regulation may reflect the light/oxidative stress experienced by Paulinella chromatophora as a consequence of the as yet incomplete integration of host and chromatophore metabolisms.

    View details for DOI 10.1111/tpj.13488

    View details for Web of Science ID 000398909800001

    View details for PubMedID 28182317

  • Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei BMC EVOLUTIONARY BIOLOGY Morales, J., Kokkori, S., Weidauer, D., Chapman, J., Goltsman, E., Rokhsar, D., Grossman, A. R., Nowack, E. C. 2016; 16

    Abstract

    Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems.Here we show that the trypanosomatid Angomonas deanei, which harbors a β-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei.After previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.

    View details for DOI 10.1186/s12862-016-0820-z

    View details for Web of Science ID 000387727400001

    View details for PubMedID 27835948

    View details for PubMedCentralID PMC5106770

  • Patterned Nanowire Electrode Array for Direct Extraction of Photosynthetic Electrons from Multiple Living Algal Cells ADVANCED FUNCTIONAL MATERIALS Kim, L. H., Kim, Y. J., Hong, H., Yang, D., Han, M., Yoo, G., Song, H. W., Chae, Y., Pyun, J., Grossman, A. R., Ryu, W. 2016; 26 (42): 7679-7689
  • Gene transfers from diverse bacteria compensate for reductive genome evolution in the chromatophore of Paulinella chromatophora PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nowack, E. C., Price, D. C., Bhattacharya, D., Singer, A., Melkonian, M., Grossman, A. R. 2016; 113 (43): 12214-12219

    Abstract

    Plastids, the photosynthetic organelles, originated >1 billion y ago via the endosymbiosis of a cyanobacterium. The resulting proliferation of primary producers fundamentally changed global ecology. Endosymbiotic gene transfer (EGT) from the intracellular cyanobacterium to the nucleus is widely recognized as a critical factor in the evolution of photosynthetic eukaryotes. The contribution of horizontal gene transfers (HGTs) from other bacteria to plastid establishment remains more controversial. A novel perspective on this issue is provided by the amoeba Paulinella chromatophora, which contains photosynthetic organelles (chromatophores) that are only 60-200 million years old. Chromatophore genome reduction entailed the loss of many biosynthetic pathways including those for numerous amino acids and cofactors. How the host cell compensates for these losses remains unknown, because the presence of bacteria in all available P. chromatophora cultures excluded elucidation of the full metabolic capacity and occurrence of HGT in this species. Here we generated a high-quality transcriptome and draft genome assembly from the first bacteria-free P. chromatophora culture to deduce rules that govern organelle integration into cellular metabolism. Our analyses revealed that nuclear and chromatophore gene inventories provide highly complementary functions. At least 229 nuclear genes were acquired via HGT from various bacteria, of which only 25% putatively arose through EGT from the chromatophore genome. Many HGT-derived bacterial genes encode proteins that fill gaps in critical chromatophore pathways/processes. Our results demonstrate a dominant role for HGT in compensating for organelle genome reduction and suggest that phagotrophy may be a major driver of HGT.

    View details for DOI 10.1073/pnas.1608016113

    View details for Web of Science ID 000386087100068

    View details for PubMedID 27791007

    View details for PubMedCentralID PMC5087059

  • Relative Contributions of Various Cellular Mechanisms to Loss of Algae during Cnidarian Bleaching PLOS ONE Bieri, T., Onishi, M., Xiang, T., Grossman, A. R., Pringle, J. R. 2016; 11 (4)

    Abstract

    When exposed to stress such as high seawater temperature, corals and other cnidarians can bleach due to loss of symbiotic algae from the host tissue and/or loss of pigments from the algae. Although the environmental conditions that trigger bleaching are reasonably well known, its cellular and molecular mechanisms are not well understood. Previous studies have reported the occurrence of at least four different cellular mechanisms for the loss of symbiotic algae from the host tissue: in situ degradation of algae, exocytic release of algae from the host, detachment of host cells containing algae, and death of host cells containing algae. The relative contributions of these several mechanisms to bleaching remain unclear, and it is also not known whether these relative contributions change in animals subjected to different types and/or durations of stresses. In this study, we used a clonal population of the small sea anemone Aiptasia, exposed individuals to various precisely controlled stress conditions, and quantitatively assessed the several possible bleaching mechanisms in parallel. Under all stress conditions tested, except for acute cold shock at 4°C, expulsion of intact algae from the host cells appeared to be by far the predominant mechanism of bleaching. During acute cold shock, in situ degradation of algae and host-cell detachment also became quantitatively significant, and the algae released under these conditions appeared to be severely damaged.

    View details for DOI 10.1371/journal.pone.0152693

    View details for Web of Science ID 000374976200010

    View details for PubMedID 27119147

    View details for PubMedCentralID PMC4847765

  • The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow, Energy-Dependent Quenching, and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation PLANT PHYSIOLOGY Saroussi, S. I., Wittkopp, T. M., Grossman, A. R. 2016; 170 (4): 1975-1988

    Abstract

    When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H(+) gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF The H(+) gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes.

    View details for DOI 10.1104/pp.15.02014

    View details for Web of Science ID 000375424200008

    View details for PubMedID 26858365

    View details for PubMedCentralID PMC4825143

  • Genome Analysis of Planctomycetes Inhabiting Blades of the Red Alga Porphyra umbilicalis PLOS ONE Kim, J. W., Brawley, S. H., Prochnik, S., Chovatia, M., Grimwood, J., Jenkins, J., LaButti, K., Mavromatis, K., Nolan, M., Zane, M., Schmutz, J., Stiller, J. W., Grossman, A. R. 2016; 11 (3)

    Abstract

    Porphyra is a macrophytic red alga of the Bangiales that is important ecologically and economically. We describe the genomes of three bacteria in the phylum Planctomycetes (designated P1, P2 and P3) that were isolated from blades of Porphyra umbilicalis (P.um.1). These three Operational Taxonomic Units (OTUs) belong to distinct genera; P2 belongs to the genus Rhodopirellula, while P1 and P3 represent undescribed genera within the Planctomycetes. Comparative analyses of the P1, P2 and P3 genomes show large expansions of distinct gene families, which can be widespread throughout the Planctomycetes (e.g., protein kinases, sensors/response regulators) and may relate to specific habitat (e.g., sulfatase gene expansions in marine Planctomycetes) or phylogenetic position. Notably, there are major differences among the Planctomycetes in the numbers and sub-functional diversity of enzymes (e.g., sulfatases, glycoside hydrolases, polysaccharide lyases) that allow these bacteria to access a range of sulfated polysaccharides in macroalgal cell walls. These differences suggest that the microbes have varied capacities for feeding on fixed carbon in the cell walls of P.um.1 and other macrophytic algae, although the activities among the various bacteria might be functionally complementary in situ. Additionally, phylogenetic analyses indicate augmentation of gene functions through expansions arising from gene duplications and horizontal gene transfers; examples include genes involved in cell wall degradation (e.g., κ-carrageenase, alginate lyase, fucosidase) and stress responses (e.g., efflux pump, amino acid transporter). Finally P1 and P2 contain various genes encoding selenoproteins, many of which are enzymes that ameliorate the impact of environmental stresses that occur in the intertidal habitat.

    View details for DOI 10.1371/journal.pone.0151883

    View details for Web of Science ID 000372708900033

    View details for PubMedCentralID PMC4807772

  • Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Heinnickel, M., Kim, R. G., Wittkopp, T. M., Yang, W., Walters, K. A., Herbert, S. K., Grossman, A. R. 2016; 113 (10): 2774-2779

    Abstract

    A Chlamydomonas reinhardtii mutant lacking CGL71, a thylakoid membrane protein previously shown to be involved in photosystem I (PSI) accumulation, exhibited photosensitivity and highly reduced abundance of PSI under photoheterotrophic conditions. Remarkably, the PSI content of this mutant declined to nearly undetectable levels under dark, oxic conditions, demonstrating that reduced PSI accumulation in the mutant is not strictly the result of photodamage. Furthermore, PSI returns to nearly wild-type levels when the O2 concentration in the medium is lowered. Overall, our results suggest that the accumulation of PSI in the mutant correlates with the redox state of the stroma rather than photodamage and that CGL71 functions under atmospheric O2 conditions to allow stable assembly of PSI. These findings may reflect the history of the Earth's atmosphere as it transitioned from anoxic to highly oxic (1-2 billion years ago), a change that required organisms to evolve mechanisms to assist in the assembly and stability of proteins or complexes with O2-sensitive cofactors.

    View details for DOI 10.1073/pnas.1524040113

    View details for Web of Science ID 000372013300056

    View details for PubMedID 26903622

    View details for PubMedCentralID PMC4791029

  • Gordon research conference on the dynamics and regulation of photosynthesis: from the origin of bio-catalysis to innovative solar conversion. Photosynthesis research Govindjee, Grossman, A. R., Bhaya, D. 2016; 127 (3): 379-389

    Abstract

    We provide here a news report on the 2015 Gordon Research Conference "Dynamics and regulation of photosynthesis: from the origin of biocatalysis to innovative solar conversion.'' It was held at Bentley University, Waltham, MA, USA, June 28-July 3, 2015, and offered a mix of traditional and emerging areas that highlighted new directions and methods of analyses. A major innovation was short (1 min) poster highlights that added an exciting dynamic to the interactions. Following the end of the formal sessions, three young scientists (Andrian Gutu, of Harvard University, USA; Alizée Malnoë, of University of California, Berkeley, USA; and Yuval Mazor of Tel Aviv University, Israel) were recognized for their research; they also each received a recent volume of "Advances in photosynthesis and respiration including bioenergy and related processes" from Govindjee. We also provide at the end a brief report on the Gordon Research Seminar that preceded the conference.

    View details for DOI 10.1007/s11120-015-0187-9

    View details for PubMedID 26338068

  • Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism. Proceedings of the National Academy of Sciences of the United States of America Yang, W., Wittkopp, T. M., Li, X., Warakanont, J., Dubini, A., Catalanotti, C., Kim, R. G., Nowack, E. C., Mackinder, L. C., Aksoy, M., Page, M. D., D'Adamo, S., Saroussi, S., Heinnickel, M., Johnson, X., Richaud, P., Alric, J., Boehm, M., Jonikas, M. C., Benning, C., Merchant, S. S., Posewitz, M. C., Grossman, A. R. 2015; 112 (48): 14978-14983

    Abstract

    Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

    View details for DOI 10.1073/pnas.1515240112

    View details for PubMedID 26627249

    View details for PubMedCentralID PMC4672766

  • The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes. Plant physiology Phuthong, W., Huang, Z., Wittkopp, T. M., Sznee, K., Heinnickel, M. L., Dekker, J. P., Frese, R. N., Prinz, F. B., Grossman, A. R. 2015; 169 (2): 1318-1332

    Abstract

    To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes associated with photosystem II (PSII-OEC) on spinach (Spinacia oleracea) grana membranes were examined using Contact Mode Atomic Force Microscopy (CM-AFM). This study represents the use of AFM to distinguish the putative large extrinsic loop of CP47 from the putative PsbO/PsbP/PsbQ/large extrinsic loop of CP43 in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles which, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I (PSI) that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze fracture electron microscopy, as well as previous AFM studies.

    View details for DOI 10.1104/pp.15.00706

    View details for PubMedID 26220954

  • Algae after dark: mechanisms to cope with anoxic/hypoxic conditions PLANT JOURNAL Yang, W., Catalanotti, C., Wittkopp, T. M., Posewitz, M. C., Grossman, A. R. 2015; 82 (3): 481-503

    Abstract

    Chlamydomonas reinhardtii is a unicellular, soil-dwelling (and aquatic) green alga that has significant metabolic flexibility for balancing redox equivalents and generating ATP when it experiences hypoxic/anoxic conditions. The diversity of pathways available to ferment sugars is often revealed in mutants in which the activities of specific branches of fermentative metabolism have been eliminated; compensatory pathways that have little activity in parental strains under standard laboratory fermentative conditions are often activated. The ways in which these pathways are regulated and integrated have not been extensively explored. In this review, we primarily discuss the intricacies of dark anoxic metabolism in Chlamydomonas, but also discuss aspects of dark oxic metabolism, the utilization of acetate, and the relatively uncharacterized but critical interactions that link chloroplastic and mitochondrial metabolic networks.

    View details for DOI 10.1111/tpj.12823

    View details for Web of Science ID 000353500000009

    View details for PubMedID 25752440

  • Symbiodinium transcriptome and global responses of cells to immediate changes in light intensity when grown under autotrophic or mixotrophic conditions PLANT JOURNAL Xiang, T., Nelson, W., Rodriguez, J., Tolleter, D., Grossman, A. R. 2015; 82 (1): 67-80

    Abstract

    Symbiosis between unicellular dinoflagellates (genus Symbiodinium) and their cnidarian hosts (e.g. corals, sea anemones) is the foundation of coral reef ecosystems. Dysfunction of this symbiosis under changing environmental conditions has led to global reef decline. Little information is known about Symbiodinium gene expression and mechanisms by which light impacts host-symbiont associations. To address these issues, we generated a transcriptome from axenic Symbiodinium strain SSB01. Here we report features of the transcriptome, including occurrence and length distribution of spliced leader sequences, the functional landscape of encoded proteins and the impact of light on gene expression. Expression of many Symbiodinium genes appears to be significantly impacted by light. Transcript encoding cryptochrome 2 declined in high light while some transcripts for Regulators of Chromatin Condensation (RCC1) declined in the dark. We also identified a transcript encoding a light harvesting AcpPC protein with homology to Chlamydomonas LHCSR2. The level of this transcript increased in high light autotrophic conditions, suggesting that it is involved in photo-protection and the dissipation of excess absorbed light energy. The most extensive changes in transcript abundances occurred when the algae were transferred from low light to darkness. Interestingly, transcripts encoding several cell adhesion proteins rapidly declined following movement of cultures to the dark, which correlated with a dramatic change in cell surface morphology, likely reflecting the complexity of the extracellular matrix. Thus, light-sensitive cell adhesion proteins may play a role in establishing surface architecture, which may in turn alter interactions between the endosymbiont and its host.

    View details for DOI 10.1111/tpj.12789

    View details for Web of Science ID 000351682300006

    View details for PubMedID 25664570

  • Profiling Chlamydomonas Metabolism under Dark, Anoxic H-2-Producing Conditions Using a Combined Proteomic, Transcriptomic, and Metabolomic Approach JOURNAL OF PROTEOME RESEARCH Subramanian, V., Dubini, A., Astling, D. P., Laurens, L. M., Old, W. M., Grossman, A. R., Posewitz, M. C., Seibert, M. 2014; 13 (12): 5431-5451

    Abstract

    Chlamydomonas reinhardtii is well adapted to survive under different environmental conditions due to the unique flexibility of its metabolism. Here we report metabolic pathways that are active during acclimation to anoxia, but were previously not thoroughly studied under dark, anoxic H2-producing conditions in this model green alga. Proteomic analyses, using 2D-differential in-gel electrophoresis in combination with shotgun mass fingerprinting, revealed increased levels of proteins involved in the glycolytic pathway downstream of 3-phosphoglycerate, the glyoxylate pathway, and steps of the tricarboxylic acid (TCA) reactions. Upregulation of the enzyme, isocitrate lyase (ICL), was observed, which was accompanied by increased intracellular succinate levels, suggesting the functioning of glyoxylate pathway reactions. The ICL-inhibitor study revealed presence of reverse TCA reactions under these conditions. Contributions of the serine-isocitrate lyase pathway, glycine cleavage system, and c1-THF/serine hydroxymethyltransferase pathway in the acclimation to dark anoxia were found. We also observed increased levels of amino acids (AAs) suggesting nitrogen reorganization in the form of de novo AA biosynthesis during anoxia. Overall, novel routes for reductant utilization, in combination with redistribution of carbon and nitrogen, are used by this alga during acclimation to O2 deprivation in the dark.

    View details for DOI 10.1021/pr500342j

    View details for Web of Science ID 000346039400014

    View details for PubMedID 25333711

  • Alternative Acetate Production Pathways in Chlamydomonas reinhardtii during Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production. Plant cell Yang, W., Catalanotti, C., D'Adamo, S., Wittkopp, T. M., Ingram-Smith, C. J., Mackinder, L., Miller, T. E., Heuberger, A. L., Peers, G., Smith, K. S., Jonikas, M. C., Grossman, A. R., Posewitz, M. C. 2014; 26 (11): 4499-4518

    Abstract

    Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

    View details for DOI 10.1105/tpc.114.129965

    View details for PubMedID 25381350

    View details for PubMedCentralID PMC4277214

  • Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation PLANT CELL Aksoy, M., Pootakham, W., Grossman, A. R. 2014; 26 (10): 4214-4229

    Abstract

    Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.

    View details for DOI 10.1105/tpc.114.129270

    View details for Web of Science ID 000345920900031

    View details for PubMedID 25281687

  • The Chlamydomonas genome project: a decade on TRENDS IN PLANT SCIENCE Blaby, I. K., Blaby-Haas, C. E., Tourasse, N., Hom, E. F., Lopez, D., Aksoy, M., Grossman, A., Umen, J., Dutcher, S., Porter, M., King, S., Witman, G. B., Stanke, M., Harris, E. H., Goodstein, D., Grimwood, J., Schmutz, J., Vallon, O., Merchant, S. S., Prochnik, S. 2014; 19 (10): 672-680

    Abstract

    The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis, and micronutrient homeostasis. Ten years since its genome project was initiated an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the omics era. Housed at Phytozome, the plant genomics portal of the Joint Genome Institute (JGI), the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of whole transcriptome sequencing (RNA-Seq) data. We present here the past, present, and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions, and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.

    View details for DOI 10.1016/j.tplants.2014.05.008

    View details for Web of Science ID 000343359900011

    View details for PubMedID 24950814

    View details for PubMedCentralID PMC4185214

  • 16S AND 23S PLASTID RDNA PHYLOGENIES OF PROTOTHECA SPECIES AND THEIR AUXANOGRAPHIC PHENOTYPES JOURNAL OF PHYCOLOGY Ewing, A., Brubaker, S., Somanchi, A., Yu, E., Rudenko, G., Reyes, N., Espina, K., Grossman, A., Franklin, S. 2014; 50 (4): 765-769

    Abstract

    Because algae have become more accepted as sources of human nutrition, phylogenetic analysis can help resolve the taxonomy of taxa that have not been well studied. This can help establish algal evolutionary relationships. Here, we compare Auxenochlorella protothecoides and 23 strains of Prototheca based on their complete 16S and partial 23S plastid rDNA sequences along with nutrient utilization (auxanographic) profiles. These data demonstrate that some of the species groupings are not in agreement with the molecular phylogenetic analyses and that auxanographic profiles are poor predictors of phylogenetic relationships.

    View details for DOI 10.1111/jpy.12209

    View details for Web of Science ID 000340460100017

    View details for PubMedCentralID PMC4373152

  • Proton Gradient Regulation 5-Mediated Cyclic Electron Flow under ATP- or Redox-Limited Conditions: A Study of Delta ATPase pgr5 and Delta rbcL pgr5 Mutants in the Green Alga Chlamydomonas reinhardtii PLANT PHYSIOLOGY Johnson, X., Steinbeck, J., Dent, R. M., Takahashi, H., Richaud, P., Ozawa, S., Houille-Vernes, L., Petroutsos, D., Rappaport, F., Grossman, A. R., Niyogi, K. K., Hippler, M., Alric, J. 2014; 165 (1): 438-452
  • Proton gradient regulation 5-mediated cyclic electron flow under ATP- or redox-limited conditions: a study of ?ATpase pgr5 and ?rbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii. Plant physiology Johnson, X., Steinbeck, J., Dent, R. M., Takahashi, H., Richaud, P., Ozawa, S., Houille-Vernes, L., Petroutsos, D., Rappaport, F., Grossman, A. R., Niyogi, K. K., Hippler, M., Alric, J. 2014; 165 (1): 438-452

    Abstract

    The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

    View details for DOI 10.1104/pp.113.233593

    View details for PubMedID 24623849

  • Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism PLANT CELL Schmollinger, S., Muehlhaus, T., Boyle, N. R., Blaby, I. K., Casero, D., Mettler, T., Moseley, J. L., Kropat, J., Sommer, F., Strenkert, D., Hemme, D., Pellegrini, M., Grossman, A. R., Stitt, M., Schroda, M., Merchant, S. S. 2014; 26 (4): 1410-1435
  • The GreenCut: re-evaluation of physiological role of previously studied proteins and potential novel protein functions. Photosynthesis research Heinnickel, M. L., Grossman, A. R. 2013; 116 (2-3): 427-436

    Abstract

    Based on comparative genomics, a list of proteins present in the green algal, flowering and nonflowering plant lineages, but not detected in nonphotosynthetic organisms, was assembled (Merchant et al., Science 318:245-250, 2007; Karpowicz et al., J Biol Chem 286:21427-21439, 2011). This protein grouping, previously designated the GreenCut, was established using stringent comparative genomic criteria; they are those Chlamydomonas reinhardtii proteins with orthologs in Arabidopsis thaliana, Physcomitrella patens, Oryza sativa, Populus tricocarpa and at least one of the three Ostreococcus species with fully sequenced genomes, but not in bacteria, yeast, fungi or mammals. Many GreenCut proteins are also present in red algae and diatoms and a subset of 189 have been identified as encoded on nearly all cyanobacterial genomes. Of the current GreenCut proteins (597 in total), approximately half have been studied previously. The functions or activities of a number of these proteins have been deduced from phenotypic analyses of mutants (defective for genes encoding specific GreenCut proteins) of A. thaliana, and in many cases the assigned functions do not exist in C. reinhardtii. Therefore, precise physiological functions of several previously studied GreenCut proteins are still not clear. The GreenCut also contains a number of proteins with certain conserved domains. Three of the most highly conserved domains are the FK506 binding, cyclophilin and PAP fibrillin domains; most members of these gene families are not well characterized. In general, our analysis of the GreenCut indicates that many processes critical to green lineage organisms remain unstudied or poorly characterized. We have begun to examine the functions of some GreenCut proteins in detail. For example, our work on the CPLD38 protein has demonstrated that it has an essential role in photosynthetic function and the stability of the cytochrome b 6 f complex.

    View details for DOI 10.1007/s11120-013-9882-6

    View details for PubMedID 23873414

  • The metabolic status drives acclimation of iron deficiency responses in Chlamydomonas reinhardtii as revealed by proteomics based hierarchical clustering and reverse genetics. Molecular & cellular proteomics Höhner, R., Barth, J., Magneschi, L., Jaeger, D., Niehues, A., Bald, T., Grossman, A., Fufezan, C., Hippler, M. 2013; 12 (10): 2774-2790

    Abstract

    Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different "distance-linkage combinations" and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.

    View details for DOI 10.1074/mcp.M113.029991

    View details for PubMedID 23820728

  • The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics MOLECULAR & CELLULAR PROTEOMICS Hoehner, R., Barth, J., Magneschi, L., Jaeger, D., Niehues, A., Bald, T., Grossman, A., Fufezan, C., Hippler, M. 2013; 12 (10): 2774-2790
  • Effect of Temperature on Photosynthesis and Growth in Marine Synechococcus spp. PLANT PHYSIOLOGY Mackey, K. R., Paytan, A., Caldeira, K., Grossman, A. R., Moran, D., McIlvin, M., Saito, M. A. 2013; 163 (2): 815-829

    Abstract

    In this study, we develop a mechanistic understanding of how temperature affects growth and photosynthesis in 10 geographically and physiologically diverse strains of Synechococcus spp. We found that Synechococcus spp. are able to regulate photochemistry over a range of temperatures by using state transitions and altering the abundance of photosynthetic proteins. These strategies minimize photosystem II (PSII) photodamage by keeping the photosynthetic electron transport chain (ETC), and hence PSII reaction centers, more oxidized. At temperatures that approach the optimal growth temperature of each strain when cellular demand for reduced nicotinamide adenine dinucleotide phosphate (NADPH) is greatest, the phycobilisome (PBS) antenna associates with PSII, increasing the flux of electrons into the ETC. By contrast, under low temperature, when slow growth lowers the demand for NADPH and linear ETC declines, the PBS associates with photosystem I. This favors oxidation of PSII and potential increase in cyclic electron flow. For Synechococcus sp. WH8102, growth at higher temperatures led to an increase in the abundance of PBS pigment proteins, as well as higher abundance of subunits of the PSII, photosystem I, and cytochrome b6f complexes. This would allow cells to increase photosynthetic electron flux to meet the metabolic requirement for NADPH during rapid growth. These PBS-based temperature acclimation strategies may underlie the larger geographic range of this group relative to Prochlorococcus spp., which lack a PBS.

    View details for DOI 10.1104/pp.113.221937

    View details for Web of Science ID 000325554100033

    View details for PubMedID 23950220

  • Coral bleaching independent of photosynthetic activity. Current biology Tolleter, D., Seneca, F. O., DeNofrio, J. C., Krediet, C. J., Palumbi, S. R., Pringle, J. R., Grossman, A. R. 2013; 23 (18): 1782-1786

    Abstract

    The global decline of reef-building corals is due in part to the loss of algal symbionts, or "bleaching," during the increasingly frequent periods of high seawater temperatures [1, 2]. During bleaching, endosymbiotic dinoflagellate algae (Symbiodinium spp.) either are lost from the animal tissue or lose their photosynthetic pigments, resulting in host mortality if the Symbiodinium populations fail to recover [3]. The >1,000 studies of the causes of heat-induced bleaching have focused overwhelmingly on the consequences of damage to algal photosynthetic processes [4-6], and the prevailing model for bleaching invokes a light-dependent generation of toxic reactive oxygen species (ROS) by heat-damaged chloroplasts as the primary trigger [6-8]. However, the precise mechanisms of bleaching remain unknown, and there is evidence for involvement of multiple cellular processes [9, 10]. In this study, we asked the simple question of whether bleaching can be triggered by heat in the dark, in the absence of photosynthetically derived ROS. We used both the sea anemone model system Aiptasia [11, 12] and several species of reef-building corals to demonstrate that symbiont loss can occur rapidly during heat stress in complete darkness. Furthermore, we observed damage to the photosynthetic apparatus under these conditions in both Aiptasia endosymbionts and cultured Symbiodinium. These results do not directly contradict the view that light-stimulated ROS production is important in bleaching, but they do show that there must be another pathway leading to bleaching. Elucidation of this pathway should help to clarify bleaching mechanisms under the more usual conditions of heat stress in the light.

    View details for DOI 10.1016/j.cub.2013.07.041

    View details for PubMedID 24012312

  • Role of Polyphosphate in Thermophilic Synechococcus sp from Microbial Mats JOURNAL OF BACTERIOLOGY Gomez-Garcia, M. R., Fazeli, F., Grote, A., Grossman, A. R., Bhaya, D. 2013; 195 (15): 3309-3319

    Abstract

    Synechococcus OS-B' , a thermophilic unicellular cyanobacterium, recently isolated from the microbial mats in Octopus Spring (Yellowstone National Park), induces a suite of genes, including phosphatases and transporters, in response to phosphorus (P) starvation. Here we describe two different approaches to examine the ability of Synechococcus OS-B' to synthesize and breakdown polyphosphate (poly P), a key storage compound in many prokaryotes. First, we developed a transformation protocol to create mutants in the polyphosphate kinase (ppk), the major enzyme responsible for the synthesis of poly P. The ppk mutant exhibited a pleiotropic phenotype with defects in poly P accumulation, aberrant levels of pho regulon transcripts, growth defects and changes in cell size and exopolysaccharide levels, among others. Second, we measured transcripts of ppk and ppx (encoding the polyphosphatase) directly from mat samples and found that the levels varied dramatically over a diel cycle. We also used Western blot analysis to quantify levels of PPK and PPX and found that these enzymes differentially accumulated during the diel cycle. Levels of polyphosphate kinase peaked at night, while polyphosphatase levels were highest during the early morning hours. We hypothesize that the opposing activities of these two enzymes allow cells to store and utilize poly P to optimize growth over a diel cycle.

    View details for DOI 10.1128/JB.00207-13

    View details for Web of Science ID 000321559400002

    View details for PubMedID 23687278

  • Isolation of clonal axenic strains of the symbiotic dinoflagellate Symbiodinium and their growth and host specificity JOURNAL OF PHYCOLOGY Xiang, T., Hambleton, E. A., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. 2013; 49 (3): 447-458

    Abstract

    The cnidarian-dinoflagellate mutualism is integral to the survival of the coral-reef ecosystem. Despite the enormous ecological and economic importance of corals, their cellular and molecular biology and the ways in which they respond to environmental change are still poorly understood. We have been developing a proxy system for examining the coral mutualism in which the dinoflagellate symbiont Symbiodinium is introduced into a clonal population of the host Aiptasia, a small sea anemone closely related to corals. To further develop the tools for this system, we generated five clonal, axenic strains of Symbiodinium and verified the lack of contaminants by growth on rich medium, microscopic examination, and PCR analysis. These strains were assigned to clades A (two strains), B, E, and F based on their chloroplast 23S rDNA sequences. Growth studies in liquid cultures showed that the clade B strain and one of the clade A strains were able to grow photoautotrophically (in light with no fixed carbon), mixotrophically (in light with fixed carbon), or heterotrophically (in dark with fixed carbon). The clade E strain, thought to be free-living, was able to grow photoautotrophically but not heterotrophically. Infection of an aposymbiotic Aiptasia host with the axenic strains showed consistent patterns of specificity, with only the clade B and one of the clade A strains able to successfully establish symbiosis. Overall, the Aiptasia-Symbiodinium association represents an important model system for dissecting aspects of the physiology and cellular and molecular biology of cnidarian-dinoflagellate mutualism and exploring issues that bear directly on coral bleaching.

    View details for DOI 10.1111/jpy.12055

    View details for Web of Science ID 000319874900003

  • Isolation of clonal axenic strains of the symbiotic dinoflagellate Symbiodinium and their growth and host specificity(1). Journal of phycology Xiang, T., Hambleton, E. A., DeNofrio, J. C., Pringle, J. R., Grossman, A. R. 2013; 49 (3): 447-58

    Abstract

    The cnidarian-dinoflagellate mutualism is integral to the survival of the coral-reef ecosystem. Despite the enormous ecological and economic importance of corals, their cellular and molecular biology and the ways in which they respond to environmental change are still poorly understood. We have been developing a proxy system for examining the coral mutualism in which the dinoflagellate symbiont Symbiodinium is introduced into a clonal population of the host Aiptasia, a small sea anemone closely related to corals. To further develop the tools for this system, we generated five clonal, axenic strains of Symbiodinium and verified the lack of contaminants by growth on rich medium, microscopic examination, and PCR analysis. These strains were assigned to clades A (two strains), B, E, and F based on their chloroplast 23S rDNA sequences. Growth studies in liquid cultures showed that the clade B strain and one of the clade A strains were able to grow photoautotrophically (in light with no fixed carbon), mixotrophically (in light with fixed carbon), or heterotrophically (in dark with fixed carbon). The clade E strain, thought to be free-living, was able to grow photoautotrophically but not heterotrophically. Infection of an aposymbiotic Aiptasia host with the axenic strains showed consistent patterns of specificity, with only the clade B and one of the clade A strains able to successfully establish symbiosis. Overall, the Aiptasia-Symbiodinium association represents an important model system for dissecting aspects of the physiology and cellular and molecular biology of cnidarian-dinoflagellate mutualism and exploring issues that bear directly on coral bleaching.

    View details for DOI 10.1111/jpy.12055

    View details for PubMedID 27007034

  • Tiered Regulation of Sulfur Deprivation Responses in Chlamydomonas reinhardtii and Identification of an Associated Regulatory Factor PLANT PHYSIOLOGY Aksoy, M., Pootakham, W., Pollock, S. V., Moseley, J. L., Gonzalez-Ballester, D., Grossman, A. R. 2013; 162 (1): 195-211

    Abstract

    During sulfur (S) deprivation, the unicellular alga Chlamydomonas reinhardtii exhibits increased expression of numerous genes. These genes encode proteins associated with sulfate (SO4(2-)) acquisition and assimilation, alterations in cellular metabolism, and internal S recycling. Administration of the cytoplasmic translational inhibitor cycloheximide prevents S deprivation-triggered accumulation of transcripts encoding arylsulfatases (ARS), an extracellular polypeptide that may be important for cell wall biosynthesis (ECP76), a light-harvesting protein (LHCBM9), the selenium-binding protein, and the haloperoxidase (HAP2). In contrast, the rapid accumulation of transcripts encoding high-affinity SO4(2-) transporters is not affected. These results suggest that there are two tiers of transcriptional regulation associated with S deprivation responses: the first is protein synthesis independent, while the second requires de novo protein synthesis. A mutant designated ars73a exhibited low ARS activity and failed to show increases in ECP76, LHCBM9, and HAP2 transcripts (among others) in response to S deprivation; increases in transcripts encoding the SO4(2-) transporters were not affected. These results suggest that the ARS73a protein, which has no known activity but might be a transcriptional regulator, is required for the expression of genes associated with the second tier of transcriptional regulation. Analysis of the ars73a strain has helped us generate a model that incorporates a number of complexities associated with S deprivation responses in C. reinhardtii.

    View details for DOI 10.1104/pp.113.214593

    View details for Web of Science ID 000318547900016

    View details for PubMedID 23482872

  • Diversity and Abundance of the Bacterial Community of the Red Macroalga Porphyra umbilicalis: Did Bacterial Farmers Produce Macroalgae? PLOS ONE Miranda, L. N., Hutchison, K., Grossman, A. R., Brawley, S. H. 2013; 8 (3)

    Abstract

    Macroalgae harbor microbial communities whose bacterial biodiversity remains largely uncharacterized. The goals of this study were 1) to examine the composition of the bacterial community associated with Porphyra umbilicalis Kützing from Schoodic Point, ME, 2) determine whether there are seasonal trends in species diversity but a core group of bacteria that are always present, and 3) to determine how the microbial community associated with a laboratory strain (P.um.1) established in the presence of antibiotics has changed. P. umbilicalis blades (n = 5, fall 2010; n = 5, winter 2011; n = 2, clonal P.um.1) were analyzed by pyrosequencing over two variable regions of the 16 S rDNA (V5-V6 and V8; 147,880 total reads). The bacterial taxa present were classified at an 80% confidence threshold into eight phyla (Bacteroidetes, Proteobacteria, Planctomycetes, Chloroflexi, Actinobacteria, Deinococcus-Thermus, Firmicutes, and the candidate division TM7). The Bacteroidetes comprised the majority of bacterial sequences on both field and lab blades, but the Proteobacteria (Alphaproteobacteria, Gammaproteobacteria) were also abundant. Sphingobacteria (Bacteroidetes) and Flavobacteria (Bacteroidetes) had inverse abundances on natural versus P.um.1 blades. Bacterial communities were richer and more diverse on blades sampled in fall compared to winter. Significant differences were observed between microbial communities among all three groups of blades examined. Only two OTUs were found on all 12 blades, and only one of these, belonging to the Saprospiraceae (Bacteroidetes), was abundant. Lewinella (as 66 OTUs) was found on all field blades and was the most abundant genus. Bacteria from the Bacteroidetes, Proteobacteria and Planctomycetes that are known to digest the galactan sulfates of red algal cell walls were well-represented. Some of these taxa likely provide essential morphogenetic and beneficial nutritive factors to P. umbilicalis and may have had unexpected effects upon evolution of macroalgal form as well as function.

    View details for DOI 10.1371/journal.pone.0058269

    View details for Web of Science ID 000317562600016

    View details for PubMedID 23526971

  • Novel Thylakoid Membrane GreenCut Protein CPLD38 Impacts Accumulation of the Cytochrome b(6)f Complex and Associated Regulatory Processes JOURNAL OF BIOLOGICAL CHEMISTRY Heinnickel, M. L., Alric, J., Wittkopp, T., Yang, W., Catalanotti, C., Dent, R., Niyogi, K. K., Wollman, F., Grossman, A. R. 2013; 288 (10): 7024-7036

    Abstract

    Based on previous comparative genomic analyses, a set of nearly 600 polypeptides was identified that is present in green algae and flowering and nonflowering plants but is not present (or is highly diverged) in nonphotosynthetic organisms. The gene encoding one of these "GreenCut" proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; the NdhL protein is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii does not grow on minimal medium, is high light-sensitive under photoheterotrophic conditions, has lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700(+), and reduced photochemical efficiency of photosystem II (ΦPSII); these phenotypes are rescued by a wild-type copy of CPLD38. Single turnover flash experiments and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and the levels of transcripts and polypeptide subunits of the cytochrome b6f complex were also significantly lower in the cpld38 mutant. Furthermore, subunits of the cytochrome b6f complex in mutant cells turned over much more rapidly than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutants relative to wild-type cells, suggesting a shift in the cpld38 mutant from photosynthesis toward chlororespiratory metabolism, which is supported by experiments that quantify the reduction state of the plastoquinone pool. Together, these findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and possibly plays a role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.

    View details for DOI 10.1074/jbc.M112.427476

    View details for Web of Science ID 000316002400024

    View details for PubMedID 23303190

  • Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Duanmu, D., Casero, D., Dent, R. M., Gallaher, S., Yang, W., Rockwell, N. C., Martin, S. S., Pellegrini, M., Niyogi, K. K., Merchant, S. S., Grossman, A. R., Lagarias, J. C. 2013; 110 (9): 3621-3626

    Abstract

    The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period.

    View details for DOI 10.1073/pnas.1222375110

    View details for Web of Science ID 000315841900082

    View details for PubMedID 23345435

  • Fermentation metabolism and its evolution in algae. Frontiers in plant science Catalanotti, C., Yang, W., Posewitz, M. C., Grossman, A. R. 2013; 4: 150-?

    Abstract

    Fermentation or anoxic metabolism allows unicellular organisms to colonize environments that become anoxic. Free-living unicellular algae capable of a photoautotrophic lifestyle can also use a range of metabolic circuitry associated with different branches of fermentation metabolism. While algae that perform mixed-acid fermentation are widespread, the use of anaerobic respiration is more typical of eukaryotic heterotrophs. The occurrence of a core set of fermentation pathways among the algae provides insights into the evolutionary origins of these pathways, which were likely derived from a common ancestral eukaryote. Based on genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism has been examined in more detail in Chlamydomonas reinhardtii (Chlamydomonas) than in any other photosynthetic protist. This green alga is metabolically flexible and can sustain energy generation and maintain cellular redox balance under a variety of different environmental conditions. Fermentation metabolism in Chlamydomonas appears to be highly controlled, and the flexible use of the different branches of fermentation metabolism has been demonstrated in studies of various metabolic mutants. Additionally, when Chlamydomonas ferments polysaccharides, it has the ability to eliminate part of the reductant (to sustain glycolysis) through the production of H2, a molecule that can be developed as a source of renewable energy. To date, little is known about the specific role(s) of the different branches of fermentation metabolism, how photosynthetic eukaryotes sense changes in environmental O2 levels, and the mechanisms involved in controlling these responses, at both the transcriptional and post-transcriptional levels. In this review, we focus on fermentation metabolism in Chlamydomonas and other protists, with only a brief discussion of plant fermentation when relevant, since it is thoroughly discussed in other articles in this volume.

    View details for DOI 10.3389/fpls.2013.00150

    View details for PubMedID 23734158

  • Porphyra (Bangiophyceae) Transcriptomes Provide Insights Into Red Algal Development And Metabolism JOURNAL OF PHYCOLOGY Chan, C. X., Blouin, N. A., Zhuang, Y., Zaeuner, S., Prochnik, S. E., Lindquist, E., Lin, S., Benning, C., Lohr, M., Yarish, C., Gantt, E., Grossman, A. R., Lu, S., Mueller, K., Stiller, J. W., Brawley, S. H., Bhattacharya, D. 2012; 48 (6): 1328-1342
  • A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii PLANT CELL Beel, B., Prager, K., Spexard, M., Sasso, S., Weiss, D., Mueller, N., Heinnickel, M., Dewez, D., Ikoma, D., Grossman, A. R., Kottke, T., Mittag, M. 2012; 24 (7): 2992-3008

    Abstract

    Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light-activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities.

    View details for DOI 10.1105/tpc.112.098947

    View details for Web of Science ID 000308352800023

    View details for PubMedID 22773746

  • Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas JOURNAL OF BIOLOGICAL CHEMISTRY Boyle, N. R., Page, M. D., Liu, B., Blaby, I. K., Casero, D., Kropat, J., Cokus, S. J., Hong-Hermesdorf, A., Shaw, J., Karpowicz, S. J., Gallaher, S. D., Johnson, S., Benning, C., Pellegrini, M., Grossman, A., Merchant, S. S. 2012; 287 (19): 15811-15825

    Abstract

    Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.

    View details for DOI 10.1074/jbc.M111.334052

    View details for Web of Science ID 000304006300061

    View details for PubMedID 22403401

  • Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nowack, E. C., Grossman, A. R. 2012; 109 (14): 5340-5345

    Abstract

    Endosymbiotic acquisition of bacteria by a protist, with subsequent evolution of the bacteria into mitochondria and plastids, had a transformative impact on eukaryotic biology. Reconstructing events that created a stable association between endosymbiont and host during the process of organellogenesis--including establishment of regulated protein import into nascent organelles--is difficult because they date back more than 1 billion years. The amoeba Paulinella chromatophora contains nascent photosynthetic organelles of more recent evolutionary origin (∼60 Mya) termed chromatophores (CRs). After the initial endosymbiotic event, the CR genome was reduced to approximately 30% of its presumed original size and more than 30 expressed genes were transferred from the CR to the amoebal nuclear genome. Three transferred genes--psaE, psaK1, and psaK2--encode subunits of photosystem I. Here we report biochemical evidence that PsaE, PsaK1, and PsaK2 are synthesized in the amoeba cytoplasm and traffic into CRs, where they assemble with CR-encoded subunits into photosystem I complexes. Additionally, our data suggest that proteins routed to CRs pass through the Golgi apparatus. Whereas genome reduction and transfer of genes from bacterial to host genome have been reported to occur in other obligate bacterial endosymbioses, this report outlines the import of proteins encoded by such transferred genes into the compartment derived from the bacterial endosymbiont. Our study showcases P. chromatophora as an exceptional model in which to study early events in organellogenesis, and suggests that protein import into bacterial endosymbionts might be a phenomenon much more widespread than currently assumed.

    View details for DOI 10.1073/pnas.1118800109

    View details for Web of Science ID 000302294700050

    View details for PubMedID 22371600

  • Analysis of Porphyra Membrane Transporters Demonstrates Gene Transfer among Photosynthetic Eukaryotes and Numerous Sodium-Coupled Transport Systems PLANT PHYSIOLOGY Chan, C. X., Zaeuner, S., Wheeler, G., Grossman, A. R., Prochnik, S. E., Blouin, N. A., Zhuang, Y., Benning, C., Berg, G. M., Yarish, C., Eriksen, R. L., Klein, A. S., Lin, S., Levine, I., Brawley, S. H., Bhattacharya, D. 2012; 158 (4): 2001-2012

    Abstract

    Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e. the Plantae hypothesis) and 19 expressed sequence tag contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for coregulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters.

    View details for DOI 10.1104/pp.112.193896

    View details for Web of Science ID 000303001400041

    View details for PubMedID 22337920

    View details for PubMedCentralID PMC3320202

  • A Mutant in the ADH1 Gene of Chlamydomonas reinhardtii Elicits Metabolic Restructuring during Anaerobiosis PLANT PHYSIOLOGY Magneschi, L., Catalanotti, C., Subramanian, V., Dubini, A., Yang, W., Mus, F., Posewitz, M. C., Seibert, M., Perata, P., Grossman, A. R. 2012; 158 (3): 1293-1305

    Abstract

    The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO(2) was significantly reduced, but fermentative H(2) production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.

    View details for DOI 10.1104/pp.111.191569

    View details for Web of Science ID 000301280500016

    View details for PubMedID 22271746

  • Altered Fermentative Metabolism in Chlamydomonas reinhardtii Mutants Lacking Pyruvate Formate Lyase and Both Pyruvate Formate Lyase and Alcohol Dehydrogenase PLANT CELL Catalanotti, C., Dubini, A., Subramanian, V., Yang, W., Magneschi, L., Mus, F., Seibert, M., Posewitz, M. C., Grossman, A. R. 2012; 24 (2): 692-707

    Abstract

    Chlamydomonas reinhardtii, a unicellular green alga, often experiences hypoxic/anoxic soil conditions that activate fermentation metabolism. We isolated three Chlamydomonas mutants disrupted for the pyruvate formate lyase (PFL1) gene; the encoded PFL1 protein catalyzes a major fermentative pathway in wild-type Chlamydomonas cells. When the pfl1 mutants were subjected to dark fermentative conditions, they displayed an increased flux of pyruvate to lactate, elevated pyruvate decarboxylation, ethanol accumulation, diminished pyruvate oxidation by pyruvate ferredoxin oxidoreductase, and lowered H(2) production. The pfl1-1 mutant also accumulated high intracellular levels of lactate, succinate, alanine, malate, and fumarate. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but it also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars and a decrease in dark, fermentative H(2) production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant reroutes glycolytic carbon to lactate and glycerol. Although the metabolic adjustments observed in the mutants facilitate NADH reoxidation and sustained glycolysis under dark, anoxic conditions, the observed changes could not have been predicted given our current knowledge of the regulation of fermentation metabolism.

    View details for DOI 10.1105/tpc.111.093146

    View details for Web of Science ID 000302131000023

    View details for PubMedID 22353371

  • Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H-2 production BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Meuser, J. E., D'Adamo, S., Jinkerson, R. E., Mus, F., Yang, W., Ghirardi, M. L., Seibert, M., Grossman, A. R., Posewitz, M. C. 2012; 417 (2): 704-709

    Abstract

    Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.

    View details for DOI 10.1016/j.bbrc.2011.12.002

    View details for Web of Science ID 000299610200009

    View details for PubMedID 22177948

  • Homomeric Interaction of the STAS Domain in Sultr1;2 8th International Workshop on Sulfur Metabolism in Higher Plants Shibagaki, N., Grossman, A. R. SPRINGER. 2012: 109–114
  • In vivo O-2 measurement inside single photosynthetic cells BIOTECHNOLOGY LETTERS Bai, S., Ryu, W., Fasching, R. J., Grossman, A. R., Prinz, F. B. 2011; 33 (8): 1675-1681

    Abstract

    The oxygen evolution of single cells was investigated using a nano-probe with an ultra-micro electrode (UME) in a submicron sized system in combination with a micro-fluidic system. A single cell was immobilized in the micro-fluidic system and a nano-probe was inserted into the cytosolic space of the single cell. Then, the UME was used for an in vivo amperometric experiment at a fixed potential and electrochemical impedance spectroscopy to detect oxygen evolution of the single cell under various light intensities.

    View details for DOI 10.1007/s10529-011-0604-x

    View details for Web of Science ID 000293752000025

    View details for PubMedID 21476096

  • Community ecology of hot spring cyanobacterial mats: predominant populations and their functional potential ISME JOURNAL Klatt, C. G., Wood, J. M., Rusch, D. B., Bateson, M. M., Hamamura, N., Heidelberg, J. F., Grossman, A. R., Bhaya, D., Cohan, F. M., Kuhl, M., Bryant, D. A., Ward, D. M. 2011; 5 (8): 1262-1278

    Abstract

    Phototrophic microbial mat communities from 60°C and 65°C regions in the effluent channels of Mushroom and Octopus Springs (Yellowstone National Park, WY, USA) were investigated by shotgun metagenomic sequencing. Analyses of assembled metagenomic sequences resolved six dominant chlorophototrophic populations and permitted the discovery and characterization of undescribed but predominant community members and their physiological potential. Linkage of phylogenetic marker genes and functional genes showed novel chlorophototrophic bacteria belonging to uncharacterized lineages within the order Chlorobiales and within the Kingdom Chloroflexi. The latter is the first chlorophototrophic member of Kingdom Chloroflexi that lies outside the monophyletic group of chlorophototrophs of the Order Chloroflexales. Direct comparison of unassembled metagenomic sequences to genomes of representative isolates showed extensive genetic diversity, genomic rearrangements and novel physiological potential in native populations as compared with genomic references. Synechococcus spp. metagenomic sequences showed a high degree of synteny with the reference genomes of Synechococcus spp. strains A and B', but synteny declined with decreasing sequence relatedness to these references. There was evidence of horizontal gene transfer among native populations, but the frequency of these events was inversely proportional to phylogenetic relatedness.

    View details for DOI 10.1038/ismej.2011.73

    View details for Web of Science ID 000295782200004

    View details for PubMedID 21697961

    View details for PubMedCentralID PMC3146275

  • Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants PLANT METHODS Gonzalez-Ballester, D., Pootakham, W., Mus, F., Yang, W., Catalanotti, C., Magneschi, L., de Montaigu, A., Higuera, J. J., Prior, M., Galvan, A., Fernandez, E., Grossman, A. R. 2011; 7

    Abstract

    A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

    View details for DOI 10.1186/1746-4811-7-24

    View details for Web of Science ID 000294348500001

    View details for PubMedID 21794168

    View details for PubMedCentralID PMC3161022

  • The GreenCut2 Resource, a Phylogenomically Derived Inventory of Proteins Specific to the Plant Lineage JOURNAL OF BIOLOGICAL CHEMISTRY Karpowicz, S. J., Prochnik, S. E., Grossman, A. R., Merchant, S. S. 2011; 286 (24): 21427-21439

    Abstract

    The plastid is a defining structure of photosynthetic eukaryotes and houses many plant-specific processes, including the light reactions, carbon fixation, pigment synthesis, and other primary metabolic processes. Identifying proteins associated with catalytic, structural, and regulatory functions that are unique to plastid-containing organisms is necessary to fully define the scope of plant biochemistry. Here, we performed phylogenomics on 20 genomes to compile a new inventory of 597 nucleus-encoded proteins conserved in plants and green algae but not in non-photosynthetic organisms. 286 of these proteins are of known function, whereas 311 are not characterized. This inventory was validated as applicable and relevant to diverse photosynthetic eukaryotes using an additional eight genomes from distantly related plants (including Micromonas, Selaginella, and soybean). Manual curation of the known proteins in the inventory established its importance to plastid biochemistry. To predict functions for the 52% of proteins of unknown function, we used sequence motifs, subcellular localization, co-expression analysis, and RNA abundance data. We demonstrate that 18% of the proteins in the inventory have functions outside the plastid and/or beyond green tissues. Although 32% of proteins in the inventory have homologs in all cyanobacteria, unexpectedly, 30% are eukaryote-specific. Finally, 8% of the proteins of unknown function share no similarity to any characterized protein and are plant lineage-specific. We present this annotated inventory of 597 proteins as a resource for functional analyses of plant-specific biochemistry.

    View details for DOI 10.1074/jbc.M111.233734

    View details for Web of Science ID 000291464700041

    View details for PubMedID 21515685

  • In situ dynamics of O-2, pH and cyanobacterial transcripts associated with CCM, photosynthesis and detoxification of ROS ISME JOURNAL Jensen, S. I., Steunou, A., Bhaya, D., Kuehl, M., Grossman, A. R. 2011; 5 (2): 317-328

    Abstract

    The relative abundance of transcripts encoding proteins involved in inorganic carbon concentrating mechanisms (CCM), detoxification of reactive oxygen species (ROS) and photosynthesis in the thermophilic cyanobacterium Synechococcus OS-B' was measured in hot spring microbial mats over two diel cycles, and was coupled with in situ determinations of incoming irradiance and microenvironmental dynamics of O(2) and pH. Fluctuations in pH and O(2) in the mats were largely driven by the diel cycle of solar irradiance, with a pH variation from ∼7.0 to ∼9.5, and O(2) levels ranging from anoxia to supersaturation during night and day, respectively. Levels of various transcripts from mat cyanobacteria revealed several patterns that correlated with incident irradiance, O(2) and pH within the mat matrix. Transcript abundances for most genes increased during the morning dark-light transition. Some transcripts remained at a near constant level throughout the light period, whereas others showed an additional increase in abundance as the mat underwent transition from low-to-high light (potentially reflecting changes in O(2) concentration and pH), followed by either a decreased abundance in the early afternoon, or a gradual decline during the early afternoon and into the evening. One specific transcipt, psbA1, was the lowest during mid-day under high irradiance and increased when the light levels declined. We discuss these complex in situ transcriptional patterns with respect to environmental and endogenous cues that might impact and regulate transcription over the diel cycle.

    View details for DOI 10.1038/ismej.2010.131

    View details for Web of Science ID 000290020000015

    View details for PubMedID 20740024

    View details for PubMedCentralID PMC3105686

  • Alternative pathways for phosphonate metabolism in thermophilic cyanobacteria from microbial mats ISME JOURNAL Gomez-Garcia, M. R., Davison, M., Blain-Hartnung, M., Grossman, A. R., Bhaya, D. 2011; 5 (1): 141-149

    Abstract

    Synechococcus sp. represents an ecologically diverse group of cyanobacteria found in numerous environments, including hot-spring microbial mats, where they are spatially distributed along thermal, light and oxygen gradients. These thermophiles engage in photosynthesis and aerobic respiration during the day, but switch to fermentative metabolism and nitrogen fixation at night. The genome of Synechococcus OS-B', isolated from Octopus Spring (Yellowstone National Park) contains a phn gene cluster encoding a phosphonate (Phn) transporter and a C-P lyase. A closely related isolate, Synechococcus OS-A, lacks this cluster, but contains genes encoding putative phosphonatases (Phnases) that appear to be active only in the presence of the Phn substrate. Both isolates grow well on several different Phns as a sole phosphorus (P) source. Interestingly, Synechococcus OS-B' can use the organic carbon backbones of Phns for heterotrophic growth in the dark, whereas in the light this strain releases organic carbon from Phn as ethane or methane (depending on the specific Phn available); Synechococcus OS-A has neither of these capabilities. These differences in metabolic strategies for assimilating the P and C of Phn by two closely related Synechococcus spp. are suggestive of niche-specific constraints in the evolution of nutrient assimilation pathways and syntrophic relationships among the microbial populations of the hot-spring mats. Thus, it is critical to evaluate levels of various P sources, including Phn, in thermally active habitats and the potential importance of these compounds in the biogeochemical cycling of P and C (some Phn compounds also contain N) in diverse terrestrial environments.

    View details for DOI 10.1038/ismej.2010.96

    View details for Web of Science ID 000285845200014

    View details for PubMedID 20631809

    View details for PubMedCentralID PMC3105666

  • Endosymbiotic Gene Transfer and Transcriptional Regulation of Transferred Genes in Paulinella chromatophora MOLECULAR BIOLOGY AND EVOLUTION Nowack, E. C., Vogel, H., Groth, M., Grossman, A. R., Melkonian, M., Gloeckner, G. 2011; 28 (1): 407-422

    Abstract

    Paulinella chromatophora is a cercozoan amoeba that contains "chromatophores," which are photosynthetic inclusions of cyanobacterial origin. The recent discovery that chromatophores evolved independently of plastids, underwent major genome reduction, and transferred at least two genes to the host nucleus has highlighted P. chromatophora as a model to infer early steps in the evolution of photosynthetic organelles. However, owing to the paucity of nuclear genome sequence data, the extent of endosymbiotic gene transfer (EGT) and host symbiont regulation are currently unknown. A combination of 454 and Illumina next generation sequencing enabled us to generate a comprehensive reference transcriptome data set for P. chromatophora on which we mapped short Illumina cDNA reads generated from cultures from the dark and light phases of a diel cycle. Combined with extensive phylogenetic analyses of the deduced protein sequences, these data revealed that 1) about 0.3-0.8% of the nuclear genes were obtained by EGT compared with 11-14% in the Plantae, 2) transferred genes show a distinct bias in that many encode small proteins involved in photosynthesis and photoacclimation, 3) host cells established control over expression of transferred genes, and 4) not only EGT, but to a minor extent also horizontal gene transfer from organisms that presumably served as food sources, helped to shape the nuclear genome of P. chromatophora. The identification of a significant number of transferred genes involved in photosynthesis and photoacclimation of thylakoid membranes as well as the observed transcriptional regulation of these genes strongly implies import of the encoded gene products into chromatophores, a feature previously thought to be restricted to canonical organelles. Thus, a possible mechanism by which P. chromatophora exerts control over the performance of its newly acquired photosynthetic organelle may involve controlling the expression of nuclear-encoded chromatophore-targeted regulatory components of the thylakoid membranes.

    View details for DOI 10.1093/molbev/msq209

    View details for Web of Science ID 000285418600043

    View details for PubMedID 20702568

  • Porphyra: a marine crop shaped by stress TRENDS IN PLANT SCIENCE Blouin, N. A., Brodie, J. A., Grossman, A. C., Xu, P., Brawley, S. H. 2011; 16 (1): 29-37

    Abstract

    The marine red alga Porphyra is an important marine crop, worth ∼US$1.3 billion per year. Cultivation research now includes farm ecology, breeding, strain conservation and new net-seeding technologies. The success of cultivation is due, in part, to the high stress tolerance of Porphyra. Many species of Porphyra lose 85-95% of their cellular water during the daytime low tide, when they are also exposed to high light and temperature stress. Antioxidant and mycosporine-like amino acid activities have been partially characterized in Porphyra, but, as we discuss here, the Porphyra umbilicalis genome project will further elucidate proteins associated with stress tolerance. Furthermore, phylogenomic and transcriptomic investigations of Porphyra sensu lato could elucidate tradeoffs made during physiological acclimation and factors associated with life-history evolution in this ancient lineage.

    View details for DOI 10.1016/j.tplants.2010.10.004

    View details for Web of Science ID 000286718200005

    View details for PubMedID 21067966

  • Responses of psbA, hli and ptox genes to changes in irradiance in marine Synechococcus and Prochlorococcus AQUATIC MICROBIAL ECOLOGY Berg, G. M., Shrager, J., van Dijken, G., Mills, M. M., Arrigo, K. R., Grossman, A. R. 2011; 65 (1): 1-14

    View details for DOI 10.3354/ame01528

    View details for Web of Science ID 000297117200001

  • Multiple facets of anoxic metabolism and hydrogen production in the unicellular green alga Chlamydomonas reinhardtii NEW PHYTOLOGIST Grossman, A. R., Catalanotti, C., Yang, W., Dubini, A., Magneschi, L., Subramanian, V., Posewitz, M. C., Seibert, M. 2011; 190 (2): 279-288

    Abstract

    Many microbes in the soil environment experience micro-oxic or anoxic conditions for much of the late afternoon and night, which inhibit or prevent respiratory metabolism. To sustain the production of energy and maintain vital cellular processes during the night, organisms have developed numerous pathways for fermentative metabolism. This review discusses fermentation pathways identified for the soil-dwelling model alga Chlamydomonas reinhardtii, its ability to produce molecular hydrogen under anoxic conditions through the activity of hydrogenases, and the molecular flexibility associated with fermentative metabolism that has only recently been revealed through the analysis of specific mutant strains.

    View details for DOI 10.1111/j.1469-8137.2010.03534.x

    View details for Web of Science ID 000288863000004

    View details for PubMedID 21563367

  • Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation PHOTOSYNTHESIS RESEARCH Grossman, A. R., Karpowicz, S. J., Heinnickel, M., Dewez, D., Hamel, B., Dent, R., Niyogi, K. K., Johnson, X., Alric, J., Wollman, F., Li, H., Merchant, S. S. 2010; 106 (1-2): 3-17

    Abstract

    Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.

    View details for DOI 10.1007/s11120-010-9555-7

    View details for Web of Science ID 000283516500002

    View details for PubMedID 20490922

  • Binding of Cysteine Synthase to the STAS Domain of Sulfate Transporter and Its Regulatory Consequences JOURNAL OF BIOLOGICAL CHEMISTRY Shibagaki, N., Grossman, A. R. 2010; 285 (32): 25094-25102

    Abstract

    The sulfate ion (SO(4)(2-)) is transported into plant root cells by SO(4)(2-) transporters and then mostly reduced to sulfide (S(2-)). The S(2-) is then bonded to O-acetylserine through the activity of cysteine synthase (O-acetylserine (thiol)lyase or OASTL) to form cysteine, the first organic molecule of the SO(4)(2-) assimilation pathway. Here, we show that a root plasma membrane SO(4)(2-) transporter of Arabidopsis, SULTR1;2, physically interacts with OASTL. The interaction was initially demonstrated using a yeast two-hybrid system and corroborated by both in vivo and in vitro binding assays. The domain of SULTR1;2 shown to be important for association with OASTL is called the STAS domain. This domain is at the C terminus of the transporter and extends from the plasma membrane into the cytoplasm. The functional relevance of the OASTL-STAS interaction was investigated using yeast mutant cells devoid of endogenous SO(4)(2-) uptake activity but co-expressing SULTR1;2 and OASTL. The analysis of SO(4)(2-) transport in these cells suggests that the binding of OASTL to the STAS domain in this heterologous system negatively impacts transporter activity. In contrast, the activity of purified OASTL measured in vitro was enhanced by co-incubation with the STAS domain of SULTR1;2 but not with the analogous domain of the SO(4)(2-) transporter isoform SULTR1;1, even though the SULTR1;1 STAS peptide also interacts with OASTL based on the yeast two-hybrid system and in vitro binding assays. These observations suggest a regulatory model in which interactions between SULTR1;2 and OASTL coordinate internalization of SO(4)(2-) with the energetic/metabolic state of plant root cells.

    View details for DOI 10.1074/jbc.M110.126888

    View details for Web of Science ID 000280542100082

    View details for PubMedID 20529854

  • Identification and Regulation of Plasma Membrane Sulfate Transporters in Chlamydomonas PLANT PHYSIOLOGY Pootakham, W., Gonzalez-Ballester, D., Grossman, A. R. 2010; 153 (4): 1653-1668

    Abstract

    Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO(4)(2-)). Aspects of SO(4)(2-) transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO(4)(2-) transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO(4)(2-) transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO(4)(2-) transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO(4)(2-) into S-deprived cells.

    View details for DOI 10.1104/pp.110.157875

    View details for Web of Science ID 000280566000018

    View details for PubMedID 20498339

    View details for PubMedCentralID PMC2923900

  • A PERSPECTIVE ON PHOTOSYNTHESIS IN THE OLIGOTROPHIC OCEANS: HYPOTHESES CONCERNING ALTERNATE ROUTES OF ELECTRON FLOW1 JOURNAL OF PHYCOLOGY Grossman, A. R., Mackey, K. R., Bailey, S. 2010; 46 (4): 629-634
  • RNA-Seq Analysis of Sulfur-Deprived Chlamydomonas Cells Reveals Aspects of Acclimation Critical for Cell Survival PLANT CELL Gonzalez-Ballester, D., Casero, D., Cokus, S., Pellegrini, M., Merchant, S. S., Grossman, A. R. 2010; 22 (6): 2058-2084

    Abstract

    The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival.

    View details for DOI 10.1105/tpc.109.071167

    View details for Web of Science ID 000280505300029

    View details for PubMedID 20587772

  • Direct Extraction of Photosynthetic Electrons from Single Algal Cells by Nanoprobing System NANO LETTERS Ryu, W., Bai, S., Park, J. S., Huang, Z., Moseley, J., Fabian, T., Fasching, R. J., Grossman, A. R., Prinz, F. B. 2010; 10 (4): 1137-1143

    Abstract

    There are numerous sources of bioenergy that are generated by photosynthetic processes, for example, lipids, alcohols, hydrogen, and polysaccharides. However, generally only a small fraction of solar energy absorbed by photosynthetic organisms is converted to a form of energy that can be readily exploited. To more efficiently use the solar energy harvested by photosynthetic organisms, we evaluated the feasibility of generating bioelectricity by directly extracting electrons from the photosynthetic electron transport chain before they are used to fix CO(2) into sugars and polysaccharides. From a living algal cell, Chlamydomonas reinhardtii, photosynthetic electrons (1.2 pA at 6000 mA/m(2)) were directly extracted without a mediator electron carrier by inserting a nanoelectrode into the algal chloroplast and applying an overvoltage. This result may represent an initial step in generating "high efficiency" bioelectricity by directly harvesting high energy photosynthetic electrons.

    View details for DOI 10.1021/nl903141j

    View details for Web of Science ID 000276557100007

    View details for PubMedID 20201533

  • An ancient light-harvesting protein is critical for the regulation of algal photosynthesis NATURE Peers, G., Truong, T. B., Ostendorf, E., Busch, A., Elrad, D., Grossman, A. R., Hippler, M., Niyogi, K. K. 2009; 462 (7272): 518-U215

    Abstract

    Light is necessary for photosynthesis, but its absorption by pigment molecules such as chlorophyll can cause severe oxidative damage and result in cell death. The excess absorption of light energy by photosynthetic pigments has led to the evolution of protective mechanisms that operate on the timescale of seconds to minutes and involve feedback-regulated de-excitation of chlorophyll molecules in photosystem II (qE). Despite the significant contribution of eukaryotic algae to global primary production, little is known about their qE mechanism, in contrast to that in flowering plants. Here we show that a qE-deficient mutant of the unicellular green alga Chlamydomonas reinhardtii, npq4, lacks two of the three genes encoding LHCSR (formerly called LI818). This protein is an ancient member of the light-harvesting complex superfamily, and orthologues are found throughout photosynthetic eukaryote taxa, except in red algae and vascular plants. The qE capacity of Chlamydomonas is dependent on environmental conditions and is inducible by growth under high light conditions. We show that the fitness of the npq4 mutant in a shifting light environment is reduced compared to wild-type cells, demonstrating that LHCSR is required for survival in a dynamic light environment. Thus, these data indicate that plants and algae use different proteins to dissipate harmful excess light energy and protect the photosynthetic apparatus from damage.

    View details for DOI 10.1038/nature08587

    View details for Web of Science ID 000272144200047

    View details for PubMedID 19940928

  • Picophytoplankton responses to changing nutrient and light regimes during a bloom MARINE BIOLOGY Mackey, K. R., Rivlin, T., Grossman, A. R., Post, A. F., Paytan, A. 2009; 156 (8): 1531-1546
  • Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity JOURNAL OF BIOLOGICAL CHEMISTRY Dubini, A., Mus, F., Seibert, M., Grossman, A. R., Posewitz, M. C. 2009; 284 (11): 7201-7213

    Abstract

    The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O2. Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malate-forming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.

    View details for DOI 10.1074/jbc.M803917200

    View details for Web of Science ID 000263919000067

    View details for PubMedID 19117946

  • Genetic Interactions Between Regulators of Chlamydomonas Phosphorus and Sulfur Deprivation Responses GENETICS Moseley, J. L., Gonzalez-Ballester, D., Pootakham, W., Bailey, S., Grossman, A. R. 2009; 181 (3): 889-905

    Abstract

    The Chlamydomonas reinhardtii PSR1 gene is required for proper acclimation of the cells to phosphorus (P) deficiency. P-starved psr1 mutants show signs of secondary sulfur (S) starvation, exemplified by the synthesis of extracellular arylsulfatase and the accumulation of transcripts encoding proteins involved in S scavenging and assimilation. Epistasis analysis reveals that induction of the S-starvation responses in P-limited psr1 cells requires the regulatory protein kinase SNRK2.1, but bypasses the membrane-targeted activator, SAC1. The inhibitory kinase SNRK2.2 is necessary for repression of S-starvation responses during both nutrient-replete growth and P limitation; arylsulfatase activity and S deficiency-responsive genes are partially induced in the P-deficient snrk2.2 mutants and become fully activated in the P-deficient psr1snrk2.2 double mutant. During P starvation, the sac1snrk2.2 double mutants or the psr1sac1snrk2.2 triple mutants exhibit reduced arylsulfatase activity compared to snrk2.2 or psr1snrk2.2, respectively, but the sac1 mutation has little effect on the abundance of S deficiency-responsive transcripts in these strains, suggesting a post-transcriptional role for SAC1 in elicitation of S-starvation responses. Interestingly, P-starved psr1snrk2.2 cells bleach and die more rapidly than wild-type or psr1 strains, suggesting that activation of S-starvation responses during P deprivation is deleterious to the cell. From these results we infer that (i) P-deficient growth causes some internal S limitation, but the S-deficiency responses are normally inhibited during acclimation to P deprivation; (ii) the S-deficiency responses are not completely suppressed in P-deficient psr1 cells and consequently these cells synthesize some arylsulfatase and exhibit elevated levels of transcripts for S-deprivation genes; and (iii) this increased expression is controlled by regulators that modulate transcription of S-responsive genes during S-deprivation conditions. Overall, the work strongly suggests integration of the different circuits that control nutrient-deprivation responses in Chlamydomonas.

    View details for DOI 10.1534/genetics.108.099382

    View details for Web of Science ID 000270213500010

    View details for PubMedID 19087952

  • Ancient Recruitment by Chromists of Green Algal Genes Encoding Enzymes for Carotenoid Biosynthesis MOLECULAR BIOLOGY AND EVOLUTION Frommolt, R., Werner, S., Paulsen, H., Goss, R., Wilhelm, C., Zauner, S., Maier, U. G., Grossman, A. R., Bhattacharya, D., Lohr, M. 2008; 25 (12): 2653-2667

    Abstract

    Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-encoded chromist proteins should be most closely related to their red algal homologs. Here we analyzed the biosynthetic pathway of carotenoids that are essential components of all photosynthetic eukaryotes and find a mosaic evolutionary origin of these enzymes in chromists. Surprisingly, about one-third (5/16) of the proteins are most closely related to green algal homologs with three branching within or sister to the early-diverging Prasinophyceae. This phylogenetic association is corroborated by shared diagnostic indels and the syntenic arrangement of a specific gene pair involved in the photoprotective xanthophyll cycle. The combined data suggest that the prasinophyte genes may have been acquired before the ancient split of stramenopiles, haptophytes, cryptophytes, and putatively also dinoflagellates. The latter point is supported by the observed monophyly of alveolates and stramenopiles in most molecular trees. One possible explanation for our results is that the green genes are remnants of a cryptic endosymbiosis that occurred early in chromalveolate evolution; that is, prior to the postulated split of stramenopiles, alveolates, haptophytes, and cryptophytes. The subsequent red algal capture would have led to the loss or replacement of most green genes via intracellular gene transfer from the new endosymbiont. We argue that the prasinophyte genes were retained because they enhance photosynthetic performance in chromalveolates, thus extending the niches available to these organisms. The alternate explanation of green gene origin via serial endosymbiotic or horizontal gene transfers is also plausible, but the latter would require the independent origins of the same five genes in some or all the different chromalveolate lineages.

    View details for DOI 10.1093/molbev/msn206

    View details for Web of Science ID 000260977300015

    View details for PubMedID 18799712

  • Phosphorus Deprivation Responses and Phosphonate Utilization in a Thermophilic Synechococcus sp from Microbial Mats JOURNAL OF BACTERIOLOGY Adams, M. M., Gomez-Garcia, M. R., Grossman, A. R., Bhaya, D. 2008; 190 (24): 8171-8184

    Abstract

    The genomes of two closely related thermophilic cyanobacterial isolates, designated Synechococcus isolate OS-A and Synechococcus isolate OS-B', from the microbial mats of Octopus Spring (Yellowstone National Park) have been sequenced. An extensive suite of genes that are controlled by phosphate levels constitute the putative Pho regulon in these cyanobacteria. We examined physiological responses of an axenic OS-B' isolate as well as transcript abundances of Pho regulon genes as the cells acclimated to phosphorus-limiting conditions. Upon imposition of phosphorus deprivation, OS-B' stopped dividing after three to four doublings, and absorbance spectra measurements indicated that the cells had lost most of their phycobiliproteins and chlorophyll a. Alkaline phosphatase activity peaked and remained high after 48 h of phosphorus starvation, and there was an accumulation of transcripts from putative Pho regulon genes. Interestingly, the genome of Synechococcus isolate OS-B' harbors a cluster of phn genes that are not present in OS-A isolates. The proteins encoded by the phn genes function in the transport and metabolism of phosphonates, which could serve as an alternative phosphorus source when exogenous phosphate is low. The phn genes were upregulated within a day of eliminating the source of phosphate from the medium. However, the ability of OS-B' to utilize methylphosphonate as a sole phosphorus source occurred only after an extensive period of exposure to the substrate. Once acclimated, the cells grew rapidly in fresh medium with methylphosphonate as the only source of phosphorus. The possible implications of these results are discussed with respect to the ecophysiology of the microbial mats.

    View details for DOI 10.1128/JB.01011-08

    View details for Web of Science ID 000261217900036

    View details for PubMedID 18931115

    View details for PubMedCentralID PMC2593230

  • Photoprotection in Cyanobacteria: Regulation of Light Harvesting PHOTOCHEMISTRY AND PHOTOBIOLOGY Bailey, S., Grossman, A. 2008; 84 (6): 1410-1420

    Abstract

    To cope with a rapidly fluctuating light environment, vascular plants and algae have evolved a photoprotective mechanism that serves to downregulate the transfer of excitation energy in the light-harvesting complexes to the photosynthetic reaction centers. This process dissipates excess excitation energy in the chlorophyll pigment bed by a nonradiative pathway. Since this pathway competes with and therefore quenches chlorophyll fluoresence in a nonphotochemical manner, it has been termed Non-photochemical Quenching (NPQ). For many years, cyanobacteria were not considered capable of performing NPQ as a photoprotective mechanism. Instead, the redistribution of the phycobilisome (PBS) light-harvesting antenna between reaction centers by a process called state transitions was considered the major means of regulating the utilization of harvested light energy. Recently, it was demonstrated that cyanobacteria are able to use NPQ as one component of their photoprotective strategies. Cyanobacteria exhibit significant NPQ during nutrient-replete growth, but it becomes a more prominent means of managing absorbed excitation energy when the cells experience iron starvation. Rapid progress in understanding the molecular mechanism of cyanobacterial NPQ has revealed a process that is very distinct from the functionally analogous process in plants and algae. Cyanobacterial NPQ involves the absorption of blue light by a carotenoid binding protein, termed the Orange Carotenoid Protein, and most likely involves quenching in the PBS core. In this study, we summarize work leading to the discovery of NPQ in cyanobacteria and the elucidation of molecular mechanisms associated with this important photoprotective process.

    View details for DOI 10.1111/j.1751-1097.2008.00453.x

    View details for Web of Science ID 000261081200016

    View details for PubMedID 19067963

  • Understanding nitrogen limitation in Aureococcus anophagefferens (Pelagophyceae) through cDNA and qRT-PCR analysis JOURNAL OF PHYCOLOGY Berg, G. M., Shrager, J., Gloeckner, G., Arrigo, K. R., Grossman, A. R. 2008; 44 (5): 1235-1249

    Abstract

    Brown tides of the marine pelagophyte Aureococcus anophagefferens Hargraves et Sieburth have been investigated extensively for the past two decades. Its growth is fueled by a variety of nitrogen (N) compounds, with dissolved organic nitrogen (DON) being particularly important during blooms. Characterization of a cDNA library suggests that A. anophagefferens can assimilate eight different forms of N. Expression of genes related to the sensing, uptake, and assimilation of inorganic and organic N, as well as the catabolic process of autophagy, was assayed in cells grown on different N sources and in N-limited cells. Growth on nitrate elicited an increase in the relative expression of nitrate and ammonium transporters, a nutrient stress-induced transporter, and a sensory kinase. Growth on urea increased the relative expression of a urea and a formate/nitrite transporter, while growth on ammonium resulted in an increase in the relative expression of an ammonium transporter, a novel ATP-binding cassette (ABC) transporter and a putative high-affinity phosphate transporter. N limitation resulted in a 30- to 110-fold increase in the relative expression of nitrate, ammonium, urea, amino acid/polyamine, and formate/nitrite transporters. A. anophagefferens demonstrated the highest relative accumulation of a transcript encoding a novel purine transporter, which was highly expressed across all N sources. This finding suggests that purines are an important source of N for the growth of this organism and could possibly contribute to the initiation and maintenance of blooms in the natural environment.

    View details for DOI 10.1111/j.1529-8817.2008.00571.x

    View details for Web of Science ID 000259866800015

  • UNDERSTANDING NITROGEN LIMITATION IN AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE) THROUGH cDNA AND qRT-PCR ANALYSIS(1). Journal of phycology Berg, G. M., Shrager, J., Glöckner, G., Arrigo, K. R., Grossman, A. R. 2008; 44 (5): 1235-1249

    Abstract

    Brown tides of the marine pelagophyte Aureococcus anophagefferens Hargraves et Sieburth have been investigated extensively for the past two decades. Its growth is fueled by a variety of nitrogen (N) compounds, with dissolved organic nitrogen (DON) being particularly important during blooms. Characterization of a cDNA library suggests that A. anophagefferens can assimilate eight different forms of N. Expression of genes related to the sensing, uptake, and assimilation of inorganic and organic N, as well as the catabolic process of autophagy, was assayed in cells grown on different N sources and in N-limited cells. Growth on nitrate elicited an increase in the relative expression of nitrate and ammonium transporters, a nutrient stress-induced transporter, and a sensory kinase. Growth on urea increased the relative expression of a urea and a formate/nitrite transporter, while growth on ammonium resulted in an increase in the relative expression of an ammonium transporter, a novel ATP-binding cassette (ABC) transporter and a putative high-affinity phosphate transporter. N limitation resulted in a 30- to 110-fold increase in the relative expression of nitrate, ammonium, urea, amino acid/polyamine, and formate/nitrite transporters. A. anophagefferens demonstrated the highest relative accumulation of a transcript encoding a novel purine transporter, which was highly expressed across all N sources. This finding suggests that purines are an important source of N for the growth of this organism and could possibly contribute to the initiation and maintenance of blooms in the natural environment.

    View details for DOI 10.1111/j.1529-8817.2008.00571.x

    View details for PubMedID 27041720

  • Open micro-fluidic system for atomic force microscopy-guided in situ electrochemical probing of a single cell LAB ON A CHIP Ryu, W., Huang, Z., Park, J. S., Moseley, J., Grossman, A. R., Fasching, R. J., Prinz, F. B. 2008; 8 (9): 1460-1467

    Abstract

    Ultra-sharp nano-probes and customized atomic force microscopy (AFM) have previously been developed in our laboratory for in situ sub-cellular probing of electrochemical phenomena in living plant cells during their photosynthesis. However, this AFM-based electrochemical probing still has numerous engineering challenges such as immobilization of the live cells, compatibility of the immobilization procedure with AFM manipulation of the probe, maintenance of biological activity of the cells for an extended time while performing the measurements, and minimization of electrochemical noise. Thus, we have developed an open micro-fluidic channel system (OMFC) in which individual cells can be immobilized in micro-traps by capillary flow. This system affords easy AFM access and allows for maintenance of the cells in a well-defined chemical environment, which sustains their biological activity. The use of micro-channels for making the electrochemical measurements significantly reduces parasitic electrical capacitances and allows for current detection in the sub-pico-ampere range at high signal bandwidths. The OMFC was further studied using simulation packages for optimal design conditions. This system was successfully used to measure light-dependent oxidation currents of a few pico-amperes from the green alga Chlamydomonas reinhardtii.

    View details for DOI 10.1039/b803450h

    View details for Web of Science ID 000259676000007

    View details for PubMedID 18818800

  • An original adaptation of photosynthesis in the marine green alga Ostreococcus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cardol, P., Bailleul, B., Rappaport, F., Derelle, E., Beal, D., Breyton, C., Bailey, S., Wollman, F. A., Grossman, A., Moreau, H., Finazzi, G. 2008; 105 (22): 7881-7886

    Abstract

    Adaptation of photosynthesis in marine environment has been examined in two strains of the green, picoeukaryote Ostreococcus: OTH95, a surface/high-light strain, and RCC809, a deep-sea/low-light strain. Differences between the two strains include changes in the light-harvesting capacity, which is lower in OTH95, and in the photoprotection capacity, which is enhanced in OTH95. Furthermore, RCC809 has a reduced maximum rate of O(2) evolution, which is limited by its decreased photosystem I (PSI) level, a possible adaptation to Fe limitation in the open oceans. This decrease is, however, accompanied by a substantial rerouting of the electron flow to establish an H(2)O-to-H(2)O cycle, involving PSII and a potential plastid plastoquinol terminal oxidase. This pathway bypasses electron transfer through the cytochrome b(6)f complex and allows the pumping of "extra" protons into the thylakoid lumen. By promoting the generation of a large DeltapH, it facilitates ATP synthesis and nonphotochemical quenching when RCC809 cells are exposed to excess excitation energy. We propose that the diversion of electrons to oxygen downstream of PSII, but before PSI, reflects a common and compulsory strategy in marine phytoplankton to bypass the constraints imposed by light and/or nutrient limitation and allow successful colonization of the open-ocean marine environment.

    View details for DOI 10.1072/pnas.0802762105

    View details for Web of Science ID 000256648600046

    View details for PubMedID 18511560

  • The central role of a SNRK2 kinase in sulfur deprivation responses PLANT PHYSIOLOGY Gonzalez-Ballester, D., Pollock, S. V., Pootakham, W., Grossman, A. R. 2008; 147 (1): 216-227

    Abstract

    In the absence of sulfur (S), Chlamydomonas reinhardtii increases the abundance of several transcripts encoding proteins associated with S acquisition and assimilation, conserves S amino acids, and acclimates to suboptimal growth conditions. A positive regulator, SAC1 (for sulfur acclimation protein 1), and a negative regulator, SAC3, were shown to participate in the control of these processes. In this study, we investigated two allelic mutants (ars11 and ars44) affected in a gene encoding a SNRK2 (for SNF1-related protein kinase 2) kinase designated SNRK2.1. Like the sac1 mutant, both snrk2.1 mutants were deficient in the expression of S-responsive genes. Furthermore, the mutant cells bleached more rapidly than wild-type cells during S deprivation, although the phenotypes of ars11 and ars44 were not identical: ars11 exhibited a more severe phenotype than either ars44 or sac1. The phenotypic differences between the ars11 and ars44 mutants reflected distinct alterations of SNRK2.1 mRNA splicing caused by insertion of the marker gene. The ars11 phenotype could be rescued by complementation with SNRK2.1 cDNA. In contrast to the nonepistatic relationship between SAC3 and SAC1, characterization of the sac3 ars11 double mutant showed that SNRK2.1 is epistatic to SAC3. These data reveal the crucial regulatory role of SNRK2.1 in the signaling cascade critical for eliciting S deprivation responses in Chlamydomonas. The phylogenetic relationships and structures of the eight members of the SNRK2 family in Chlamydomonas are discussed.

    View details for DOI 10.1104/pp.108.116137

    View details for Web of Science ID 000256419400021

    View details for PubMedID 18326790

  • A photosynthetic strategy for coping in a high-light, low-nutrient environment LIMNOLOGY AND OCEANOGRAPHY Mackey, K. R., Paytan, A., Grossman, A. R., Bailey, S. 2008; 53 (3): 900-913
  • Regulation of nif gene expression and the energetics of N-2 fixation over the diel cycle in a hot spring microbial mat ISME JOURNAL Steunou, A., Jensen, S. I., Brecht, E., Becraft, E. D., Bateson, M. M., Kilian, O., Bhaya, D., Ward, D. M., Peters, J. W., Grossman, A. R., Kuhl, M. 2008; 2 (4): 364-378

    Abstract

    Nitrogen fixation, a prokaryotic, O2-inhibited process that reduces N2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O2 consumption by respiration still exceeded the rate of O2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N2 fixation in hot spring mats and factors that can markedly influence the extent of N2 fixation over the diel cycle.

    View details for DOI 10.1038/ismej.2007.117

    View details for Web of Science ID 000255288700003

    View details for PubMedID 18323780

  • Alternative photosynthetic electron flow to oxygen in marine Synechococcus BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS Bailey, S., Melis, A., Mackey, K. R., Cardol, P., Finazzi, G., van Dijken, G., Berg, G. M., Arrigo, K., Shrager, J., Grossman, A. 2008; 1777 (3): 269-276

    Abstract

    Cyanobacteria dominate the world's oceans where iron is often barely detectable. One manifestation of low iron adaptation in the oligotrophic marine environment is a decrease in levels of iron-rich photosynthetic components, including the reaction center of photosystem I and the cytochrome b6f complex [R.F. Strzepek and P.J. Harrison, Photosynthetic architecture differs in coastal and oceanic diatoms, Nature 431 (2004) 689-692.]. These thylakoid membrane components have well characterised roles in linear and cyclic photosynthetic electron transport and their low abundance creates potential impediments to photosynthetic function. Here we show that the marine cyanobacterium Synechococcus WH8102 exhibits significant alternative electron flow to O2, a potential adaptation to the low iron environment in oligotrophic oceans. This alternative electron flow appears to extract electrons from the intersystem electron transport chain, prior to photosystem I. Inhibitor studies demonstrate that a propyl gallate-sensitive oxidase mediates this flow of electrons to oxygen, which in turn alleviates excessive photosystem II excitation pressure that can often occur even at relatively low irradiance. These findings are also discussed in the context of satisfying the energetic requirements of the cell when photosystem I abundance is low.

    View details for DOI 10.1016/j.bbabio.2008.01.002

    View details for Web of Science ID 000254674600004

    View details for PubMedID 18241667

  • Genomics, environmental genomics and the issue of microbial species HEREDITY Ward, D. M., Cohan, F. M., Bhaya, D., Heidelberg, J. F., Kuhl, M., Grossman, A. 2008; 100 (2): 207-219

    Abstract

    A microbial species concept is crucial for interpreting the variation detected by genomics and environmental genomics among cultivated microorganisms and within natural microbial populations. Comparative genomic analyses of prokaryotic species as they are presently described and named have led to the provocative idea that prokaryotes may not form species as we think about them for plants and animals. There are good reasons to doubt whether presently recognized prokaryotic species are truly species. To achieve a better understanding of microbial species, we believe it is necessary to (i) re-evaluate traditional approaches in light of evolutionary and ecological theory, (ii) consider that different microbial species may have evolved in different ways and (iii) integrate genomic, metagenomic and genome-wide expression approaches with ecological and evolutionary theory. Here, we outline how we are using genomic methods to (i) identify ecologically distinct populations (ecotypes) predicted by theory to be species-like fundamental units of microbial communities, and (ii) test their species-like character through in situ distribution and gene expression studies. By comparing metagenomic sequences obtained from well-studied hot spring cyanobacterial mats with genomic sequences of two cultivated cyanobacterial ecotypes, closely related to predominant native populations, we can conduct in situ population genetics studies that identify putative ecotypes and functional genes that determine the ecotypes' ecological distinctness. If individuals within microbial communities are found to be grouped into ecologically distinct, species-like populations, knowing about such populations should guide us to a better understanding of how genomic variation is linked to community function.

    View details for DOI 10.1038/sj.hdy.6801011

    View details for Web of Science ID 000252585800014

    View details for PubMedID 17551524

  • A novel two domain-fusion protein in cyanobacteria with similarity to the CAB/ELIP/HLIP superfamily: Evolutionary implications and regulation MOLECULAR PLANT Kilian, O., Steunou, A. S., Grossman, A. R., Bhaya, D. 2008; 1 (1): 155-166

    Abstract

    Vascular plants contain abundant, light-harvesting complexes in the thylakoid membrane that are non-covalently associated with chlorophylls and carotenoids. These light-harvesting chlorophyll a/b binding (LHC) proteins are members of an extended CAB/ELIP/HLIP superfamily of distantly related polypeptides, which have between one and four transmembrane helices (TMH). This superfamily includes the single TMH, high-light-inducible proteins (Hlips), found in cyanobacteria that are induced by various stress conditions, including high light, and are considered ancestral to the LHC proteins. The roles of, and evolutionary relationships between, these superfamily members are of particular interest, since they function in both light harvesting and photoprotection and may have evolved through tandem gene duplication and fusion events. We have investigated the Hlips (hli gene family) in the thermophilic unicellular cyanobacterium Synechococcus OS-B'. The five hli genes present on the genome of Synechococcus OS-B' are relatively similar, but transcript analyses indicate that there are different patterns of transcript accumulation when the cells are exposed to various growth conditions, suggesting that different Hlips may have specific functions. Hlip5 has an additional TMH at the N-terminus as a result of a novel fusion event. This additional TMH is very similar to a conserved hypothetical, single membrane-spanning polypeptide present in most cyanobacteria. The evolutionary significance of these results is discussed.

    View details for DOI 10.1093/mp/ssm019

    View details for Web of Science ID 000259068900015

    View details for PubMedID 20031922

  • Reversible oxidation of spinach ferredoxin at surface-modified electrodes JOURNAL OF THE ELECTROCHEMICAL SOCIETY Komadina, J., Walch, S., Fasching, R., Grossman, A., Prinz, F. B. 2008; 155 (10): B1008-B1012

    View details for DOI 10.1149/1.2962768

    View details for Web of Science ID 000258976500020

  • Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses ISME JOURNAL Bhaya, D., Grossman, A. R., Steunou, A., Khuri, N., Cohan, F. M., Hamamura, N., Melendrez, M. C., Bateson, M. M., Ward, D. M., Heidelberg, J. F. 2007; 1 (8): 703-713

    Abstract

    In microbial mat communities of Yellowstone hot springs, ribosomal RNA (rRNA) sequence diversity patterns indicate the presence of closely related bacterial populations along environmental gradients of temperature and light. To identify the functional bases for adaptation, we sequenced the genomes of two cyanobacterial (Synechococcus OS-A and OS-B') isolates representing ecologically distinct populations that dominate at different temperatures and are major primary producers in the mat. There was a marked lack of conserved large-scale gene order between the two Synechococcus genomes, indicative of extensive genomic rearrangements. Comparative genomic analyses showed that the isolates shared a large fraction of their gene content at high identity, yet, differences in phosphate and nitrogen utilization pathways indicated that they have adapted differentially to nutrient fluxes, possibly by the acquisition of genes by lateral gene transfer or their loss in certain populations. Comparisons of the Synechococcus genomes to metagenomic sequences derived from mats where these Synechococcus stains were originally isolated, revealed new facets of microbial diversity. First, Synechococcus populations at the lower temperature regions of the mat showed greater sequence diversity than those at high temperatures, consistent with a greater number of ecologically distinct populations at the lower temperature. Second, we found evidence of a specialized population that is apparently very closely related to Synechococcus OS-B', but contains genes that function in the uptake of reduced ferrous iron. In situ expression studies demonstrated that these genes are differentially expressed over the diel cycle, with highest expression when the mats are anoxic and iron may be in the reduced state. Genomic information from these mat-specific isolates and metagenomic information can be coupled to detect naturally occurring populations that are associated with different functionalities, not always represented by isolates, but which may nevertheless be important for niche partitioning and the establishment of microbial community structure.

    View details for DOI 10.1038/ismej.2007.46

    View details for Web of Science ID 000251946500004

    View details for PubMedID 18059494

  • The Chlamydomonas genome reveals the evolution of key animal and plant functions SCIENCE Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., Terry, A., Salamov, A., Fritz-Laylin, L. K., Marechal-Drouard, L., Marshall, W. F., Qu, L., Nelson, D. R., Sanderfoot, A. A., Spalding, M. H., Kapitonov, V. V., Ren, Q., Ferris, P., Lindquist, E., Shapiro, H., Lucas, S. M., Grimwood, J., Schmutz, J., Cardol, P., Cerutti, H., Chanfreau, G., Chen, C., Cognat, V., Croft, M. T., Dent, R., Dutcher, S., Fernandez, E., Fukuzawa, H., Gonzalez-Ballester, D., Gonzalez-Halphen, D., Hallmann, A., Hanikenne, M., Hippler, M., Inwood, W., Jabbari, K., Kalanon, M., Kuras, R., Lefebvre, P. A., Lemaire, S. D., Lobanov, A. V., Lohr, M., Manuell, A., Meier, I., Mets, L., Mittag, M., Mittelmeier, T., Moroney, J. V., Moseley, J., Napoli, C., Nedelcu, A. M., Niyogi, K., Novoselov, S. V., Paulsen, I. T., Pazour, G., Purton, S., Ral, J., Riano-Pachon, D. M., Riekhof, W., Rymarquis, L., Schroda, M., Stern, D., Umen, J., Willows, R., Wilson, N., Zimmer, S. L., Allmer, J., Balk, J., Bisova, K., Chen, C., Elias, M., Gendler, K., Hauser, C., Lamb, M. R., Ledford, H., Long, J. C., Minagawa, J., Page, M. D., Pan, J., Pootakham, W., Roje, S., Rose, A., Stahlberg, E., Terauchi, A. M., Yang, P., Ball, S., Bowler, C., Dieckmann, C. L., Gladyshev, V. N., Green, P., Jorgensen, R., Mayfield, S., Mueller-Roeber, B., Rajamani, S., Sayre, R. T., Brokstein, P., Dubchak, I., Goodstein, D., Hornick, L., Huang, Y. W., Jhaveri, J., Luo, Y., Martinez, D., Ngau, W. C., Otillar, B., Poliakov, A., Porter, A., Szajkowski, L., Werner, G., Zhou, K., Grigoriev, I. V., Rokhsar, D. S., Grossman, A. R. 2007; 318 (5848): 245-251

    Abstract

    Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

    View details for DOI 10.1126/science.1143609

    View details for Web of Science ID 000250086100040

    View details for PubMedID 17932292

    View details for PubMedCentralID PMC2875087

  • Anaerobic acclimation in Chlamydomonas reinhardtii - Anoxic gene expression, hydrogenase induction, and metabolic pathways JOURNAL OF BIOLOGICAL CHEMISTRY Mus, F., Dubini, A., Seibert, M., Posewitz, M. C., Grossman, A. R. 2007; 282 (35): 25475-25486

    Abstract

    Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production.

    View details for DOI 10.1074/jbc.M701415200

    View details for Web of Science ID 000249014100033

    View details for PubMedID 17565990

  • Responses of a thermophilic Synechococcus isolate from the microbial mat of octopus spring to light APPLIED AND ENVIRONMENTAL MICROBIOLOGY Kilian, O., Steunou, A., Fazeli, F., Bailey, S., Bhaya, D., Grossman, A. R. 2007; 73 (13): 4268-4278

    Abstract

    Thermophilic cyanobacteria of the genus Synechococcus are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park. Synechococcus OS-B' was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 micromol photons m(-2) s(-1) but dies when the irradiance is increased to 400 micromol photons m(-2) s(-1). At 200 micromol photons m(-2) s(-1) (high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid. Synechococcus OS-B' synthesizes the carotenoids zeaxanthin and beta,beta-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that Synechococcus OS-B' accumulates very small amounts of photosystem II relative to that of photosystem I. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (psbAI and psbAII/psbAIII, respectively) were also regulated according to the light regimen. The results are discussed in the context of how Synechococcus OS-B' may cope with high light irradiances in the high-temperature environment of the microbial mat.

    View details for DOI 10.1128/AEM.00201-07

    View details for Web of Science ID 000248070000023

    View details for PubMedID 17483258

    View details for PubMedCentralID PMC1932787

  • Novel metabolism in Chlamydomonas through the lens of genomics CURRENT OPINION IN PLANT BIOLOGY Grossman, A. R., Croft, M., Gladyshev, V. N., Merchant, S. S., Posewitz, M. C., Prochnik, S., Spalding, M. H. 2007; 10 (2): 190-198

    Abstract

    Chlamydomonas has traditionally been exploited as an organism that is associated with sophisticated physiological, genetic and molecular analyses, all of which have been used to elucidate several biological processes, especially photosynthesis and flagella function and assembly. Recently, the genomics of Chlamydomonas has been combined with other technologies to unveil new aspects of metabolism, including inorganic carbon utilization, anaerobic fermentation, the suite and functions of selenoproteins, and the regulation of vitamin biosynthesis. These initial findings represent the first glimpse through a genomic window onto the highly complex metabolisms that characterize a unicellular, photosynthetic eukaryote that has maintained both plant-like and animal-like characteristics over evolutionary time.

    View details for DOI 10.1016/j.pbi.2007.01.012

    View details for Web of Science ID 000245573100014

    View details for PubMedID 17291820

  • EST assembly supported by a draft genome sequence: an analysis of the Chlamydomonas reinhardtii transcriptome NUCLEIC ACIDS RESEARCH Jain, M., Shrager, J., Harris, E. H., Halbrook, R., Grossman, A. R., Hauser, C., Vallon, O. 2007; 35 (6): 2074-2083

    Abstract

    Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12,063 ACEGs (assembly of contiguous ESTs based on genome) and generated 15,857 contigs of average length 934 nt. We predict that roughly 3000 of our contigs represent full-length transcripts. Compared to previous assemblies, ACEGs show extended contig length, increased accuracy and a reduction in redundancy. Because our assembly protocol also uses ESTs with no corresponding genomic sequences, it provides sequence information for genes interrupted by sequence gaps. Detailed analysis of randomly sampled ACEGs reveals several hundred putative cases of alternative splicing, many overlapping transcription units and new genes not identified by gene prediction algorithms. Our protocol, although developed for and tailored to the C. reinhardtii dataset, can be exploited by any eukaryotic genome project for which both a draft genome sequence and ESTs are available.

    View details for DOI 10.1093/nar/gkm081

    View details for Web of Science ID 000246123600038

    View details for PubMedID 17355987

    View details for PubMedCentralID PMC1874618

  • A novel analytical method for in vivo phosphate tracking (vol 580, pg 5885, 2006) FEBS LETTERS Gu, H., Lalonde, S., Okumoto, S., Looger, L. L., Scharff-Poulsen, A. M., Grossman, A. R., Kossmann, J., Jakobsen, I., Frommer, W. B. 2007; 581 (3): 579-579
  • Counting low-copy number proteins in a single cell SCIENCE Huang, B., Wu, H., Bhaya, D., Grossman, A., Granier, S., Kobilka, B. K., Zare, R. N. 2007; 315 (5808): 81-84

    Abstract

    We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.

    View details for DOI 10.1126/science.1133992

    View details for Web of Science ID 000243259100039

    View details for PubMedID 17204646

  • In the grip of algal genomics TRANSGENIC MICROALGAE AS GREEN CELL FACTORIES Grossman, A. R. 2007; 616: 54-76

    Abstract

    Algae are dominant primary producers on the Earth and have a major impact on global productivity and biogeochemical cycling. There are still few algal genomes that have been completely characterized, and resources directed toward algal genomic sequencing are limited. However, it is also becoming evident that algae and prokaryotic picoplankton have a critical role in the fixation and sequestration of carbon, and so the interest in algal genomics is expanding. There are some algae for which full or near-full genome sequences have been secured; these genomes include those of the red alga Cyanidioschyzon merolae, the green algae or chlorophytes Chlamydomonas reinhardtii and Volvox carteri, the marine picoeukaryote Ostreococcus tauri (two different strains of O. tauri have been sequenced), the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum, and the haptophyte Emiliania huxleyi. There is also a full sequence for the vestigal 'red' algal genome of the nucleomorph of the Cyptomonad Guillardia theta. In addition, numerous genomes of photosynthetic microbes, including marine Synechococcus and Prochlorococcus species have been sequenced. There have also been projects developed to define algal transcriptomes as determined by cDNA analysis, full genome sequences of numerous plastids, and the genomes of a variety of viruses that infect marine and freshwater algae. The recent efforts focused on acquiring and analyzing algal genome sequences have generated an influx of exciting data to a field that is in its infancy. In this review I discuss potential criteria for determining which organisms should be targeted for genome projects, successful forays into algal genomic sequencing, and some of the inferences generated from the analysis of the sequence information.

    View details for Web of Science ID 000251745700006

    View details for PubMedID 18161491

  • AFM/EC nano probing of single cells and organelles 6th IEEE Sensors Conference Fasching, R., Ryu, W., Bai, S., Park, J., Fabian, T., Moseley, J., Grossman, A., Prinz, F. IEEE. 2007: 699–702
  • A novel analytical method for in vivo phosphate tracking FEBS LETTERS Gu, H., Lalonde, S., Okumoto, S., Looger, L. L., Scharff-Poulsen, A. M., Grossman, A. R., Kossmann, J., Jakobsen, I., Frommer, W. B. 2006; 580 (25): 5885-5893

    Abstract

    Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P(i)) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P(i)-dependent increases in FRET efficiency. FLIPPi affinity mutants cover P(i) changes over eight orders of magnitude. COS-7 cells co-expressing a low-affinity FLIPPi and a Na(+)/P(i) co-transporter exhibited FRET changes when perfused with 100 microM P(i), demonstrating concentrative P(i) uptake by PiT2. FLIPPi sensors are suitable for real-time monitoring of P(i) metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P(i) during cell migration.

    View details for DOI 10.1016/j.febslet.2006.09.048

    View details for Web of Science ID 000241707100014

    View details for PubMedID 17034793

  • Phototropin involvement in the expression of genes encoding chlorophyll and carotenoid biosynthesis enzymes and LHC apoproteins in Chlamydomonas reinhardtii PLANT JOURNAL Im, C., Eberhard, S., Huang, K., Beck, C. F., Grossman, A. R. 2006; 48 (1): 1-16

    Abstract

    Phototropin (PHOT) is a photoreceptor involved in a variety of blue-light-elicited physiological processes including phototropism, chloroplast movement and stomatal opening in plants. The work presented here tests whether PHOT is involved in expression of light-regulated genes in Chlamydomonas reinhardtii. When C. reinhardtii was transferred from the dark to very low-fluence rate white light, there was a substantial increase in the level of transcripts encoding glutamate-1-semialdehyde aminotransferase (GSAT), phytoene desaturase (PDS) and light-harvesting polypeptides (e.g. LHCBM6). Increased levels of these transcripts were also elicited by low-intensity blue light, and this blue-light stimulation was suppressed in three different RNAi strains that synthesize low levels of PHOT. The levels of GSAT and LHCBM6 transcripts also increased following exposure of algal cells to low-intensity red light (RL). The red-light-dependent increase in transcript abundance was not affected by the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, implying that the influence of RL on transcript accumulation was not controlled by cytoplasmic redox conditions, and that a red-light photoreceptor(s) may be involved in regulating the levels of transcripts from specific photosynthesis-related genes in C. reinhardtii. Interestingly, elevated GSAT and LHCBM6 transcript levels in RL were significantly reduced in the PHOT RNAi strains, which raises the possibility of co-action between blue and RL signaling pathways. Microarray experiments indicated that the levels of several transcripts for photosystem (PS) I and II polypeptides were also modulated by PHOT. These data suggest that, in C. reinhardtii, (i) PHOT is involved in blue-light-mediated changes in transcript accumulation, (ii) synchronization of the synthesis of chlorophylls (Chl), carotenoids, Chl-binding proteins and other components of the photosynthetic apparatus is achieved, at least in part, through PHOT-mediated signaling, and (iii) a red-light photoreceptor can also influence levels of certain transcripts associated with photosynthetic function, although its action requires normal levels of PHOT.

    View details for DOI 10.1111/j.1365-313X.2006.02852.x

    View details for Web of Science ID 000240440400001

    View details for PubMedID 16972865

  • The role of the STAS domain in the function and biogenesis of a sulfate transporter as probed by random mutagenesis JOURNAL OF BIOLOGICAL CHEMISTRY Shibagaki, N., Grossman, A. R. 2006; 281 (32): 22964-22973

    Abstract

    Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the beta-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second alpha-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasma membrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.

    View details for DOI 10.1074/jbc.M603462200

    View details for Web of Science ID 000239542600058

    View details for PubMedID 16754669

  • Examination of diel changes in global transcript accumulation in Synechocystis (cyanobacteria) JOURNAL OF PHYCOLOGY Labiosa, R. G., Arrigo, K. R., Tu, C. J., Bhaya, D., Bay, S., Grossman, A. R., Shrager, J. 2006; 42 (3): 622-636
  • In situ analysis of nitrogen fixation and metabolic switching in unicellular thermophilic cyanobacteria inhabiting hot spring microbial mats PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Steunou, A. S., Bhaya, D., Bateson, M. M., Melendrez, M. C., Ward, D. M., Brecht, E., Peters, J. W., Kuhl, M., Grossman, A. R. 2006; 103 (7): 2398-2403

    Abstract

    Genome sequences of two Synechococcus ecotypes inhabiting the Octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C); transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at >50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (ldh, pdhB, ald, and ackA) decreased in the evening, whereas others (pflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.

    View details for DOI 10.1073/pnas.0507513103

    View details for Web of Science ID 000235411600071

    View details for PubMedID 16467157

    View details for PubMedCentralID PMC1413695

  • Generation of an oligonucleotide array for analysis of gene expression in Chlamydomonas reinhardtii CURRENT GENETICS Eberhard, S., Jain, M., Im, C. S., Pollock, S., Shrager, J., Lin, Y. A., Peek, A. S., Grossman, A. R. 2006; 49 (2): 106-124

    Abstract

    The availability of genome sequences makes it possible to develop microarrays that can be used for profiling gene expression over developmental time, as organisms respond to environmental challenges, and for comparison between wild-type and mutant strains under various conditions. The desired characteristics of microarrays (intense signals, hybridization specificity and extensive coverage of the transcriptome) were not fully met by the previous Chlamydomonas reinhardtii microarray: probes derived from cDNA sequences (approximately 300 bp) were prone to some nonspecific cross-hybridization and coverage of the transcriptome was only approximately 20%. The near completion of the C. reinhardtii nuclear genome sequence and the availability of extensive cDNA information have made it feasible to improve upon these aspects. After developing a protocol for selecting a high-quality unigene set representing all known expressed sequences, oligonucleotides were designed and a microarray with approximately 10,000 unique array elements (approximately 70 bp) covering 87% of the known transcriptome was developed. This microarray will enable researchers to generate a global view of gene expression in C. reinhardtii. Furthermore, the detailed description of the protocol for selecting a unigene set and the design of oligonucleotides may be of interest for laboratories interested in developing microarrays for organisms whose genome sequences are not yet completed (but are nearing completion).

    View details for DOI 10.1007/s00294-005-0041-2

    View details for Web of Science ID 000234906200004

    View details for PubMedID 16333659

  • Regeneration of a cell from protoplasm JOURNAL OF PHYCOLOGY Grossman, A. 2006; 42 (1): 1-5
  • Genome-based approaches to understanding phosphorus deprivation responses and PSR1 control in Chlamydomonas reinhardtii EUKARYOTIC CELL Moseley, J. L., Chang, C. W., Grossman, A. R. 2006; 5 (1): 26-44

    Abstract

    The Chlamydomonas reinhardtii transcription factor PSR1 is required for the control of activities involved in scavenging phosphate from the environment during periods of phosphorus limitation. Increased scavenging activity reflects the development of high-affinity phosphate transport and the expression of extracellular phosphatases that can cleave phosphate from organic compounds in the environment. A comparison of gene expression patterns using microarray analyses and quantitative PCRs with wild-type and psr1 mutant cells deprived of phosphorus has revealed that PSR1 also controls genes encoding proteins with potential "electron valve" functions--these proteins can serve as alternative electron acceptors that help prevent photodamage caused by overexcitation of the photosynthetic electron transport system. In accordance with this finding, phosphorus-starved psr1 mutants die when subjected to elevated light intensities; at these intensities, the wild-type cells still exhibit rapid growth. Acclimation to phosphorus deprivation also involves a reduction in the levels of transcripts encoding proteins involved in photosynthesis and both cytoplasmic and chloroplast translation as well as an increase in the levels of transcripts encoding stress-associated chaperones and proteases. Surprisingly, phosphorus-deficient psr1 cells (but not wild-type cells) also display expression patterns associated with specific responses to sulfur deprivation, suggesting a hitherto unsuspected link between the signal transduction pathways involved in controlling phosphorus and sulfur starvation responses. Together, these results demonstrate that PSR1 is critical for the survival of cells under conditions of suboptimal phosphorus availability and that it plays a key role in controlling both scavenging responses and the ability of the cell to manage excess absorbed excitation energy.

    View details for DOI 10.1128/EC.5.1.26-44.2006

    View details for Web of Science ID 000234725100002

    View details for PubMedID 16400166

  • A hybrid, recursive algorithm for clustering expressed sequence tags in Chlamydomonas reinhardtii 18th International Conference on Pattern Recognition (ICPR 2006) Jain, M., Holz, H., Shrager, J., Vallon, O., Hauser, C., Grossman, A. IEEE COMPUTER SOC. 2006: 404–407
  • Insights into the acclimation of Chlamydomonas reinhardtii to sulfur deprivation PHOTOSYNTHESIS RESEARCH Pollock, S. V., Pootakham, W., Shibagaki, N., Moseley, J. L., Grossman, A. R. 2005; 86 (3): 475-489

    Abstract

    During sulfur deprivation, the photosynthetic green alga Chlamydomonas reinhardtii develops a high-affinity sulfate uptake system and increases the expression of genes encoding proteins involved in sulfur assimilation. Although two regulatory elements, SAC1 and SAC3, have been shown to be required for normal acclimation of C. reinhardtii to sulfur deprivation, a number of other regulatory elements appear to also be involved. The molecular mechanisms by which these regulatory elements function are largely unknown. This manuscript presents our current knowledge of sulfur deprivation responses and the regulation of these responses in C. reinhardtii. In addition, we present preliminary results of a sub-saturation screen for novel sulfur acclimation mutants of C. reinhardtii. A speculative model, incorporating the activities of established regulatory elements with putative novel components of the signal transduction pathway(s) is discussed.

    View details for DOI 10.1007/s11120-005-4048-9

    View details for Web of Science ID 000235165000014

    View details for PubMedID 16307308

  • Analyses of CIA5, the master regulator of the carbon-concentrating mechanism in Chlamydomonas reinhardtii, and its control of gene expression 5th International Symposium on Inorganic Carbon Utilization by Aquatic Photosynthetic Organisms Wang, Y., Sun, Z. H., Horken, K. M., Im, C. S., Xiang, Y. B., Grossman, A. R., Weeks, D. P. CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS. 2005: 765–79

    View details for DOI 10.1139/B05-062

    View details for Web of Science ID 000231985400009

  • The LPB1 gene is important for acclimation of Chlamydomonas reinhardtii to phosphorus and sulfur deprivation PLANT PHYSIOLOGY Chang, C. W., Moseley, J. L., Wykoff, D., Grossman, A. R. 2005; 138 (1): 319-329

    Abstract

    Organisms exhibit a diverse set of responses when exposed to low-phosphate conditions. Some of these responses are specific for phosphorus limitation, including responses that enable cells to efficiently scavenge phosphate from internal and external stores via the production of high-affinity phosphate transporters and the synthesis of intracellular and extracellular phosphatases. Other responses are general and occur under a number of different environmental stresses, helping coordinate cellular metabolism and cell division with the growth potential of the cell. In this article, we describe the isolation and characterization of a mutant of Chlamydomonas reinhardtii, low-phosphate bleaching (lpb1), which dies more rapidly than wild-type cells during phosphorus limitation. The responses of this mutant to nitrogen limitation appear normal, although the strain is also somewhat more sensitive than wild-type cells to sulfur deprivation. Interestingly, depriving the cells of both nutrients simultaneously allows for sustained survival that is similar to that observed with wild-type cells. Furthermore, upon phosphorus deprivation, the lpb1 mutant, like wild-type cells, exhibits increased levels of mRNA encoding the PHOX alkaline phosphatase, the PTB2 phosphate transporter, and the regulatory element PSR1. The mutant strain is also able to synthesize the extracellular alkaline phosphatase activity upon phosphorus deprivation and the arylsulfatase upon sulfur deprivation, suggesting that the specific responses to phosphorus and sulfur deprivation are normal. The LPB1 gene was tagged by insertion of the ARG7 gene, which facilitated its isolation and characterization. This gene encodes a protein with strong similarity to expressed proteins in Arabidopsis (Arabidopsis thaliana) and predicted proteins in Oryza sativa and Parachlamydia. A domain in the protein contains some similarity to the superfamily of nucleotide-diphospho-sugar transferases, and it is likely to be localized to the chloroplast or mitochondrion based on programs that predict subcellular localization. While the precise catalytic role and physiological function of the putative protein is not known, it may function in some aspect of polysaccharide metabolism and/or influence phosphorus metabolism (either structural or regulatory) in a way that is critical for allowing the cells to acclimate to nutrient limitation conditions.

    View details for DOI 10.1104/pp.105.059550

    View details for Web of Science ID 000229023100032

    View details for PubMedID 15849300

  • Genome-based examination of chlorophyll and carotenoid biosynthesis in Chlamydomonas reinhardtii PLANT PHYSIOLOGY Lohr, M., Im, C. S., Grossman, A. R. 2005; 138 (1): 490-515

    Abstract

    The unicellular green alga Chlamydomonas reinhardtii is a particularly important model organism for the study of photosynthesis since this alga can grow heterotrophically, and mutants in photosynthesis are therefore conditional rather than lethal. The recently developed tools for genomic analyses of this organism have allowed us to identify most of the genes required for chlorophyll and carotenoid biosynthesis and to examine their phylogenetic relationships with homologous genes from vascular plants, other algae, and cyanobacteria. Comparative genome analyses revealed some intriguing features associated with pigment biosynthesis in C. reinhardtii; in some cases, there are additional conserved domains in the algal and plant but not the cyanobacterial proteins that may directly influence their activity, assembly, or regulation. For some steps in the chlorophyll biosynthetic pathway, we found multiple gene copies encoding putative isozymes. Phylogenetic studies, theoretical evaluation of gene expression through analysis of expressed sequence tag data and codon bias of each gene, enabled us to generate hypotheses concerning the function and regulation of the individual genes, and to propose targets for future research. We have also used quantitative polymerase chain reaction to examine the effect of low fluence light on the level of mRNA accumulation encoding key proteins of the biosynthetic pathways and examined differential expression of those genes encoding isozymes that function in the pathways. This work is directing us toward the exploration of the role of specific photoreceptors in the biosynthesis of pigments and the coordination of pigment biosynthesis with the synthesis of proteins of the photosynthetic apparatus.

    View details for DOI 10.1104/pp.104.056069

    View details for Web of Science ID 000229023100048

    View details for PubMedID 15849308

  • Paths toward algal genomics PLANT PHYSIOLOGY Grossman, A. R. 2005; 137 (2): 410-427

    View details for DOI 10.1104/pp.104.053447

    View details for Web of Science ID 000227116900003

    View details for PubMedID 15710682

  • Approaches using yeast cells to probe the function of STAS domain in SULTR1;2 6th International Workshop on Plant Sulfur Metabolism Shibagaki, N., Grossman, A. R. BACKHUYS PUBLISHERS. 2005: 33–35
  • AplA, a member of a new class of phycobiliproteins lacking a traditional role in photosynthetic light harvesting JOURNAL OF BACTERIOLOGY Montgomery, B. L., Casey, E. S., Grossman, A. R., Kehoe, D. M. 2004; 186 (21): 7420-7428

    Abstract

    All known phycobiliproteins have light-harvesting roles during photosynthesis and are found in water-soluble phycobilisomes, the light-harvesting complexes of cyanobacteria, cyanelles, and red algae. Phycobiliproteins are chromophore-bearing proteins that exist as heterodimers of alpha and beta subunits, possess a number of highly conserved amino acid residues important for dimerization and chromophore binding, and are invariably 160 to 180 amino acids long. A new and unusual group of proteins that is most closely related to the allophycocyanin members of the phycobiliprotein superfamily has been identified. Each of these proteins, which have been named allophycocyanin-like (Apl) proteins, apparently contains a 28-amino-acid extension at its amino terminus relative to allophycocyanins. Apl family members possess the residues critical for chromophore interactions, but substitutions are present at positions implicated in maintaining the proper alpha-beta subunit interactions and tertiary structure of phycobiliproteins, suggesting that Apl proteins are able to bind chromophores but fail to adopt typical allophycocyanin conformations. AplA isolated from the cyanobacterium Fremyella diplosiphon contained a covalently attached chromophore and, although present in the cell under a number of conditions, was not detected in phycobilisomes. Thus, Apl proteins are a new class of photoreceptors with a different cellular location and structure than any previously described members of the phycobiliprotein superfamily.

    View details for DOI 10.1128/JB.186.21.7420-7428.2004

    View details for Web of Science ID 000224575500040

    View details for PubMedID 15489454

  • Insights into the survival of Chlamydomonas reinhardtii during sulfur starvation based on microarray analysis of gene expression EUKARYOTIC CELL Zhang, Z. D., Shrager, J., Jain, M., Chang, C. W., Vallon, O., Grossman, A. R. 2004; 3 (5): 1331-1348

    Abstract

    Responses of photosynthetic organisms to sulfur starvation include (i) increasing the capacity of the cell for transporting and/or assimilating exogenous sulfate, (ii) restructuring cellular features to conserve sulfur resources, and (iii) modulating metabolic processes and rates of cell growth and division. We used microarray analyses to obtain a genome-level view of changes in mRNA abundances in the green alga Chlamydomonas reinhardtii during sulfur starvation. The work confirms and extends upon previous findings showing that sulfur deprivation elicits changes in levels of transcripts for proteins that help scavenge sulfate and economize on the use of sulfur resources. Changes in levels of transcripts encoding members of the light-harvesting polypeptide family, such as LhcSR2, suggest restructuring of the photosynthetic apparatus during sulfur deprivation. There are also significant changes in levels of transcripts encoding enzymes involved in metabolic processes (e.g., carbon metabolism), intracellular proteolysis, and the amelioration of oxidative damage; a marked and sustained increase in mRNAs for a putative vanadium chloroperoxidase and a peroxiredoxin may help prolong survival of C. reinhardtii during sulfur deprivation. Furthermore, many of the sulfur stress-regulated transcripts (encoding polypeptides associated with sulfate uptake and assimilation, oxidative stress, and photosynthetic function) are not properly regulated in the sac1 mutant of C. reinhardtii, a strain that dies much more rapidly than parental cells during sulfur deprivation. Interestingly, sulfur stress elicits dramatic changes in levels of transcripts encoding putative chloroplast-localized chaperones in the sac1 mutant but not in the parental strain. These results suggest various strategies used by photosynthetic organisms during acclimation to nutrient-limited growth.

    View details for DOI 10.1128/EC.3.5.1331-1348.2004

    View details for Web of Science ID 000224822300027

    View details for PubMedID 15470261

    View details for PubMedCentralID PMC522608

  • Probing the function of STAS domains of the Arabidopsis sulfate transporters JOURNAL OF BIOLOGICAL CHEMISTRY Shibagaki, N., Grossman, A. R. 2004; 279 (29): 30791-30799

    Abstract

    Sulfate transporters in plants and animals are structurally conserved and have an amino-terminal domain that functions in transport and a carboxyl-terminal region that has been designated the STAS domain. The STAS domain in sulfate transporters has significant similarity to bacterial anti-sigma factor antagonists. To determine if the STAS domain has a role in controlling the activity of sulfate transporters, their stability, or their localization to the plasma membrane, we examined the effect of deleting or modifying the STAS domain of dominant sulfate transporters in roots of Arabidopsis thaliana. The A. thaliana Sultr1;2 and Sultr1;1 sulfate transporters rescue the methionine-dependent growth phenotype of the yeast sulfate transporter mutant strain CP154-7B. Constructs of Sultr1;2 in which the STAS domain was deleted (DeltaSTAS) resulted in synthesis of a truncated polypeptide that was unable to rescue the CP154-7B phenotype. The inability of these constructs to rescue the mutant phenotype probably reflected both low level cellular accumulation of the transporter and the inability of the truncated protein to localize to the plasma membrane. Fusing the STAS domain from other sulfate transporters to Sultr1;2 DeltaSTAS constructs restored elevated accumulation and plasma membrane localization, although the kinetics of sulfate uptake in the transformants were markedly altered with respect to transformants synthesizing wild-type Sultr1;2 protein. These results suggest that the STAS domain is essential, either directly or indirectly, for facilitating localization of the transporters to the plasma membrane, but it also appears to influence the kinetic properties of the catalytic domain of transporters.

    View details for DOI 10.1074/jbc.M403248200

    View details for Web of Science ID 000222531900110

    View details for PubMedID 15136568

  • Consequences of a deletion in dspA on transcript accumulation in Synechocystis sp strain PCC6803 JOURNAL OF BACTERIOLOGY Tu, C. J., Shrager, J., Burnap, R. L., Postier, B. L., Grossman, A. R. 2004; 186 (12): 3889-3902

    Abstract

    A sensor histidine kinase of Synechococcus sp. strain PCC7942, designated nblS, was previously identified and shown to be critical for the acclimation of cells to high-light and nutrient limitation conditions and to influence the expression of a number of light-responsive genes. The nblS orthologue in Synechocystis sp. strain PCC6803 is designated dspA (also called hik33). We have generated a dspA null mutant and analyzed global gene expression in both the mutant and wild-type strains under high- and low-light conditions. The mutant is aberrant for the expression of many genes encoding proteins critical for photosynthesis, phosphate and carbon acquisition, and the amelioration of stress conditions. Furthermore, transcripts from a number of genes normally detected only during exposure of wild-type cells to high-light conditions become partially constitutive in the low-light-grown dspA mutant. Other genes for which transcripts decline upon exposure of wild-type cells to high light are already lower in the mutant during growth in low light. These results suggest that DspA may influence gene expression in both a positive and a negative manner and that the dspA mutant behaves as if it were experiencing stress conditions (e.g., high-light exposure) even when maintained at near-optimal growth conditions for wild-type cells. This is discussed with respect to the importance of DspA for regulating the responses of the cell to environmental cues.

    View details for Web of Science ID 000221869100026

    View details for PubMedID 15175303

    View details for PubMedCentralID PMC419946

  • Control of photosynthetic and high-light-responsive genes by the histidine kinase DspA: Negative and positive regulation and interactions between signal transduction pathways JOURNAL OF BACTERIOLOGY Hsiao, H. Y., He, Q. F., van Waasbergen, L. G., Grossman, A. R. 2004; 186 (12): 3882-3888

    Abstract

    We have deleted a gene for a sensor histidine kinase, dspA (or hik33), in the cyanobacterium Synechocystis sp. strain PCC6803. In low and moderate light, the mutant grew slowly under photoautotrophic conditions, with a doubling time of approximately 40 h, and had severely reduced photosynthetic oxygen evolution. When the mutant was maintained in low or moderate light in the presence of glucose, its growth rate was only somewhat lower than that of wild-type cells. However, the mutant was light sensitive and rapidly died in high light. Furthermore, levels of many transcripts encoding genes associated with photosynthesis were altered in the mutant relative to wild-type Synechocystis sp. strain PCC6803 both in low light and following exposure to high light. There was constitutive expression of several high-light-inducible genes, including hli, psbAIII, and gpx2; there was little increased accumulation of sodB mRNA in high light; and the cells failed to accumulate cpcBA and psaAB mRNAs in low light in the presence of glucose, although a normal decline in the levels of these mRNAs was observed during exposure to high light. These results suggest that DspA is involved in controlling sets of photosynthetic and high-light-responsive genes, either directly or indirectly. These and other results, some of which are presented in a companion paper (C.-J. Tu, J. Shrager, R. Burnap, B. L. Postier, and A. R. Grossman, J. Bacteriol. 186:3889-3902, 2004), suggest that DspA acts as a global regulator that helps coordinate cellular metabolism with growth limitations imposed by environmental conditions.

    View details for DOI 10.1128/JB.186.12.3882-3888.2004

    View details for Web of Science ID 000221869100025

    View details for PubMedID 15175302

  • Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp PCC 6803 and Prochlorococcus MED4 and MIT9313 8th International Workshop on Opportunistic Protists/International Conference on Anaerobic Protists Mary, I., Tu, C. J., Grossman, A., Vaulot, D. SOC GENERAL MICROBIOLOGY. 2004: 1271–1281

    Abstract

    Cyanobacteria constitute an ancient, diverse and ecologically important bacterial group. The responses of these organisms to light and nutrient conditions are finely controlled, enabling the cells to survive a range of environmental conditions. In particular, it is important to understand how cyanobacteria acclimate to the absorption of excess excitation energy and how stress-associated transcripts accumulate following transfer of cells from low- to high-intensity light. In this study, quantitative RT-PCR was used to monitor changes in levels of transcripts encoding chaperones and stress-associated proteases in three cyanobacterial strains that inhabit different ecological niches: the freshwater strain Synechocystis sp. PCC 6803, the marine high-light-adapted strain Prochlorococcus MED4 and the marine low-light-adapted strain Prochlorococcus MIT9313. Levels of transcripts encoding stress-associated proteins were very sensitive to changes in light intensity in all of these organisms, although there were significant differences in the degree and kinetics of transcript accumulation. A specific set of genes that seemed to be associated with high-light adaptation (groEL/groES, dnaK2, dnaJ3, clpB1 and clpP1) could be targeted for more detailed studies in the future. Furthermore, the strongest responses were observed in Prochlorococcus MED4, a strain characteristic of the open ocean surface layer, where hsp genes could play a critical role in cell survival.

    View details for DOI 10.1099/mic.0.27014-0

    View details for Web of Science ID 000221538000025

    View details for PubMedID 15133090

  • A genome's-eye view of the light-harvesting polypeptides of Chlamydomonas reinhardtii CURRENT GENETICS Elrad, D., Grossman, A. R. 2004; 45 (2): 61-75

    Abstract

    Chlamydomonas reinhardtii is a valuable model system for defining the structure and function of polypeptides of the photosynthetic apparatus and the dynamic aspects of photosynthesis. Recently, a genome-wide analysis of cDNAs and a draft genome sequence that covers approximately 90% of the genome were made available, providing a clear picture of the composition of specific gene families, the relationships among the gene family members, and the location of each member on the genome. We used the available sequence information to analyze the extensive family of light-harvesting genes in C. reinhardtii. There are nine genes encoding polypeptides of the major light-harvesting complex of photosystem II, two genes encoding the minor light-harvesting polypeptides of photosystem II, and nine genes encoding polypeptides predicted to comprise the photosystem I light-harvesting complex. Furthermore, there are five genes encoding early light-induced proteins and two genes encoding LI818 polypeptides. A candidate for the PsbS gene has also been found in the raw genome sequence data (Niyogi, personal communication), although no genes encoding homologues of the Sep, or Hli polypeptides have been identified. In this manuscript, we identify and classify the family of light-harvesting polypeptides encoded on the C. reinhardtii genome. This is an important first step in designing specific genetic, biochemical, and physiological studies aimed at characterizing the composition, function, and regulation of the light-harvesting complexes.

    View details for DOI 10.1007/s00294-003-0460-x

    View details for Web of Science ID 000188748600001

    View details for PubMedID 14652691

  • Chlamydomonas reinhardtii in the landscape of pigments ANNUAL REVIEW OF GENETICS Grossman, A. R., Lohr, M., Im, C. S. 2004; 38: 119-173

    Abstract

    This review focuses on the biosynthesis of pigments in the unicellular alga Chlamydomonas reinhardtii and their physiological and regulatory functions in the context of information gathered from studies of other photosynthetic organisms. C. reinhardtii is serving as an important model organism for studies of photosynthesis and the pigments associated with the photosynthetic apparatus. Despite extensive information pertaining to the biosynthetic pathways critical for making chlorophylls and carotenoids, we are just beginning to understand the control of these pathways, the coordination between pigment and apoprotein synthesis, and the interactions between the activities of these pathways and those for other important cellular metabolites branching from these pathways. Other exciting areas relating to pigment function are also emerging: the role of intermediates of pigment biosynthesis as messengers that coordinate metabolism in the chloroplast with nuclear gene activity, and the identification of photoreceptors and their participation in critical cellular processes including phototaxis, gametogenesis, and the biogenesis of the photosynthetic machinery. These areas of research have become especially attractive for intensive development with the application of potent molecular and genomic tools currently being applied to studies of C. reinhardtii.

    View details for DOI 10.1146/annurev.genet.38.072902.092328

    View details for Web of Science ID 000226244600005

    View details for PubMedID 15568974

  • RcaE is a complementary chromatic adaptation photoreceptor required for green and red light responsiveness MOLECULAR MICROBIOLOGY Terauchi, K., Montgomery, B. L., Grossman, A. R., Lagarias, J. C., Kehoe, D. M. 2004; 51 (2): 567-577

    Abstract

    The recent discovery of large numbers of phytochrome photoreceptor genes in both photosynthetic and non-photosynthetic prokaryotes has led to efforts to understand their physiological roles in environmental acclimation. One receptor in this class, RcaE, is involved in controlling complementary chromatic adaptation, a process that regulates the transcription of operons encoding light-harvesting proteins in cyanobacteria. Although all previously identified phytochrome responses are maximally sensitive to red and far red light, complementary chromatic adaptation is unique in that it is responsive to green and red light. Here, we present biochemical and genetic evidence demonstrating that RcaE is a photoreceptor and that it requires the cysteine at position 198 to ligate an open chain tetrapyrrole covalently in a manner analogous to chromophore attachment in plant phytochromes. Furthermore, although the wild-type rcaE gene can rescue red and green light photoresponses of an rcaE null mutant, a gene in which the codon for cysteine 198 is converted to an alanine codon rescues the red light but not the green light response. Thus, RcaE is a photoreceptor that is required for both green and red light responsiveness during complementary chromatic adaptation and is the first identified phytochrome class sensor that is involved in sensing and responding to green and red light rather than red and far red light.

    View details for DOI 10.1046/j.1365-2958.2003.03853.x

    View details for Web of Science ID 000187890800022

    View details for PubMedID 14756794

  • Chlamydomonas reinhardtii at the crossroads of genomics EUKARYOTIC CELL Grossman, A. R., Harris, E. E., Hauser, C., Lefebvre, P. A., Martinez, D., Rokhsar, D., Shrager, J., Silflow, C. D., Stern, D., Vallon, O., Zhang, Z. D. 2003; 2 (6): 1137-1150

    View details for DOI 10.1128/EC.2.6.1137-1150.2003

    View details for Web of Science ID 000187363500001

    View details for PubMedID 14665449

    View details for PubMedCentralID PMC326643

  • Analysis of light and CO(2) regulation in Chlamydomonas reinhardtii using genome-wide approaches. Photosynthesis research Im, C. S., Zhang, Z., Shrager, J., Chang, C. W., Grossman, A. R. 2003; 75 (2): 111-25

    Abstract

    Over the past decade new technologies have been developed to elucidate ways in which cells acclimate to environmental change. Many of these techniques have allowed the identification of specific transcripts that change in abundance in response to particular environmental stimuli; such transcripts represent genes that are potentially differentially regulated. Two techniques that foster identification of differentially regulated genes are differential display and expression profiling using high density DNA microarrays. The former technology amplifies cDNA fragments from mRNAs that differentially accumulate under specific environmental conditions, while the latter provides a more global view of changes in gene expression in response to environmental stimuli. Coupling these technologies with the analysis of mutants aberrant for regulatory molecules that participate in acclimation processes will allow the identification of groups of genes controlled by specific regulatory elements. In this article we describe the use of differential display and DNA microarray profiling to examine environmentally-regulated gene expression. We also show specific experiments using the unicellular green alga Chlamydomonas reinhardtii, in which mRNA abundance is evaluated in response to both changing light and CO(2) conditions.

    View details for DOI 10.1023/A:1022800630430

    View details for PubMedID 16245082

  • A molecular understanding of complementary chromatic adaptation. Photosynthesis research Grossman, A. R. 2003; 76 (1-3): 207-15

    Abstract

    Photosynthetic activity and the composition of the photosynthetic apparatus are strongly regulated by environmental conditions. Some visually dramatic changes in pigmentation of cyanobacterial cells that occur during changing nutrient and light conditions reflect marked alterations in components of the major light-harvesting complex in these organisms, the phycobilisome. As noted well over 100 years ago, the pigment composition of some cyanobacteria is very sensitive to ambient wavelengths of light; this sensitivity reflects molecular changes in polypeptide constituents of the phycobilisome. The levels of different pigmented polypeptides or phycobiliproteins that become associated with the phycobilisome are adjusted to optimize absorption of excitation energy present in the environment. This process, called complementary chromatic adaptation, is controlled by a bilin-binding photoreceptor related to phytochrome of vascular plants; however, many other regulatory elements also play a role in chromatic adaptation. My perspectives and biases on the history and significance of this process are presented in this essay.

    View details for DOI 10.1023/A:1024907330878

    View details for PubMedID 16228579

  • Elimination of high-light-inducible polypeptides related to eukaryotic chlorophyll a/b-binding proteins results in aberrant photoacclimation in Synechocystis PCC6803 BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS Havaux, M., Guedeney, G., He, Q. F., Grossman, A. R. 2003; 1557 (1-3): 21-33

    Abstract

    The hli genes, present in cyanobacteria, algae and vascular plants, encode small proteins [high-light-inducible polypeptides (HLIPs)] with a single membrane-spanning alpha-helix related to the first and third helices of eukaryotic chlorophyll a/b-binding proteins. The HLIPs are present in low amounts in low light and they accumulate transiently at high light intensities. We are investigating the function of those polypeptides in a Synechocystis PCC6803 mutant lacking four of the five hli genes. Growth of the quadruple hli mutant was adversely affected by high light intensities. The most striking effect of the quadruple hli mutation was an alteration of cell pigmentation. Pigment changes associated with cell acclimation to increasing light intensity [i.e. decrease in light-harvesting pigments, accumulation of the carotenoid myxoxanthophyll and decrease in photosystem I (PSI)-associated chlorophylls] were strongly exacerbated in the quadruple hli mutant, resulting in yellowish cultures that bleached in high light and died as light intensities exceeded (>500 micromol photon m(-2) s(-1)). However, these pigment changes were not associated with an inhibition of photosynthesis, as probed by in vivo chlorophyll fluorescence, photoacoustic and O(2)-evolution measurements. On the contrary, the HLIP deficiency was accompanied by a stimulation of the photochemical activity, especially in high-light-grown cells. Western blot analyses revealed that the PSI reaction center level (PsaA/B) was noticeably reduced in the quadruple hli mutant relative to the wild type, whereas the abundance of the PSII reaction center protein D1 was comparatively little affected. The hli mutations did not enhance photoinhibition and photooxidation when cells were exposed over a short term to a very high light intensity. Together, the results of this study indicate that HLIPs are critical in the adaptation of the cyanobacterium to variations in light intensity. The data are consistent with the idea that HLIPs are involved, through a direct or indirect means, in nonphotochemical dissipation of absorbed light energy.

    View details for DOI 10.1016/S0005-2728(02)00391-2

    View details for Web of Science ID 000181495600003

    View details for PubMedID 12615345

  • Multiple light inputs control phototaxis in Synechocystis sp strain PCC6803 JOURNAL OF BACTERIOLOGY Ng, W. O., Grossman, A. R., Bhaya, D. 2003; 185 (5): 1599-1607

    Abstract

    The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.

    View details for DOI 10.1128/JB.185.5.1599-1607.2003

    View details for Web of Science ID 000181151200015

    View details for PubMedID 12591877

    View details for PubMedCentralID PMC148062

  • Chlamydomonas reinhardtii genome project. A guide to the generation and use of the cDNA information PLANT PHYSIOLOGY Shrager, J., Hauser, C., Chang, C. W., Harris, E. H., DAVIES, J., McDermott, J., Tamse, R., Zhang, Z. D., Grossman, A. R. 2003; 131 (2): 401-408

    Abstract

    The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

    View details for DOI 10.1104/pp.016899

    View details for Web of Science ID 000181005000003

    View details for PubMedID 12586865

    View details for PubMedCentralID PMC166817

  • A molecular understanding of complementary chromatic adaptation PHOTOSYNTHESIS RESEARCH Grossman, A. R. 2003; 76 (1-3): 207-215
  • Analysis of light and CO2 regulation in Chlamydomonas reinhardtii using genome-wide approaches PHOTOSYNTHESIS RESEARCH Im, C. S., Zhang, Z. D., Shrager, J., Chang, C. W., Grossman, A. R. 2003; 75 (2): 111-125

    Abstract

    Over the past decade new technologies have been developed to elucidate ways in which cells acclimate to environmental change. Many of these techniques have allowed the identification of specific transcripts that change in abundance in response to particular environmental stimuli; such transcripts represent genes that are potentially differentially regulated. Two techniques that foster identification of differentially regulated genes are differential display and expression profiling using high density DNA microarrays. The former technology amplifies cDNA fragments from mRNAs that differentially accumulate under specific environmental conditions, while the latter provides a more global view of changes in gene expression in response to environmental stimuli. Coupling these technologies with the analysis of mutants aberrant for regulatory molecules that participate in acclimation processes will allow the identification of groups of genes controlled by specific regulatory elements. In this article we describe the use of differential display and DNA microarray profiling to examine environmentally-regulated gene expression. We also show specific experiments using the unicellular green alga Chlamydomonas reinhardtii, in which mRNA abundance is evaluated in response to both changing light and CO(2) conditions.

    View details for Web of Science ID 000181506700002

  • In vivo characterization of diatom multipartite plastid targeting signals JOURNAL OF CELL SCIENCE Apt, K. E., Zaslavkaia, L., Lippmeier, J. C., Lang, M., Kilian, O., Wetherbee, R., Grossman, A. R., Kroth, P. G. 2002; 115 (21): 4061-4069

    Abstract

    Plastids of diatoms and related algae are delineated by four membranes: the outermost membrane (CER) is continuous with the endoplasmic reticulum while the inner two membranes are homologous to plastid envelope membranes of vascular plants and green algae. Proteins are transported into these plastids by pre-sequences that have two recognizable domains. To characterize targeting of polypeptides within diatom cells, we generated constructs encoding green fluorecent protein (GFP) fused to leader sequences. A fusion of GFP to the pre-sequence of BiP [an endoplasmic reticulum (ER)-localized chaperone] resulted in accumulation of GFP within the ER; a construct encoding the pre-sequence of a plastid protein fused to GFP was directed into the plastids. Additional constructs demonstrated that the N-terminal region of the bipartite plastid targeting pre-sequence was necessary for transport of polypeptides to the lumen of the ER, while the C-terminal region was shown to enable the proteins to traverse the plastid double envelope membrane. Our data strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae and raises questions about the continuity of the ER and CER and the function of the latter in polypeptide trafficking.

    View details for DOI 10.1242/jcs.00092

    View details for Web of Science ID 000179254000009

    View details for PubMedID 12356911

  • Analysis of the hli gene family in marine and freshwater cyanobacteria FEMS MICROBIOLOGY LETTERS Bhaya, D., Dufresne, A., Vaulot, D., Grossman, A. 2002; 215 (2): 209-219

    Abstract

    Certain cyanobacteria thrive in natural habitats in which light intensities can reach 2000 micromol photon m(-2) s(-1) and nutrient levels are extremely low. Recently, a family of genes designated hli was demonstrated to be important for survival of cyanobacteria during exposure to high light. In this study we have identified members of the hli gene family in seven cyanobacterial genomes, including those of a marine cyanobacterium adapted to high-light growth in surface waters of the open ocean (Prochlorococcus sp. strain Med4), three marine cyanobacteria adapted to growth in moderate- or low-light (Prochlorococcus sp. strain MIT9313, Prochlorococcus marinus SS120, and Synechococcus WH8102), and three freshwater strains (the unicellular Synechocystis sp. strain PCC6803 and the filamentous species Nostoc punctiforme strain ATCC29133 and Anabaena sp. [Nostoc] strain PCC7120). The high-light-adapted Prochlorococcus Med4 has the smallest genome (1.7 Mb), yet it has more than twice as many hli genes as any of the other six cyanobacterial species, some of which appear to have arisen from recent duplication events. Based on cluster analysis, some groups of hli genes appear to be specific to either marine or freshwater cyanobacteria. This information is discussed with respect to the role of hli genes in the acclimation of cyanobacteria to high light, and the possible relationships among members of this diverse gene family.

    View details for Web of Science ID 000178923300007

    View details for PubMedID 12399037

  • A major light-harvesting polypeptide of photosystem II functions in thermal dissipation PLANT CELL Elrad, D., Niyogi, K. K., Grossman, A. R. 2002; 14 (8): 1801-1816

    Abstract

    Under high-light conditions, photoprotective mechanisms minimize the damaging effects of excess light. A primary photoprotective mechanism is thermal dissipation of excess excitation energy within the light-harvesting complex of photosystem II (LHCII). Although roles for both carotenoids and specific polypeptides in thermal dissipation have been reported, neither the site nor the mechanism of this process has been defined precisely. Here, we describe the physiological and molecular characteristics of the Chlamydomonas reinhardtii npq5 mutant, a strain that exhibits little thermal dissipation. This strain is normal for state transition, high light-induced violaxanthin deepoxidation, and low light growth, but it is more sensitive to photoinhibition than the wild type. Furthermore, both pigment data and measurements of photosynthesis suggest that the photosystem II antenna in the npq5 mutant has one-third fewer light-harvesting trimers than do wild-type cells. The npq5 mutant is null for a gene designated Lhcbm1, which encodes a light-harvesting polypeptide present in the trimers of the photosystem II antennae. Based on sequence data, the Lhcbm1 gene is 1 of 10 genes that encode the major LHCII polypeptides in Chlamydomonas. Amino acid alignments demonstrate that these predicted polypeptides display a high degree of sequence identity but maintain specific differences in their N-terminal regions. Both physiological and molecular characterization of the npq5 mutant suggest that most thermal dissipation within LHCII of Chlamydomonas is dependent on the peripherally associated trimeric LHC polypeptides.

    View details for DOI 10.1005/tpc.002154

    View details for Web of Science ID 000177604700010

    View details for PubMedID 12172023

    View details for PubMedCentralID PMC151466

  • Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii PLANT JOURNAL Im, C. S., Grossman, A. R. 2002; 30 (3): 301-313

    Abstract

    We have used restriction fragment differential display for isolating genes of the unicellular green alga Chlamydomonas reinhardtii that exhibit elevated expression on exposure of cells to high light. Some of the high light-activated genes were also controlled by CO2 concentration. Genes requiring both elevated light and low CO2 levels for activation encoded both novel polypeptides and those that function in concentrating inorganic carbon (extracellular carbonic anhydrase, low CO2-induced protein, ABC transporter of the MRP subfamily). All the genes in this category were shown to be under the control of Cia5, a protein that regulates the responses of C. reinhardtii to low-CO2 conditions. Genes specifically activated by high light, even under high-CO2 conditions, encoded a 30 kDa chloroplast membrane protein, a serine hydroxymethyltransferase, a nuclease, and two proteins of unknown function. Experiments using DCMU, an inhibitor of photosynthetic electron transport, and mutants devoid of either photosystem I or photosystem II activity, showed aberrant expression of all the genes regulated by both CO2 and high light, suggesting that redox plays a role in controlling their expression. In contrast, there was little effect of DCMU or lesions that block photosynthetic electron transport on the activity of genes that were specifically controlled by high light.

    View details for Web of Science ID 000175531600004

    View details for PubMedID 12000678

  • nblS, a gene involved in controlling photosynthesis-related gene expression during high light and nutrient stress in Synechococcus elongatus PCC 7942 JOURNAL OF BACTERIOLOGY van Waasbergen, L. G., Dolganov, N., Grossman, A. R. 2002; 184 (9): 2481-2490

    Abstract

    The HliA protein of the cyanobacterium Synechococcus elongatus PCC 7942 is a small, thylakoid-associated protein that appears to play a role in photoprotection; its transcript rapidly accumulates in response to high-intensity light (HL) and the hli gene family is required for survival of cells in high light. In order to discover regulatory factors involved in HL acclimation in cyanobacteria, a screen was performed for chemically generated mutants unable to properly control expression of the hliA gene in response to HL. One such mutant was identified, and complementation analysis led to the identification of the affected gene, designated nblS. Based on its deduced protein sequence, NblS appears to be a membrane-bound, PAS-domain-bearing, sensor histidine kinase of two-component regulatory systems in bacteria. The nblS mutant was unable to properly control light intensity-mediated expression of several other photosynthesis-related genes, including all three psbA genes and the cpcBA genes. The mutant was also unable to control expression of the hliA and psbA genes in response to low-intensity blue/UV-A light, a response that may be related to the HL-mediated regulation of the genes. Additionally, in response to nutrient deprivation, the nblS mutant was unable to properly control accumulation of the nblA transcript and associated degradation of the light-harvesting phycobilisomes. The nblS mutant dies more rapidly than wild-type cells following exposure to HL or nutrient deprivation, likely due to its inability to properly acclimate to these stress conditions. Thus, the NblS protein is involved in the control of a number of processes critical for altering the photosynthetic apparatus in response to both HL and nutrient stress conditions.

    View details for DOI 10.1128/JB.184.9.2481-2490.2002

    View details for Web of Science ID 000175000400019

    View details for PubMedID 11948163

  • Novel motility mutants of Synechocystis strain PCC 6803 generated by in vitro transposon mutagenesis JOURNAL OF BACTERIOLOGY Bhaya, D., Takahashi, A., Shahi, P., Grossman, A. R. 2001; 183 (20): 6140-6143

    Abstract

    We screened for transposon-generated mutants of Synechocystis sp. strain PCC 6803 that exhibited aberrant phototactic movement. Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes. These novel genes and their possible roles in motility are discussed.

    View details for Web of Science ID 000171267100040

    View details for PubMedID 11567015

    View details for PubMedCentralID PMC99694

  • Sulfur economy and cell wall biosynthesis during sulfur limitation of Chlamydomonas reinhardtii PLANT PHYSIOLOGY Takahashi, H., Braby, C. E., Grossman, A. R. 2001; 127 (2): 665-673

    Abstract

    We have identified two novel periplasmic/cell wall polypeptides that specifically accumulate during sulfur limitation of Chlamydomonas reinhardtii. These polypeptides, present at high levels in the extracellular polypeptide fraction from a sulfur-deprived, cell wall-minus C. reinhardtii strain, have apparent molecular masses of 76 and 88 kD and are designated Ecp76 and Ecp88. N-terminal sequences of these polypeptides facilitated the isolation of full-length Ecp76 and Ecp88 cDNAs. Ecp76 and Ecp88 polypeptides are deduced to be 583 and 595 amino acids, respectively. Their amino acid sequences are similar to each other, with features characteristic of cell wall-localized hydroxyproline-rich glycoproteins; the N terminus of each polypeptide contains a predicted signal sequence, whereas the C terminus is rich in proline, alanine, and serine. Ecp76 and Ecp88 have either no (Ecp88) or one (Ecp76) sulfur-containing amino acid and transcripts encoding these polypeptides are not detected in cultures maintained on complete medium, but accumulate when cells are deprived of sulfur. This accumulation is temporally delayed relative to the accumulation of sulfur stress-induced arylsulfatase and ATP sulfurylase transcripts. The addition of sulfate back to sulfur-starved cultures caused a rapid decline in Ecp76 and Ecp88 mRNAs (half lives < 10 min). Furthermore, the C. reinhardtii sac1 mutant, which lacks a regulatory protein critical for acclimation to sulfur limitation, does not accumulate Ecp76 or Ecp88 transcripts. These results suggest that the Ecp76 and Ecp88 genes are under SacI control, and that restructuring of the C. reinhardtii cell wall during sulfur limitation may be important for redistribution of internal and efficient utilization of environmental sulfur-containing molecules.

    View details for Web of Science ID 000172144500031

    View details for PubMedID 11598240

  • Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bhaya, D., Takahashi, A., Grossman, A. R. 2001; 98 (13): 7540-7545

    Abstract

    To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an "inverted motility response" (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

    View details for Web of Science ID 000169456600098

    View details for PubMedID 11404477

    View details for PubMedCentralID PMC34704

  • Trophic obligate conversion of an photoautotrophic organism through metabolic engineering SCIENCE Zaslavskaia, L. A., Lippmeier, J. C., Shih, C., Ehrhardt, D., Grossman, A. R., Apt, K. E. 2001; 292 (5524): 2073-2075

    Abstract

    Most microalgae are obligate photoautotrophs and their growth is strictly dependent on the generation of photosynthetically derived energy. We show that the microalga Phaeodactylum tricornutum can be genetically engineered to thrive on exogenous glucose in the absence of light through the introduction of a gene encoding a glucose transporter (glut1 or hup1). This demonstrates that a fundamental change in the metabolism of an organism can be accomplished through the introduction of a single gene. This also represents progress toward the use of fermentation technology for large-scale commercial exploitation of algae by reducing limitations associated with light-dependent growth.

    View details for Web of Science ID 000169284700046

    View details for PubMedID 11408656

  • Genes essential to iron transport in the cyanobacterium Synechocystis sp strain PCC 6803 JOURNAL OF BACTERIOLOGY Katoh, H., Hagino, N., Grossman, A. R., Ogawa, T. 2001; 183 (9): 2779-2784

    Abstract

    Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in Synechocystis sp. strain PCC 6803 were identified. These genes are slr1295, slr0513, slr0327, and recently reported sll1878 (Katoh et al., J. Bacteriol. 182:6523-6524, 2000) and were designated futA1, futA2, futB, and futC, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or futA1 and futA2 together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the fut genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The futA1 and futA2 genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of futB and futC genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of slr1392, a homologue of feoB in Escherichia coli, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the fut-less mutants grown in complete medium.

    View details for Web of Science ID 000168082000008

    View details for PubMedID 11292796

  • Tracking the light environment by cyanobacteria and the dynamic nature of light harvesting JOURNAL OF BIOLOGICAL CHEMISTRY Grossman, A. R., Bhaya, D., He, Q. F. 2001; 276 (15): 11449-11452

    View details for Web of Science ID 000168081800002

    View details for PubMedID 11279225

  • Light minireview series JOURNAL OF BIOLOGICAL CHEMISTRY Simoni, R. D., Grossman, A. R. 2001; 276 (15): 11447-11448
  • Highly expressed and alien genes of the Synechocystis genome NUCLEIC ACIDS RESEARCH Mrazek, J., Bhaya, D., Grossman, A. R., Karlin, S. 2001; 29 (7): 1590-1601

    Abstract

    Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the SYNECHOCYSTIS: PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In SYNECHOCYSTIS:, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO(2) fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.

    View details for Web of Science ID 000167970300024

    View details for PubMedID 11266562

    View details for PubMedCentralID PMC31270

  • The gamma subunits of phycoerythrin from a red alga: Position in phycobilisomes and sequence characterization JOURNAL OF PHYCOLOGY Apt, K. E., Metzner, S., Grossman, A. R. 2001; 37 (1): 64-70
  • The high light-inducible polypeptides in Synechocystis PCC6803 - Expression and function in high light JOURNAL OF BIOLOGICAL CHEMISTRY He, Q. F., Dolganov, N., Bjorkman, O., Grossman, A. R. 2001; 276 (1): 306-314

    Abstract

    There are five Synechocystis PCC6803 genes encoding polypeptides with similarity to the Lhc polypeptides of plants. Four of the polypeptides, designated HliA-D (Dolganov, N. A. M., Bhaya, D., and Grossman, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 636-640) (corresponding to ScpC, ScpD, ScpB, and ScpE in Funk, C., and Vermaas, W. (1999) Biochemistry 38, 9397-9404) contain a single transmembrane domain. The fifth polypeptide (HemH) represents a fusion between a ferrochelatase and an Hli-like polypeptide. By using an epitope tag to identify specifically the different Hli polypeptides, the accumulation of each (excluding HemH) was examined under various environmental conditions. The levels of all of the Hli polypeptides were elevated in high light and during nitrogen limitation, whereas HliA, HliB, and HliC also accumulated to high levels following exposure to sulfur deprivation and low temperature. The temporal pattern of accumulation was significantly different among the different Hli polypeptides. HliC rapidly accumulated in high light, and its level remained high for at least 24 h. HliA and HliB also accumulated rapidly, but their levels began to decline 9-12 h following the imposition of high light. HliD was transiently expressed in high light and was not detected 24 h after the initiation of high light exposure. These results demonstrate that there is specificity to the accumulation of the Hli polypeptides under a diverse range of environmental conditions. Furthermore, mutants for the individual and combinations of the hli genes were evaluated for their fitness to grow in high light. Although all of the mutants grew as fast as wild-type cells in low light, strains inactivated for hliA or hliC/hliD were unable to compete with wild-type cells during co-cultivation in high light. A mutant lacking all four hli genes gradually lost its photosynthesis capacity and died in high light. Hence, the Hli polypeptides are critical for survival when Synechocystis PCC6803 is absorbing excess excitation energy and may allow the cells to cope more effectively with the production of reactive oxygen species.

    View details for Web of Science ID 000166280700042

    View details for PubMedID 11024039

  • Introduction. Photosynthesis research Berry, J. A., Field, C. B., Grossman, A. R. 2001; 67 (1-2): 1-3

    View details for PubMedID 16228311

  • Special issue in honour of Olle Bjorkman: Plants and their light environment - Introduction PHOTOSYNTHESIS RESEARCH Berry, J. A., Field, C. B., Grossman, A. R. 2001; 67 (1-2): 1-3
  • Macronutrient utilization by photosynthetic eukaryotes and the fabric of interactions ANNUAL REVIEW OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY Grossman, A., Takahashi, H. 2001; 52: 163-210

    Abstract

    Organisms acclimate to a continually fluctuating nutrient environment. Acclimation involves responses specific for the limiting nutrient as well as responses that are more general and occur when an organism experiences different stress conditions. Specific responses enable organisms to efficiently scavenge the limiting nutrient and may involve the induction of high-affinity transport systems and the synthesis of hydrolytic enzymes that facilitate the release of the nutrient from extracellular organic molecules or from internal reserves. General responses include changes in cell division rates and global alterations in metabolic activities. In photosynthetic organisms there must be precise regulation of photosynthetic activity since when severe nutrient limitation prevents continued cell growth, excitation of photosynthetic pigments could result in the formation of reactive oxygen species, which can severely damage structural and functional features of the cell. This review focuses on ways that photosynthetic eukaryotes assimilate the macronutrients nitrogen, sulfur, and phosphorus, and the mechanisms that govern assimilatory activities. Also discussed are molecular responses to macronutrient limitation and the elicitation of those responses through integration of environmental and cellular cues.

    View details for Web of Science ID 000169615600008

  • A gene of Synechocystis sp strain PCC 6803 encoding a novel iron transporter JOURNAL OF BACTERIOLOGY Katoh, H., Grossman, A. R., Hagino, N., Ogawa, T. 2000; 182 (22): 6523-6524

    Abstract

    A mutant of Synechocystis sp. strain PCC 6803 disrupted for sll1878 exhibited greatly reduced Fe(3+) transport activity. The K(m) value of sll1878-dependent Fe(3+) transport in cells grown in iron-replete medium was 0.5 microM. Both the maximal rate and K(m) value were increased in iron-starved cells.

    View details for Web of Science ID 000090111400032

    View details for PubMedID 11053401

  • Isolation of regulated genes of the cyanobacterium Synechocystis sp strain PCC 6803 by differential display JOURNAL OF BACTERIOLOGY Bhaya, D., Vaulot, D., Amin, P., Takahashi, A. W., Grossman, A. R. 2000; 182 (20): 5692-5699

    Abstract

    Global identification of differentially regulated genes in prokaryotes is constrained because the mRNA does not have a 3' polyadenylation extension; this precludes specific separation of mRNA from rRNA and tRNA and synthesis of cDNAs from the entire mRNA population. Knowledge of the entire genome sequence of Synechocystis sp. strain PCC 6803 has enabled us to develop a differential display procedure that takes advantage of a short palindromic sequence that is dispersed throughout the Synechocystis sp. strain PCC 6803 genome. This sequence, designated the HIP (highly iterated palindrome) element, occurs in approximately half of the Synechocystis sp. strain PCC 6803 genes but is absent in rRNA and tRNA genes. To determine the feasibility of exploiting the HIP element, alone or in combination with specific primer subsets, for analyzing differential gene expression, we used HIP-based primers to identify light intensity-regulated genes. Several gene fragments, including those encoding ribosomal proteins and phycobiliprotein subunits, were differentially amplified from RNA templates derived from cells grown in low light or exposed to high light for 3 h. One novel finding was that expression of certain genes of the pho regulon, which are under the control of environmental phosphate levels, were markedly elevated in high light. High-light activation of pho regulon genes correlated with elevated growth rates that occur when the cells are transferred from low to high light. These results suggest that in high light, the rate of growth of Synechocystis sp. strain PCC 6803 exceeds its capacity to assimilate phosphate, which, in turn, may trigger a phosphate starvation response and activation of the pho regulon.

    View details for Web of Science ID 000089576300007

    View details for PubMedID 11004166

    View details for PubMedCentralID PMC94689

  • Acclimation of Chlamydomonas reinhardtii to its nutrient environment PROTIST Grossman, A. 2000; 151 (3): 201-224

    Abstract

    To cope with low nutrient availability in nature, organisms have evolved inducible systems that enable them to scavenge and efficiently utilize the limiting nutrient. Furthermore, organisms must have the capacity to adjust their rate of metabolism and make specific alterations in metabolic pathways that favor survival when the potential for cell growth and division is reduced. In this article I will focus on the acclimation of Chlamydomonas reinhardtii, a unicellular, eukaryotic green alga to conditions of nitrogen, sulfur and phosphorus deprivation. This organism has a distinguished history as a model for classical genetic analyses, but it has recently been developed for exploitation using an array of molecular and genomic tools. The application of these tools to the analyses of nutrient limitation responses (and other biological processes) is revealing mechanisms that enable Chlamydomonas to survive harsh environmental conditions and establishing relationships between the responses of this morphologically simple, photosynthetic eukaryote and those of both nonphotosynthetic organisms and vascular plants.

    View details for Web of Science ID 000090155400001

    View details for PubMedID 11079767

  • Type IV pilus biogenesis and motility in the cyanobacterium Synechocystis sp PCC6803 MOLECULAR MICROBIOLOGY Bhaya, D., Bianco, N. R., Bryant, D., Grossman, A. 2000; 37 (4): 941-951

    Abstract

    We have recently shown that phototactic movement in the unicellular cyanobacterium Synechocystis sp. PCC6803 requires type IV pilins. To elucidate further type IV pilus-dependent motility, we inactivated key genes implicated in pilus biogenesis and function. Wild-type Synechocystis sp. PCC6803 cells have two morphologically distinct pilus types (thick and thin pili). Of these, the thick pilus morphotype, absent in a mutant disrupted for the pilin-encoding pilA1 gene, appears to be required for motility. The thin pilus morphotype does not appear to be altered in the pilA1 mutant, raising the possibility that thin pili have a function distinct from that of motility. Mutants disrupted for pilA2, which encodes a second pilin-like protein, are still motile and exhibit no difference in morphology or density of cell-surface pili. In contrast, inactivation of pilD (encoding the leader peptidase) or pilC (encoding a protein required for pilus assembly) abolishes cell motility, and both pilus morphotypes are absent. Thus, the PilA1 polypeptide is required for the biogenesis of the thick pilus morphotype which, in turn, is necessary for motility (hence we refer to them as type IV pili). Furthermore, PilA2 is critical neither for motility nor for pilus biogenesis. Two genes encoding proteins with similarity to PilT, which is considered to be a component of the motor essential for type IV pilus-dependent movement, were also inactivated. A pilT1 mutant is (i) non-motile, (ii) hyperpiliated and (iii) accumulates higher than normal levels of the pilA1 transcript. In contrast, pilT2 mutants are motile, but are negatively phototactic under conditions in which wild-type cells are positively phototactic.

    View details for Web of Science ID 000089054100022

    View details for PubMedID 10972813

  • Characterization of a gene encoding the light-harvesting violaxanthin-chlorophyll protein of Nannochloropsis sp (Eustigmatophyceae) JOURNAL OF PHYCOLOGY Sukenik, A., Livne, A., Apt, K. E., Grossman, A. R. 2000; 36 (3): 563-570
  • Chlamydomonas reinhardtii and photosynthesis: genetics to genomics CURRENT OPINION IN PLANT BIOLOGY Grossman, A. R. 2000; 3 (2): 132-137

    Abstract

    Genetic and physiological features of the green alga Chlamydomonas reinhardtii have provided a useful model for elucidating the function, biogenesis and regulation of the photosynthetic apparatus. Combining these characteristics with newly developed molecular technologies for engineering Chlamydomonas and the promise of global analyses of nuclear and chloroplast gene expression will add a new perspective to views on photosynthetic function and regulation.

    View details for Web of Science ID 000086004800007

    View details for PubMedID 10712957

  • Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes JOURNAL OF PHYCOLOGY Zaslavskaia, L. A., Lippmeier, J. C., Kroth, P. G., Grossman, A. R., Apt, K. E. 2000; 36 (2): 379-386
  • A pigment-binding protein essential for regulation of photosynthetic light harvesting NATURE Li, X. P., Bjorkman, O., Shih, C., Grossman, A. R., Rosenquist, M., Jansson, S., Niyogi, K. K. 2000; 403 (6768): 391-395

    Abstract

    Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plant's capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

    View details for Web of Science ID 000085121100039

    View details for PubMedID 10667783

  • Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wykoff, D. D., Grossman, A. R., Weeks, D. P., Usuda, H., Shimogawara, K. 1999; 96 (26): 15336-15341

    Abstract

    Understanding the ways in which phosphorus metabolism is regulated in photosynthetic eukaryotes is critical for optimizing crop productivity and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene encoding a regulator of phosphorus metabolism, designated Psr1 (phosphorus starvation response), from a photosynthetic eukaryote. The Psr1 protein is critical for acclimation of the unicellular green alga Chlamydomonas reinhardtii to phosphorus starvation. The N-terminal half of Psr1 contains a region similar to myb DNA-binding domains and the C-terminal half possesses glutamine-rich sequences characteristic of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies demonstrate that this protein is nuclear-localized under both nutrient-replete and phosphorus-starvation conditions. Finally, Psr1 and angiosperm proteins have domains that are similar, suggesting a possible role for Psr1 homologs in the control of phosphorus metabolism in vascular plants. With the identification of regulators such as Psr1 it may become possible to engineer photosynthetic organisms for more efficient utilization of phosphorus and to establish better practices for the management of agricultural lands and natural ecosystems.

    View details for Web of Science ID 000084375400118

    View details for PubMedID 10611385

  • Chlamydomonas reinhardtii mutants abnormal in their responses to phosphorus deprivation PLANT PHYSIOLOGY Shimogawara, K., Wykoff, D. D., Usuda, H., Grossman, A. R. 1999; 120 (3): 685-693

    Abstract

    P-starved plants scavenge inorganic phosphate (Pi) by developing elevated rates of Pi uptake, synthesizing extracellular phosphatases, and secreting organic acids. To elucidate mechanisms controlling these acclimation responses in photosynthetic organisms, we characterized the responses of the green alga Chlamydomonas reinhardtii to P starvation and developed screens for isolating mutants (designated psr [phosphorus-stress response]) abnormal in their responses to environmental levels of Pi. The psr1-1 mutant was identified in a selection for cells that survived exposure to high concentrations of radioactive Pi. psr1-2 and psr2 were isolated as strains with aberrant levels of extracellular phosphatase activity during P-deficient or nutrient-replete growth. The psr1-1 and psr1-2 mutants were phenotypically similar, and the lesions in these strains were recessive and allelic. They exhibited no increase in extracellular phosphatase activity or Pi uptake upon starvation. Furthermore, when placed in medium devoid of P, the psr1 strains lost photosynthetic O2 evolution and stopped growing more rapidly than wild-type cells; they may not be as efficient as wild-type cells at scavenging/accessing P stores. In contrast, psr2 showed elevated extracellular phosphatase activity during growth in nutrient-replete medium, and the mutation was dominant. The mutant phenotypes and the roles of Psr1 and Psr2 in P-limitation responses are discussed.

    View details for Web of Science ID 000081437500006

    View details for PubMedID 10398703

  • Trafficking of proteins from chloroplast to vacuoles: Perspectives and directions JOURNAL OF PHYCOLOGY Hoffman, N., Grossman, A. R. 1999; 35 (3): 443-445
  • Sac3, an Snf1-like serine threonine kinase that positively and negatively regulates the responses of chlamydomonas to sulfur limitation PLANT CELL Davies, J. P., Yildiz, F. H., Grossman, A. R. 1999; 11 (6): 1179-1190

    Abstract

    The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation. In wild-type cells, arylsulfatase activity is detected only during sulfur limitation. The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity. In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system-the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation. We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene. The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK). Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants. In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region. The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed.

    View details for Web of Science ID 000081359300017

    View details for PubMedID 10368187

  • The role of an alternative sigma factor in motility and pilus formation in the cyanobacterium Synechocystis sp. strain PCC6803 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bhaya, D., Watanabe, N., Ogawa, T., Grossman, A. R. 1999; 96 (6): 3188-3193

    Abstract

    Disruption of a gene for an alternative sigma factor, designated sigF, in the freshwater, unicellular cyanobacterium Synechocystis sp. strain PCC6803 resulted in a pleiotropic phenotype. Most notably, this mutant lost phototactic movement with a concomitant loss of pili, which are abundant on the surface of wild-type cells. The sigF mutant also secreted both high levels of yellow-brown and UV-absorbing pigments and a polypeptide that is similar to a large family of extracellular proteins that includes the hemolysins. Furthermore, the sigF mutant had a dramatically reduced level of the transcript from two tandemly arranged pilA genes (sll1694 and sll1695), which encode major structural components of type IV pili. Inactivation of these pilA genes eliminated phototactic movement, though some pili were still present in this strain. Together, these results demonstrate that SigF plays a critical role in motility via the control of pili formation and is also likely to regulate other features of the cell surface. Furthermore, the data provide evidence that type IV pili are required for phototactic movement in certain cyanobacteria and suggest that different populations of pili present on the Synechocystis cell surface may perform different functions.

    View details for Web of Science ID 000079224500113

    View details for PubMedID 10077659

    View details for PubMedCentralID PMC15917

  • A proposal for extending the nomenclature of light-harvesting proteins of the three transmembrane helix type PLANT MOLECULAR BIOLOGY REPORTER Jansson, S., Green, B., Grossman, A. R., Hiller, R. 1999; 17 (3): 221-224
  • Identification of a gene involved in the acclimation of Chlamydomonas reinhardtii to phosphorus starvation 12th Annual Penn State Symposium in Plant Physiology Shimogawara, K., Wykoff, D. D., Weeks, D. P., Kovar, J. L., Tsuzuki, M., Usuda, H., Grossman, A. R. AMER SOC PLANT BIOLOGISTS. 1999: 361–364
  • A polypeptide with similarity to phycocyanin alpha-subunit phycocyanobilin lyase involved in degradation of phycobilisomes JOURNAL OF BACTERIOLOGY Dolganov, N., Grossman, A. R. 1999; 181 (2): 610-617

    Abstract

    To optimize the utilization of photosynthate and avoid damage that can result from the absorption of excess excitation energy, photosynthetic organisms must rapidly modify the synthesis and activities of components of the photosynthetic apparatus in response to environmental cues. During nutrient-limited growth, cyanobacteria degrade their light-harvesting complex, the phycobilisome, and dramatically reduce the rate of photosynthetic electron transport. In this report, we describe the isolation and characterization of a cyanobacterial mutant that does not degrade its phycobilisomes during either sulfur or nitrogen limitation and exhibits an increased ratio of phycocyanin to chlorophyll during nutrient-replete growth. The mutant phenotype was complemented by a gene encoding a polypeptide with similarities to polypeptides that catalyze covalent bond formation between linear tetrapyrrole chromophores and subunits of apophycobiliproteins. The complementing gene, designated nblB, is expressed at approximately the same level in cells grown in nutrient-replete medium and medium devoid of either sulfur or nitrogen. These results suggest that the NblB polypeptide may be a constitutive part of the machinery that coordinates phycobilisome degradation with environmental conditions.

    View details for Web of Science ID 000078040200033

    View details for PubMedID 9882677

  • The use of Chlamydomonas (Chlorophyta : Volvocales) as a model algal system for genome studies and the elucidation of photosynthetic processes JOURNAL OF PHYCOLOGY Davies, J. P., Grossman, A. R. 1998; 34 (6): 907-917
  • Bilin deletions and subunit stability in cyanobacterial light-harvesting proteins MOLECULAR MICROBIOLOGY Toole, C. M., Plank, T. L., Grossman, A. R., Anderson, L. K. 1998; 30 (3): 475-486

    Abstract

    Light-harvesting in cyanobacteria and red algae is a function of the biliproteins, which have covalently bound bilin chromophores. The biliproteins are assembled with linker proteins into the phycobilisome, a large complex that resides on the surface of the photosynthetic membranes. Early steps in the phycobilisome assembly pathway include the folding of biliprotein alpha- and beta-subunits, covalent modification of subunits by bilin attachment and formation of the primary assembly unit, the alphabeta heterodimer. The potential role of bilins in subunit structure and assembly is examined in this study by site mutagenesis of biliprotein genes. Phycocyanin subunits from Synechocystis sp. 6701 that were unable to bind chromophores at specific sites were generated by changing the codons for bilin-binding cysteines to alanine residues. The altered genes were then expressed in a phycocyanin-minus mutant of the transformable Synechocystis sp. strain 6803. Single and multiple chromophore deletions cause specific and reproducible variations in phycobilisome-associated phycocyanin that do not correlate with transcript levels. Sedimentation equilibrium studies with purified proteins showed that bilin absence reduces the strength of alphabeta interaction in the heterodimer. These results suggest that phycocyanin instability in bilin-deletion mutants is a consequence of diversion of unassembled alpha- and beta-subunits to a degradation pathway. Attachment of the central bilin, which is common to all biliprotein subunits, may facilitate alphabeta interaction by completing the final stage of subunit folding and stabilizing the contact domains of binding partners in the heterodimer.

    View details for Web of Science ID 000076893000003

    View details for PubMedID 9822814

  • A response regulator of cyanobacteria integrates diverse environmental signals and is critical for survival under extreme conditions PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schwarz, R., Grossman, A. R. 1998; 95 (18): 11008-11013

    Abstract

    Microorganisms must sense their environment and rapidly tune their metabolism to ambient conditions to efficiently use available resources. We have identified a gene encoding a response regulator, NblR, that complements a cyanobacterial mutant unable to degrade its light-harvesting complex (phycobilisome), in response to nutrient deprivation. Cells of the nblR mutant (i) have more phycobilisomes than wild-type cells during nutrient-replete growth, (ii) do not degrade phycobilisomes during sulfur, nitrogen, or phosphorus limitation, (iii) cannot properly modulate the phycobilisome level during exposure to high light, and (iv) die rapidly when starved for either sulfur or nitrogen, or when exposed to high light. Apart from regulation of phycobilisome degradation, NblR modulates additional functions critical for cell survival during nutrient-limited and high-light conditions. NblR does not appear to be involved in acclimation responses that occur only during a specific nutrient limitation. In contrast, it controls at least some of the general acclimation responses; those that occur during any of a number of different stress conditions. NblR plays a pivotal role in integrating different environmental signals that link the metabolism of the cell to light harvesting capabilities and the activities of the photosynthetic apparatus; this modulation is critical for cell survival.

    View details for Web of Science ID 000075730500118

    View details for PubMedID 9724820

  • Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion PLANT CELL Niyogi, K. K., Grossman, A. R., Bjorkman, O. 1998; 10 (7): 1121-1134

    Abstract

    A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.

    View details for Web of Science ID 000074952100007

    View details for PubMedID 9668132

  • The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii PLANT PHYSIOLOGY Wykoff, D. D., Davies, J. P., Melis, A., Grossman, A. R. 1998; 117 (1): 129-139

    Abstract

    The light-saturated rate of photosynthetic O2 evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O2 evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150-2159).

    View details for Web of Science ID 000073660500015

    View details for PubMedID 9576782

  • High-efficiency transformation of Chlamydomonas reinhardtii by electroporation GENETICS Shimogawara, K., Fujiwara, S., Grossman, A., Usuda, H. 1998; 148 (4): 1821-1828

    Abstract

    We have established a high-efficiency method for transforming the unicellular, green alga Chlamydomonas reinhardtii by electroporation. Electroporation of strains CC3395 and CC425, cell wall-less mutants devoid of argininosuccinate lyase (encoded by ARG7), in the presence of the plasmid pJD67 (which contains ARG7) was used to optimize conditions for the introduction of exogenous DNA. The conditions that were varied included osmolarity, temperature, concentration of exogenous DNA, voltage and capacitance. Following optimization, the maximum transformation frequency obtained was 2 x 10(5) transformants per microg of DNA; this frequency is two orders of magnitude higher than obtained with the current standard method using glass beads to introduce exogenous DNA. The electroporation procedure described in this article is of general utility, and makes it feasible to isolate genes by direct complementation of Chlamydomonas reinhardtii mutants.

    View details for Web of Science ID 000073187000041

    View details for PubMedID 9560396

  • Use of molecular genetics to investigate complementary chromatic adaptation: Advances in transformation and complementation PHOTOSYNTHESIS: MOLECULAR BIOLOGY OF ENERGY CAPTURE Kehoe, D. M., Grossman, A. R. 1998; 297: 279-290
  • Phycobilisome degradation and responses of cyanobacteria to nutrient limitation and high light XIth International Congress on Photosynthesis - Mechanisms and Effects Grossman, A. R., Schwarz, R., Bhaya, D., Dolganov, N. SPRINGER. 1998: 2853–2858
  • The roles of specific xanthophylls in photoprotection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Niyogi, K. K., Bjorkman, O., Grossman, A. R. 1997; 94 (25): 14162-14167

    Abstract

    Xanthophyll pigments have critical structural and functional roles in the photosynthetic light-harvesting complexes of algae and vascular plants. Genetic dissection of xanthophyll metabolism in the green alga Chlamydomonas reinhardtii revealed functions for specific xanthophylls in the nonradiative dissipation of excess absorbed light energy, measured as nonphotochemical quenching of chlorophyll fluorescence. Mutants with a defect in either the alpha- or beta-branch of carotenoid biosynthesis exhibited less nonphotochemical quenching but were still able to tolerate high light. In contrast, a double mutant that was defective in the synthesis of lutein, loroxanthin (alpha-carotene branch), zeaxanthin, and antheraxanthin (beta-carotene branch) had almost no nonphotochemical quenching and was extremely sensitive to high light. These results strongly suggest that in addition to the xanthophyll cycle pigments (zeaxanthin and antheraxanthin), alpha-carotene-derived xanthophylls such as lutein, which are structural components of the subunits of the light-harvesting complexes, contribute to the dissipation of excess absorbed light energy and the protection of plants from photo-oxidative damage.

    View details for Web of Science ID A1997YK82500143

    View details for PubMedID 9391170

  • DNA insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii PHOTOSYNTHESIS RESEARCH Zhang, L. P., Niyogi, K. K., Baroli, I., Nemson, J. A., Grossman, A. R., Melis, A. 1997; 53 (2-3): 173-184
  • Phosphorelay control of phycobilisome biogenesis during complementary chromatic adaptation PHOTOSYNTHESIS RESEARCH Grossman, A. R., Kehoe, D. M. 1997; 53 (2-3): 95-108
  • Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching. The Plant cell Niyogi, K. K., Bjorkman, O., Grossman, A. R. 1997; 9 (8): 1369-1380

    Abstract

    The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light.

    View details for DOI 10.1105/tpc.9.8.1369

    View details for PubMedID 12237386

    View details for PubMedCentralID PMC157004

  • Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching PLANT CELL Niyogi, K. K., Bjorkman, O., Grossman, A. R. 1997; 9 (8): 1369-1380
  • Suppression of mutants aberrant in light intensity responses of complementary chromatic adaptation JOURNAL OF BACTERIOLOGY Casey, E. S., Kehoe, D. M., Grossman, A. R. 1997; 179 (14): 4599-4606

    Abstract

    Complementary chromatic adaptation is a process in which cyanobacteria alter the pigment protein (phycocyanin and phycoerythrin) composition of their light-harvesting complexes, the phycobilisomes, to help optimize the absorbance of prevalent wavelengths of light in the environment. Several classes of mutants that display aberrant complementary chromatic adaptation have been isolated. One of the mutant classes, designated "blue" or FdB, accumulates high levels of the blue chromoprotein phycocyanin in low-intensity green light, a condition that normally suppresses phycocyanin synthesis. We demonstrate here that the synthesis of the phycocyanin protein and mRNA in the FdB mutants can be suppressed by increasing the intensity of green light. Hence, these mutants have a decreased sensitivity to green light with respect to suppression of phycocyanin synthesis. Although we were unable to complement the blue mutants, we did isolate genes that could suppress the mutant phenotype. These genes, which have been identified previously, encode a histidine kinase sensor and response regulator protein that play key roles in controlling complementary chromatic adaptation. These findings are discussed with respect to the mechanism by which light quality and quantity control the biosynthesis of the phycobilisome.

    View details for Web of Science ID A1997XM20800019

    View details for PubMedID 9226271

  • New classes of mutants in complementary chromatic adaptation provide evidence for a novel four-step phosphorelay system JOURNAL OF BACTERIOLOGY Kehoe, D. M., Grossman, A. R. 1997; 179 (12): 3914-3921

    Abstract

    Complementary chromatic adaptation appears to be controlled by a complex regulatory system with similarity to four-step phosphorelays. Such pathways utilize two histidine and two aspartate residues for signal transduction. Previous studies of the signaling system controlling complementary chromatic adaptation have uncovered two elements of this pathway, a putative sensor, RcaE, and a response regulator, RcaC. In this work, we describe a second response regulator controlling complementary chromatic adaptation, RcaF, and identify putative DNA binding and histidine phosphoacceptor domains within RcaC. RcaF is a small response regulator with similarity to SpoOF of Bacillus subtilis; the latter functions in the four-step phosphorelay system controlling sporulation. We have also determined that within this phosphorelay pathway, RcaE precedes RcaF, and RcaC is probably downstream of RcaE and RcaF. This signal transduction pathway is novel because it appears to use at least five, instead of four, phosphoacceptor domains in the phosphorelay circuit.

    View details for Web of Science ID A1997XE30000012

    View details for PubMedID 9190806

  • Stable nuclear transformation of the diatom Phaeodactylum tricornutum MOLECULAR & GENERAL GENETICS Apt, K. E., KROTHPANCIC, P. G., Grossman, A. R. 1996; 252 (5): 572-579

    Abstract

    A nuclear transformation system has been developed for the diatom Phaeodactylum tricornutum using microparticle bombardment to introduce the sh ble gene from Streptoalloteichus hindustanus into cells. The sh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from the P. tricornutum fcp genes were used to drive expression of sh ble. Between 10-100 transformants were recovered/10(8) cells. Transformants were able to grow on at least 500 micrograms/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations the sh ble gene was present in 1-3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1-2.0 micrograms/mg total protein). The cat reporter gene fused to a fcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed in P. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.

    View details for Web of Science ID A1996VQ51400009

    View details for PubMedID 8914518

  • Sulfur availability and the SAC1 gene control adenosine triphosphate sulfurylase gene expression in Chlamydomonas reinhardtii PLANT PHYSIOLOGY Yildiz, F. H., Davies, J. P., Grossman, A. 1996; 112 (2): 669-675

    Abstract

    A Chlamydomonas reinhardtii adenosine triphosphate (ATP) sulfurylase cDNA clone (pATS1) was selected by complementing a mutation in the ATP sulfurylase gene (cysD) of Escherichia coli. E. coli cysD strains harboring pATS1 grow on medium containing sulfate as the sole sulfur source and exhibit ATP sulfurylase activity. The amino acid sequence of the C. reinhardtii ATP sulfurylase, derived from the nucleotide sequence of the complementing gene (ATS1), is 25 to 40% identical to that of ATP sulfurylases in other eukaryotic organisms and has a putative transit peptide at its amino terminus. ATP sulfurylase mRNA was present when cells were grown in sulfur-replete medium, but accumulated to higher levels when the cells were exposed to sulfur-limiting conditions. Furthermore, sulfur-stress-induced accumulation of the ATS1 transcript was reduced in a strain defective in SAC1, a gene that is critical for acclimation to sulfur-limited growth.

    View details for Web of Science ID A1996VP17900025

    View details for PubMedID 8883379

  • Similarity of a chromatic adaptation sensor to phytochrome and ethylene receptors SCIENCE Kehoe, D. M., Grossman, A. R. 1996; 273 (5280): 1409-1412

    Abstract

    Complementary chromatic adaptation in cyanobacteria acts through photoreceptors to control the biosynthesis of light-harvesting complexes. The mutant FdBk, which appears black, cannot chromatically adapt and may contain a lesion in the apparatus that senses light quality. The complementing gene identified here, rcaE, encodes a deduced protein in which the amino-terminal region resembles the chromophore attachment domain of phytochrome photoreceptors and regions of plant ethylene receptors; the carboxyl- terminal half is similar to the histidine kinase domain of two-component sensor kinases.

    View details for Web of Science ID A1996VF61000049

    View details for PubMedID 8703080

  • Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii PLANT PHYSIOLOGY Quisel, J. D., Wykoff, D. D., Grossman, A. R. 1996; 111 (3): 839-848

    Abstract

    We have examined the extracellular phosphatases produced by the terrestrial green alga Chlamydomonas reinhardtii in response to phosphorus deprivation. Phosphorus-deprived cells increase extra-cellular alkaline phosphatase activity 300-fold relative to unstarved cells. The alkaline phosphatases are released into the medium by cell-wall-deficient strains and by wild-type cells after treatment with autolysin, indicating that they are localized to the periplasm. Anion-exchange chromatography and analysis by nondenaturing polyacrylamide gel electrophoresis revealed that there are two major inducible alkaline phosphatases. A calcium-dependent enzyme composed of 190-kD glycoprotein subunits accounts for 85 to 95% of the Alkaline phosphatase activity. This phosphatase has optimal activity at pH 9.5 and a Km of 120 to 262 microns for all physiological substrates tested, with the exception of phytic acid, which it cleaved with a 50-fold lower efficiency. An enzyme with optimal activity at pH 9 and no requirement for divalent cations accounts for 2 to 10% of the alkaline phosphatase activity. This phosphatase was only able to efficiently hydrolyze arylphosphates. The information reported here, in conjunction with the results of previous studies, defines the complement of extracellular phosphatases produced by phosphorus-deprived Chlamydomonas cells.

    View details for Web of Science ID A1996UX88100023

    View details for PubMedID 8754684

  • Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation EMBO JOURNAL Davies, J. P., Yildiz, F. H., Grossman, A. 1996; 15 (9): 2150-2159

    Abstract

    The sac1 mutant of Chlamydomonas reinhardtii is aberrant in most of the normal responses to sulfur limitation; it cannot synthesize arylsulfatase, does not take up sulfate as rapidly as wild-type cells, and does not synthesize periplasmic proteins that normally accumulate during sulfur-limited growth. Here, we show that the sac1 mutant dies much more rapidly than wild-type cells during sulfur deprivation; this emphasizes the vital role of the acclimation process. The loss of viability of the sac1 mutant during sulfur deprivation is only observed in the light and is mostly inhibited by DCMU. During sulfur-stress, wild-type cells, but not the sac1 mutant, downregulate photosynthesis. Thus, death of the sac1 mutant during sulfur deprivation is probably a consequence of its inability to downregulate photosynthesis. Furthermore, since SAC1 is necessary for the downregulation of photosynthesis, the process must be highly controlled and not simply the result of a general decrease in protein synthesis due to sulfur limitation. Genomic and cDNA copies of the SAC1 gene have been cloned. The deduced amino acid sequence of Sac1 is similar to an Escherichia coli gene that may involved in the response of E.coli to nutrient deprivation.

    View details for Web of Science ID A1996UK07400012

    View details for PubMedID 8641280

  • DISRUPTION OF A GENE ENCODING A NOVEL THIOREDOXIN-LIKE PROTEIN ALTERS THE CYANOBACTERIAL PHOTOSYNTHETIC APPARATUS JOURNAL OF BACTERIOLOGY Collier, J. L., Grossman, A. R. 1995; 177 (11): 3269-3276

    Abstract

    A gene that may encode a novel protein disulfide oxidoreductase, designated txlA (thioredoxin-like), was isolated from the cyanobacterium Synechococcus sp. strain PCC7942. Interruption of txlA near the putative thioredoxin-like active site yielded cells that grew too poorly to be analyzed. In contrast, a disruption of txlA near the C terminus that left the thioredoxin-like domain intact yielded two different mutant phenotypes. One type, designated txlXb, exhibited a slightly reduced growth rate and an increased cellular content of apparently normal phycobilisomes. The cellular content of phycobilisomes also increased in in the other mutant strain, designated txlXg. However, txlXg also exhibited a proportionate increase in chlorophyll and other components of the photosynthetic apparatus and grew as fast as wild-type cells. Both the txlXb and txlXg phenotypes were stable. The differences between the two strains may result from a genetic polymorphism extant in the original cell population. Further investigation of txlA may provide new insights into mechanisms that regulate the structure and function of the cyanobacterial photosynthetic apparatus.

    View details for Web of Science ID A1995RA62200042

    View details for PubMedID 7768827

  • EVOLUTION OF THE PHYCOBILIPROTEINS JOURNAL OF MOLECULAR BIOLOGY Apt, K. E., Collier, J. L., Grossman, A. R. 1995; 248 (1): 79-96

    Abstract

    Amino acid sequence alignments and phylogenetic analyses have been used to examine the relationships among 100 phycobiliprotein sequences. The alignments revealed a number of highly conserved amino acid residues that are involved in chromophore attachment and conformation, alpha-beta interactions and phycobilisome assembly. The phylogenetic analysis confirmed that the phycobiliprotein subfamilies, previously classified by their biochemical and spectroscopic properties, also formed coherent evolutionary groups. The alpha and beta subunits formed two distinct evolutionary lines that originate from a common ancestor. The pattern of divergence among the alpha subfamilies was identical to that of the beta subfamilies, strongly suggesting that the alpha and beta subunits of each phycobiliprotein type have coevolved. The phylogenetic data support a monophyletic separation of the eukaryotic sequences from the extant cyanobacterial sequences. The eukaryotic phycoerythrins appeared more closely related to the marine Synechococcus phycoerythrins than to the other cyanobacterial phycoerythrins. The cryptophyte phycobiliproteins formed a monophyletic group within the rhodophyte lineage. In conjunction with other phylogenetic markers, the analysis of additional phycobiliprotein sequences may help to further resolve the relationships among phycobiliprotein-containing organisms.

    View details for Web of Science ID A1995QU29900006

    View details for PubMedID 7731046

  • THE STRUCTURE OF PHYCOBILISOMES IN MUTANTS OF SYNECHOCOCCUS SP STRAIN PCC-7942 DEVOID OF SPECIFIC LINKER POLYPEPTIDES PHOTOCHEMISTRY AND PHOTOBIOLOGY Bhalerao, R. P., Collier, J. L., Gustafsson, P., Grossman, A. R. 1995; 61 (3): 298-302
  • THE GENE FAMILY ENCODING THE FUCOXANTHIN CHLOROPHYLL PROTEINS FROM THE BROWN ALGA MACROCYSTIS-PYRIFERA MOLECULAR & GENERAL GENETICS Apt, K. E., Clendennen, S. K., Powers, D. A., Grossman, A. R. 1995; 246 (4): 455-464

    Abstract

    Six members of a multigene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex from female gametophytes of the brown alga Macrocystis pyrifera have been cloned and characterized. The deduced amino acid sequences are very similar to those of fucoxanthin chlorophyll binding proteins (Fcp) from the diatom Phaeodactylum tricornutum and exhibit limited homology to chlorophyll a/b binding (Cab) polypeptides from higher plants. The primary translation products from the M. pyrifera fcp genes are synthesized as higher molecular weight precursors that are processed prior to their assembly into the Fcp complex. The presumed N-terminal 40-amino acid presequence of the Fcp precursor polypeptide has features resembling that of a signal sequence. This presequence may be required for the protein to transverse the endoplasmic reticulum that surrounds the plastid in brown algae. A subsequent targeting step would be required for the protein to cross the double membrane of the plastid envelope. M. pyrifera fcp transcripts are of two sizes, 1.2 and 1.6 kb. The size difference is accounted for by the length of the 3' untranslated region, which can be up to 1000 bases. Transcript abundance's of members of the fcp gene family are dependent on light quantity, light quality, or both. Transcript levels of one gene increased approximately five- to tenfold in thalli grown in low intensity relative to high intensity white or blue light. Transcripts from this gene also significantly increase in red light relative to blue light at equivalent light intensities.

    View details for Web of Science ID A1995QL64300007

    View details for PubMedID 7891659

  • CYANOBACTERIAL PROTEIN WITH SIMILARITY TO THE CHLOROPHYLL A/B BINDING-PROTEINS OF HIGHER-PLANTS - EVOLUTION AND REGULATION PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA DOLGANOV, N. A., Bhaya, D., Grossman, A. R. 1995; 92 (2): 636-640

    Abstract

    We have isolated, from the prokaryotic cyanobacterium Synechococcus sp. strain PCC 7942, a gene encoding a protein of 72 amino acids [designated high light inducible protein (HLIP)] with similarity to the extended family of eukaryotic chlorophyll a/b binding proteins (CABs). HLIP has a single membrane-spanning alpha-helix, whereas both the CABs and the related early light inducible proteins have three membrane-spanning helices. Hence, HLIP may represent an evolutionary progenitor of the eukaryotic members of the CAB extended family. We also show that the gene encoding HLIP is induced by high light and blue/UV-A radiation. The evolution, regulation, and potential function of HLIP are discussed.

    View details for Web of Science ID A1995QC87400063

    View details for PubMedID 7831342

    View details for PubMedCentralID PMC42797

  • The use of site directed mutagenesis in the analysis of complementary chromatic adaptation Xth International Photosynthesis Congress Kehoe, D., Grossman, A. SPRINGER. 1995: 501–504
  • Light-harvesting complexes in oxygenic photosynthesis: Diversity, control, and evolution ANNUAL REVIEW OF GENETICS Grossman, A. R., Bhaya, D., Apt, K. E., Kehoe, D. M. 1995; 29: 231-288

    Abstract

    This article focuses on light-harvesting complexes (LHCs) in oxygen evolving photosynthetic organisms. These organisms include cyanobacteria, red algae, plants, green algae, brown algae, diatoms, chrysophytes, and dinoflagellates. We highlight the diversity of pigment-protein complexes that fuel the conversion of radiant energy to chemical bond energy in land plants and the diverse groups of the algae, detail the ways in which environmental parameters (i.e. light quantity and quality, nutrients) modulate the synthesis of these complexes, and discuss the evolutionary relationships among the LHC structural polypeptides.

    View details for Web of Science ID A1995TL71900010

    View details for PubMedID 8825475

  • CHANGES IN THE CYANOBACTERIAL PHOTOSYNTHETIC APPARATUS DURING ACCLIMATION TO MACRONUTRIENT DEPRIVATION PHOTOSYNTHESIS RESEARCH Collier, J. L., Herbert, S. K., FORK, D. C., Grossman, A. R. 1994; 42 (3): 173-183
  • Complementary chromatic adaptation: photoperception to gene regulation. Seminars in cell biology Kehoe, D. M., Grossman, A. R. 1994; 5 (5): 303-313

    Abstract

    Many photosynthetic organisms can acclimate to the quantity and quality of light present in their environment. In certain cyanobacteria the wavelengths of light in the environment control the synthesis of specific polypeptides of the light harvesting antenna complex or phycobilisome. This phenomenon, called complementary chromatic adaptation, is most dramatically observed in a comparison of cyanobacteria after growth in green light and red light. In red light-grown cells the phycobilisome is largely composed of phycocyanin and its associated linker polypeptides (the latter are important for the assembly of the phycocyanin subunits and their placement within the light harvesting structure); the organisms appear blue-green in color. In green light-grown cells the phycobilisome is largely composed of phycoerythrin and its associated linker polypeptides; the organisms appear red in color. The ways in which these cyanobacteria sense their changing light environment and the regulatory elements involved in controlling the process of complementary chromatic adaptation are discussed in this review.

    View details for PubMedID 7881070

  • IN-VIVO AND IN-VITRO CHARACTERIZATION OF THE LIGHT-REGULATED CPCB2A2 PROMOTER OF FREMYELLA DIPLOSIPHON JOURNAL OF BACTERIOLOGY Casey, E. S., Grossman, A. 1994; 176 (20): 6362-6374

    Abstract

    When exposed to different spectral qualities of light, many cyanobacteria dramatically alter their phycobilisome rod composition in a process termed complementary chromatic adaptation. In the cyanobacterium Fremyella diplosiphon, this response is associated with differential expression of the cpcB2A2, cpeBA, and cpeCDE operons, which code for the phycobiliproteins phycocyanin and phycoerythrin and the phycoerythrin linker polypeptides, respectively. To define components of the signal transduction pathway involved in light-regulated expression of genes encoding phycobilisome polypeptides, we have used in vivo and in vitro techniques to identify cis-acting sequences and trans-acting factors necessary for the regulation of the red-light-inducible cpcB2A2 operon. Deletion of the cpcB2A2 upstream sequences to -76 bp with respect to the transcription start site had no effect on red-light induction of a cpcB2A2-beta-glucuronidase (GUS) chimeric gene, while deletion to -37 bp abolished GUS expression. Furthermore, a fragment of the cpcB2A2 gene from -76 to +25 bp linked to the untranslated leader of cpcB1A1 (a constitutively expressed operon encoding phycocyanin) is sufficient to drive high-level GUS expression in red light. Therefore, the sequence between positions -76 and -37 is necessary for the expression of cpcB2A2, and the region extending from -76 to +25 is sufficient for red-light induction of the operon. Attempts were made to correlate the in vivo data with protein binding in the region upstream of the transcription start site of cpcB2A2. Using in vitro analysis, we detected two protein-binding sites in the cpcB2A2 promoter which were localized to positions -162 to -122 and -37 to +25. Proteins from both red- and green-light-grown cells interacted with the former site, while only proteins present in extracts from red-light-grown cells interacted with the latter site. The data from both the in vivo and in vitro analyses suggest that while two regions upstream of the cpcB2A2 transcription initiation site specifically bind proteins, only the binding site bordering the transcription start site is important for complementary chromatic adaptation.

    View details for Web of Science ID A1994PK77200026

    View details for PubMedID 7929008

    View details for PubMedCentralID PMC196978

  • SEQUENCES CONTROLLING TRANSCRIPTION OF THE CHLAMYDOMONAS-REINHARDTII BETA(2)-TUBULIN GENE AFTER DEFLAGELLATION AND DURING THE CELL-CYCLE MOLECULAR AND CELLULAR BIOLOGY Davies, J. P., Grossman, A. R. 1994; 14 (8): 5165-5174

    Abstract

    In Chlamydomonas reinhardtii, transcripts from the beta 2-tubulin gene (tubB2), as well as those from other tubulin-encoding genes, accumulate immediately after flagellar excision as well as at a specific time in the cell cycle. Control of tubB2 transcript accumulation following deflagellation is regulated, at least partially, at the transcriptional level. We have fused the tubB2 promoter to the arylsulfatase (ars) reporter gene, introduced this construct into C. reinhardtii, and compared expression of the chimeric gene with that of the endogenous tubB2 gene. After flagellar excision, transcripts from the tubB2/ars chimeric gene accumulate with kinetics similar to those of transcripts from the endogenous tubB2 gene. The tubB2/ars transcripts also accumulate in a cell cycle-specific manner; however, chimeric transcripts are more abundant earlier in the cell cycle than the endogenous tubB2 transcripts. To elucidate transcriptional control of tubB2, we have mutated or removed sequences in the tubB2 promoter and examined the effect on transcription. The tubB2 promoter shares features with the promoters of other tubulin-encoding genes; these include a GC-rich region between the TATA box and the transcription initiation site and multiple copies of a 10-bp sequence motif that we call the tub box. The tubB2 gene contains seven tub box motifs. Changing the GC-rich region to an AT-rich region or removing three of the seven tub box motifs did not significantly affect transcription of the chimeric gene. However, removing four or five tub box motifs prevented increased transcription following deflagellation and diminished cell cycle-regulated transcription from the tubB2 promoter.

    View details for Web of Science ID A1994NY42900015

    View details for PubMedID 8035797

  • CHARACTERIZATION OF GENES ENCODING THE LIGHT-HARVESTING PROTEINS IN DIATOMS - BIOGENESIS OF THE FUCOXANTHIN CHLOROPHYLL-A/C PROTEIN COMPLEX Colloquium on Microalgal Biotechnology and Commercial Applications, at the 47th Annual Meeting of the Phycological-Society-of-America Apt, K. E., Bhaya, D., Grossman, A. R. KLUWER ACADEMIC PUBL. 1994: 225–30
  • Characterization of Sulfate Transport in Chlamydomonas reinhardtii during Sulfur-Limited and Sulfur-Sufficient Growth. Plant physiology Yildiz, F. H., Davies, J. P., Grossman, A. R. 1994; 104 (3): 981-987

    Abstract

    We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.

    View details for PubMedID 12232142

    View details for PubMedCentralID PMC160696

  • CHARACTERIZATION OF SULFATE TRANSPORT IN CHLAMYDOMONAS-REINHARDTII DURING SULFUR-LIMITED AND SULFUR-SUFFICIENT GROWTH PLANT PHYSIOLOGY Yildiz, F. H., Davies, J. P., Grossman, A. R. 1994; 104 (3): 981-987
  • A SMALL POLYPEPTIDE TRIGGERS COMPLETE DEGRADATION OF LIGHT-HARVESTING PHYCOBILIPROTEINS IN NUTRIENT-DEPRIVED CYANOBACTERIA EMBO JOURNAL Collier, J. L., Grossman, A. R. 1994; 13 (5): 1039-1047

    Abstract

    Phycobilisomes are the multiprotein complexes predominantly responsible for harvesting light energy in cyanobacteria and some eukaryotic algae. When the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient, the phycobilisomes are specifically and rapidly degraded. Degradation may be either partial (after phosphorus deprivation) or complete (after sulfur or nitrogen deprivation). We have developed a visual screen to obtain mutants unable to degrade their phycobilisomes upon nutrient starvation. Complementation of one of these mutants led to the identification of a gene, designated nblA, that encodes a 59 amino acid polypeptide essential for phycobilisome degradation. Transcription of nblA increases dramatically in sulfur- or nitrogen-deprived cells and moderately in phosphorus-deprived cells. Using the phosphorus-regulated alkaline phosphatase (phoA) promoter as a tool, we engineered constructs from which we could control the expression of either sense or antisense nblA. Increased expression of sense nbLA caused complete phycobilisome degradation during phosphorus deprivation, while expression of antisense nblA prevented phycobilisome degradation. Hence, nblA is necessary, and may be sufficient, for the degradation of phycobilisomes under adverse environmental conditions. Further investigation of the mechanism by which nblA causes phycobilisome destruction may reveal general principles that govern the specificity of macromolecular complex degradation.

    View details for Web of Science ID A1994NA28800005

    View details for PubMedID 8131738

  • Mutants of Chlamydomonas with Aberrant Responses to Sulfur Deprivation. The Plant cell Davies, J. P., Yildiz, F., Grossman, A. R. 1994; 6 (1): 53-63

    Abstract

    In the absence of sulfur, Chlamydomonas reinhardtii, a unicellular green alga, increases its rate of sulfate import and synthesizes several periplasmic proteins, including an arylsulfatase (Ars). These changes appear to help cells acclimate to a sulfur-deficient environment. The elevated rate of sulfate import results from an increase in the capacity and affinity of the transport system for sulfate. The synthesis of Ars, a periplasmic enzyme that cleaves sulfate from aromatic compounds, enables cells to use these molecules as a source of sulfur when free sulfate is not available. To characterize the ways in which C. reinhardtii perceives changes in the sulfur status of the environment and regulates its responses to these changes, we mutagenized cells and isolated strains exhibiting aberrant accumulation of Ars activity. These mutants were characterized for Ars activity, ars mRNA accumulation, periplasmic protein accumulation, and sulfate transport activity when grown in both sulfur-sufficient and sulfur-deficient conditions. All of the mutants exhibited pleiotropic effects with respect to several of these responses. Strains harboring double mutant combinations were constructed and characterized for Ars activity and ars mRNA accumulation. From the mutant phenotypes, we inferred that both positive and negative regulatory elements were involved in the acclimation process. Both the epistatic relationships among the mutations and the effects of the lesions on the responses of C. reinhardtii to sulfur limitation distinguished these mutants from similar mutants in Neurospora crassa.

    View details for DOI 10.1105/tpc.6.1.53

    View details for PubMedID 12244220

    View details for PubMedCentralID PMC160415

  • MUTANTS OF CHLAMYDOMONAS WITH ABERRANT RESPONSES TO SULFUR DEPRIVATION PLANT CELL Davies, J. P., Yildiz, F., Grossman, A. R. 1994; 6 (1): 53-63
  • SPECIFIC AND GENERAL RESPONSES OF CYANOBACTERIA TO MACRONUTRIENT DEPRIVATION International Symposium on Cellular and Molecular Biology of Phosphate and Phosphorylated Compounds in Microorganisms Grossman, A. R., Bhaya, D., Collier, J. L. AMER SOC MICROBIOLOGY. 1994: 112–118
  • INSERTIONAL INACTIVATION OF GENES TO ISOLATE MUTANTS OF SYNECHOCOCCUS SP STRAIN PCC-7942 - ISOLATION OF FILAMENTOUS STRAINS JOURNAL OF BACTERIOLOGY Dolganov, N., Grossman, A. R. 1993; 175 (23): 7644-7651

    Abstract

    We have developed a simple procedure for generating mutants of the cyanobacterium Synechococcus sp. strain PCC 7942 in which the site of the lesion can be readily identified. This procedure involves transforming Synechococcus sp. strain PCC 7942 with a library of its own DNA that was fully digested with Sau3A and ligated into the plasmid vector pUC8. The homologous integration of the recombinant plasmid into the genome will often result in the disruption of a gene and the loss of gene function. We have used this method to generate many mutants of Synechococcus sp. strain PCC 7942 which grow as multicellular filaments rather than as unicells. Since the gene harboring the lesion was tagged with pUC8, it was easily isolated. In this paper, we discuss the usefulness of this procedure for the generation of mutants, and we characterize one mutant in which the lesion may be in an operon involved in the assembly of lipopolysaccharides.

    View details for Web of Science ID A1993MH73700016

    View details for PubMedID 8244933

  • CHARACTERIZATION OF GENE CLUSTERS ENCODING THE FUCOXANTHIN CHLOROPHYLL PROTEINS OF THE DIATOM PHAEODACTYLUM-TRICORNUTUM NUCLEIC ACIDS RESEARCH Bhaya, D., Grossman, A. R. 1993; 21 (19): 4458-4466

    Abstract

    We are studying the multigene family encoding the fucoxanthin-chlorophyll binding proteins (fcp genes) that constitute the major component of the photosystem II-associated light harvesting complex in diatoms and brown algae. The characteristics of clusters of fcp genes on the genome of the diatom Phaeodactylum tricornutum are described. Sequence analysis of two genomic clones, PT5 and PT4, has demonstrated the presence of four fcp genes (fcpA, fcpB, fcpC, fcpD) on the former and two fcp genes (fcpE, fcpF) on the latter. The proteins encoded by the six characterized fcp genes range in similarity from 86% to 99%. The genes within each cluster are separated by short intergenic sequences (between 0.5 to 1.1 kb). None of these genes contain introns and all appear to be transcribed with short 5' transcribed, untranslated leader sequences; the transcription initiation sites were mapped 26 to 48 bases upstream of the ATG translation start site. Small conserved motifs are found among all of the genes just upstream of both the translation and the transcription start sites. The codon bias is similar in all of the fcp genes, with a predominance of pyrimidines in the third positions of codons of the four codon families. The two fcp genes that are most similar are fcpC and fcpD, and might represent a recent gene duplication. Southern analyses using fcp cDNAs as hybridization probes suggest that there may be additional sequences on the P. tricornutum genome that resemble the characterized fcp sequences.

    View details for Web of Science ID A1993MB07000004

    View details for PubMedID 8233779

    View details for PubMedCentralID PMC311176

  • THE PHYCOBILISOME, A LIGHT-HARVESTING COMPLEX RESPONSIVE TO ENVIRONMENTAL-CONDITIONS MICROBIOLOGICAL REVIEWS Grossman, A. R., Schaefer, M. R., Chiang, G. G., Collier, J. L. 1993; 57 (3): 725-749

    Abstract

    Photosynthetic organisms can acclimate to their environment by changing many cellular processes, including the biosynthesis of the photosynthetic apparatus. In this article we discuss the phycobilisome, the light-harvesting apparatus of cyanobacteria and red algae. Unlike most light-harvesting antenna complexes, the phycobilisome is not an integral membrane complex but is attached to the surface of the photosynthetic membranes. It is composed of both the pigmented phycobiliproteins and the nonpigmented linker polypeptides; the former are important for absorbing light energy, while the latter are important for stability and assembly of the complex. The composition of the phycobilisome is very sensitive to a number of different environmental factors. Some of the filamentous cyanobacteria can alter the composition of the phycobilisome in response to the prevalent wavelengths of light in the environment. This process, called complementary chromatic adaptation, allows these organisms to efficiently utilize available light energy to drive photosynthetic electron transport and CO2 fixation. Under conditions of macronutrient limitation, many cyanobacteria degrade their phycobilisomes in a rapid and orderly fashion. Since the phycobilisome is an abundant component of the cell, its degradation may provide a substantial amount of nitrogen to nitrogen-limited cells. Furthermore, degradation of the phycobilisome during nutrient-limited growth may prevent photodamage that would occur if the cells were to absorb light under conditions of metabolic arrest. The interplay of various environmental parameters in determining the number of phycobilisomes and their structural characteristics and the ways in which these parameters control phycobilisome biosynthesis are fertile areas for investigation.

    View details for Web of Science ID A1993LW44100010

    View details for PubMedID 8246846

  • THE GAMMA-SUBUNIT OF R-PHYCOERYTHRIN AND ITS POSSIBLE MODE OF TRANSPORT INTO THE PLASTID OF RED ALGAE JOURNAL OF BIOLOGICAL CHEMISTRY Apt, K. E., Hoffman, N. E., Grossman, A. R. 1993; 268 (22): 16208-16215

    Abstract

    R-phycoerythrin is the major light-harvesting pigment protein of most red algal phycobilisomes. It is composed of three pigmented polypeptide subunits, the alpha, beta, and gamma. While alpha and beta phycoerythrin subunits are each unique in the red alga Aglaothamnion neglectum, there are two different gamma subunits with distinct molecular masses. Both gamma subunits are pigmented by virtue of covalently attached linear tetrapyrroles. The amino acid sequence of one of the gamma subunits, as deduced from the nucleotide sequence of a cDNA clone, has no significant similarity to any known sequence in the data bases. This result is surprising, since the gamma subunit of phycoerythrin is thought to have a function that is similar to cyanobacterial linker polypeptides. The A. neglectum gamma subunit is synthesized as a 36-kDa precursor protein that is processed at the amino terminus to yield a 33-kDa mature protein. The amino-terminal extension was able to direct the pea small subunit of Rubisco into isolated pea chloroplasts. This result suggests that red algae transport proteins into the plastid by a mechanism similar to that of higher plants. There are significant changes in levels of mRNA encoding the gamma 33 subunit when A. neglectum is grown under different conditions of illumination and in nitrogen-deficient medium. These changes parallel those previously observed for transcripts encoding the alpha and beta phycoerythrin subunits. Hence, there may be coordinated expression of nuclear and plastid-encoded phycoerythrin subunit genes.

    View details for Web of Science ID A1993LQ33600021

    View details for PubMedID 8344905

  • GENES ENCODING PHYCOBILISOME LINKER POLYPEPTIDES ON THE PLASTID GENOME OF AGLAOTHAMNION-NEGLECTUM (RHODOPHYTA) PHOTOSYNTHESIS RESEARCH Apt, K. E., Grossman, A. R. 1993; 35 (3): 235-245

    Abstract

    The genes encoding the phycobilisome anchor protein (apcE) and rod-core linker (cpcG) are on the plastid genome of the red alga Aglaothamnion neglectum. The apcE gene product is 5' to and in the same operon as the α and β subunit genes of allophycocyanin. This arrangement is identical to the arrangement observed in many cyanobacteria. The cpcG gene product is 5' to the operon encoding the α and β subunits of phycoerythrin, but is transcribed from the opposite DNA strand. This gene arrangement is different from that observed in cyanobacteria.The amino acid sequences of the A. neglectum anchor protein and rod-core linker polypeptide, as deduced from the nucleotide sequences of the genes, are approximately 50% identical to analogous polypeptides from cyanobacteria and another eukaryotic alga Cyanophora paradoxa. The conserved nature of these proteins suggests that the structure of the core and the rod-core interface are very similar in phycobilisomes of cyanobacteria and eukaryotic red algae.Environmental factors such as nutrient availability and light intensity can significantly affect the levels of mRNAs encoding the anchor protein and the rod-core linker polypeptide. Most of these changes are similar to the environmentally-controlled changes in the levels of phycobiliprotein transcripts of A. neglectum (Apt and Grossman 1992b). However, unlike the mRNAs encoding other phycobilisome components, the apcE transcript remains high during growth under conditions of nutrient deprivation.

    View details for Web of Science ID A1993KR42800005

    View details for PubMedID 24318754

  • ENVIRONMENTAL-EFFECTS ON THE LIGHT-HARVESTING COMPLEX OF CYANOBACTERIA JOURNAL OF BACTERIOLOGY Grossman, A. R., Schaefer, M. R., Chiang, G. G., Collier, J. L. 1993; 175 (3): 575-582

    View details for Web of Science ID A1993KJ72200001

    View details for PubMedID 8423132

    View details for PubMedCentralID PMC196191

  • CHARACTERIZATION AND TRANSCRIPT ANALYSIS OF THE MAJOR PHYCOBILIPROTEIN SUBUNIT GENES FROM AGLAOTHAMNION-NEGLECTUM (RHODOPHYTA) PLANT MOLECULAR BIOLOGY Apt, K. E., Grossman, A. R. 1993; 21 (1): 27-38

    Abstract

    The genes encoding the alpha and beta subunits of allophycocyanin, phycocyanin and phycoerythrin from the red alga Aglaothamnion neglectum were isolated and characterized. While the operons containing the different phycobiliprotein genes are dispersed on the plastid genome, the genes encoding the alpha and beta subunits for each phycobiliprotein are contiguous. The beta subunit gene is 5' for both the phycocyanin and phycoerythrin operons, while the alpha subunit gene is 5' for the allophycocyanin operon. The amino acid sequences of A. neglectum phycobiliproteins, as deduced from the nucleotide sequences of the genes, are 65-85% identical to analogous proteins from other red algae and cyanobacteria. The conserved nature of the plastid-encoded red algal and cyanobacterial phycobiliprotein genes supports the proposed origin of red algal plastids from cyanobacterial endosymbionts. Many environmental factors effect phycobilisome biosynthesis. The effect of both nutrient availability and light quantity on the level of A. neglectum phycobiliprotein subunits and the mRNA species encoding those subunits is described.

    View details for Web of Science ID A1993KK40700003

    View details for PubMedID 7678762

  • COMPLEMENTATION OF A RED-LIGHT-INDIFFERENT CYANOBACTERIAL MUTANT PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chiang, G. G., Schaefer, M. R., Grossman, A. R. 1992; 89 (20): 9415-9419

    Abstract

    Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation.

    View details for Web of Science ID A1992JT97700011

    View details for PubMedID 1409650

  • A POLYUBIQUITIN CARRIER DNA FROM A RED ALGA PLANT PHYSIOLOGY Apt, K. E., Grossman, A. R. 1992; 99 (4): 1732-1733

    View details for Web of Science ID A1992JK72300078

    View details for PubMedID 16669105

  • CHLOROSIS INDUCED BY NUTRIENT DEPRIVATION IN SYNECHOCOCCUS SP STRAIN PCC-7942 - NOT ALL BLEACHING IS THE SAME JOURNAL OF BACTERIOLOGY Collier, J. L., Grossman, A. R. 1992; 174 (14): 4718-4726

    Abstract

    Cell coloration changes from normal blue-green to yellow or yellow-green when the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient. We found that this bleaching process (chlorosis) in cells deprived of sulfur (S) was similar to that in cells deprived of nitrogen (N), but that cells deprived of phosphorus (P) bleached differently. Cells divided once after N deprivation, twice after S deprivation, and four times after P deprivation. Chlorophyll (Chl) accumulation stopped almost immediately upon N or S deprivation but continued for several hours after P deprivation. There was no net Chl degradation during N, S, or P deprivation, although cellular Chl content decreased because cell division continued after Chl accumulation ceased. Levels of the light-harvesting phycobiliproteins declined dramatically in a rapid response to N or S deprivation, reflecting an ordered breakdown of the phycobilisomes (PBS). In contrast, P-deprived cultures continued to accumulate PBS for several hours. Whole PBS were not extensively degraded in P-deprived cells, although the PBS contents of P-deprived cells declined because of continued cell division after PBS accumulation ceased. Levels of mRNAs encoding PBS polypeptides declined by 90 to 95% in N- or S-deprived cells and by 80 to 85% in P-deprived cells. These changes in both the synthesis and stability of PBS resulted in a 90% decline in the PC/Chl ratio of N- or S-deprived cells and a 40% decline in the PC/Chl ratio of P-deprived cells. Therefore, although bleaching appears to be a general response to nutrient deprivation, it is not the same under all nutrient-limited conditions and is probably composed of independently controlled subprocesses.

    View details for Web of Science ID A1992JE39900022

    View details for PubMedID 1624459

  • EXPRESSION OF THE ARYLSULFATASE GENE FROM THE BETA-2-TUBULIN PROMOTER IN CHLAMYDOMONAS-REINHARDTII NUCLEIC ACIDS RESEARCH Davies, J. P., Weeks, D. P., Grossman, A. R. 1992; 20 (12): 2959-2965

    Abstract

    Arylsulfatase, produced by Chlamydomonas reinhardtii during sulfur-limited growth, is secreted into the periplasmic space and is readily assayed using a chromogenic substrate. To assess the usefulness of the gene encoding arylsulfatase (ars) as a reporter gene in C. reinhardtii, we have fused the promoter region of the beta 2-tubulin gene (tubB2) to the coding region of an ars genomic clone to form a tubB2/ars chimeric sequence. This construct was introduced into C. reinhardtii, strain CC425 (cw-15, arg-2), via cotransformation with the argininosuccinate lyase gene (which complements the arg-2 lesion) (1). Transformants expressing arylsulfatase (Ars) in sulfur-sufficient medium were isolated and subsequently shown to contain the tubB2/ars gene. RNA analysis determined that tubB2/ars transcripts accumulated in these cells. Abundance of the chimeric transcript increased immediately following deflagellation in a manner similar to that of the endogenous tubB2 transcript. Thus, chimeric genes incorporating ars coding sequences and heterologous promoters can be used to examine regulated gene expression in C. reinhardtii.

    View details for Web of Science ID A1992JC85000005

    View details for PubMedID 1620590

  • TRANSFORMATION OF THE FILAMENTOUS CYANOBACTERIUM FREMYELLA-DIPLOSIPHON BY CONJUGATION OR ELECTROPORATION MEETING ON THE PYRROLES OF PHOTOSYNTHETIC ORGANISMS Chiang, G. G., Schaefer, M. R., Grossman, A. R. GAUTHIER-VILLARS. 1992: 315–25
  • TARGETING PROTEINS TO DIATOM PLASTIDS INVOLVES TRANSPORT THROUGH AN ENDOPLASMIC-RETICULUM MOLECULAR & GENERAL GENETICS Bhaya, D., Grossman, A. 1991; 229 (3): 400-404

    Abstract

    Diatoms and related algae, in contrast to higher plants, have a xanthophyll-dominated light harvesting complex and an endoplasmic reticulum (ER) network surrounding the plastid. We have previously demonstrated that polypeptide constituents of the light harvesting complex from the diatom Phaeodactylum tricornutum are nuclear encoded and synthesized as higher molecular weight precursors in the cytoplasm. The amino-termini of the precursor proteins, as deduced from their gene sequences, have features of a signal peptide. Here, we show that the precursor polypeptides can be cotranslationally imported and processed by an in vitro microsomal membrane system, suggesting that cytoplasmically synthesized proteins require a signal peptide to traverse an ER before entering the plastid. These results are discussed in the context of plastid evolution.

    View details for Web of Science ID A1991GM74400011

    View details for PubMedID 1944228

  • ISOLATION, TRANSCRIPTION, AND INACTIVATION OF THE GENE FOR AN ATYPICAL ALKALINE-PHOSPHATASE OF SYNECHOCOCCUS-SP STRAIN PCC 7942 JOURNAL OF BACTERIOLOGY Ray, J. M., Bhaya, D., Block, M. A., Grossman, A. R. 1991; 173 (14): 4297-4309

    Abstract

    The alkaline phosphatase of Synechococcus sp. strain PCC 7942 is 145 kDa, which is larger than any alkaline phosphatase previously characterized and approximately three times the size of the analogous enzyme in Escherichia coli. The gene for the alkaline phosphatase, phoA, was cloned and sequenced, and the protein that it encodes was found to have little similarity to other phosphatases. Some sequence similarities were observed between the Synechococcus sp. strain PCC 7942 alkaline phosphatase, the alpha subunit of the ATPase from bacteria and chloroplasts, and the UshA sugar hydrolase of E. coli. Also, limited sequence similarity was observed between a region of the phosphatase and a motif implicated in nucleotide binding. Interestingly, although the alkaline phosphatase is transported across the inner cytoplasmic membrane and into the periplasmic space, it does not appear to have a cleavable signal sequence at its amino terminus. The half-life of the mRNA encoding the alkaline phosphatase, measured after inhibition of RNA synthesis, is approximately 5 min. Similar kinetics for the loss of alkaline phosphatase mRNA occur upon the addition of phosphate to phosphate-depleted cultures, suggesting that high levels of this nutrient inhibit transcription from phoA almost immediately. The phoA gene also appears to be the first gene of an operon; the largest detectable transcript that hybridizes to a phoA gene-specific probe is 11 kb, over twice the size needed to encode the mature protein. Other phosphate-regulated mRNAs are also transcribed upstream of the phoA gene. Insertional inactivation of phoA results in the loss of extracellular, phosphate-regulated phosphatase activity but does not alter the capacity of the cell for phosphate uptake.

    View details for Web of Science ID A1991FX32700007

    View details for PubMedID 1712356

    View details for PubMedCentralID PMC208089

  • CHARACTERIZATION AND MUTAGENESIS OF SULFUR-REGULATED GENES IN A CYANOBACTERIUM - EVIDENCE FOR FUNCTION IN SULFATE TRANSPORT JOURNAL OF BACTERIOLOGY Laudenbach, D. E., Grossman, A. R. 1991; 173 (9): 2739-2750

    Abstract

    A sulfur-regulated gene (cysA) that encodes the membrane-associated ATP-binding protein of the sulfate transport system of the cyanobacterium Synechococcus sp. strain PCC 7942 was recently isolated and sequenced. Adjacent to cysA and transcribed in the opposite direction is a gene encoding the sulfate-binding protein (sbpA). Two other genes, cysT and cysW, encode proteins that may form a channel for the transport of sulfate across the cytoplasmic membrane. A fourth gene, cysR, located between cysT, and cysW, encodes a polypeptide that has some homology to a family of prokaryotic regulatory proteins. Mutant strains in which cysA, cysT, or cysW was interrupted by a drug resistance marker were not viable when grown with sulfate as the sole sulfur source and exhibited essentially no sulfate uptake. In contrast, sbpA and cysR mutants grew on sulfate, although they did not exhibit the 20-fold increase in the Vmax (concentration of sulfate at half-maximal transport rate) for sulfate transport characteristic of wild-type cells grown under sulfur-limiting conditions. Three of the sulfur-regulated genes in Synechococcus sp. strain PCC 7942 are similar to genes encoded by the chloroplast genome of the primitive plant Marchantia polymorpha. These data suggest that a sulfate transport system similar to that of Synechococcus sp. strain PCC 7942 may exist in the chloroplast envelope of photosynthetic eukaryotes.

    View details for Web of Science ID A1991FK03800002

    View details for PubMedID 1708375

  • ISOLATION AND CHARACTERIZATION OF A SULFUR-REGULATED GENE ENCODING A PERIPLASMICALLY LOCALIZED PROTEIN WITH SEQUENCE SIMILARITY TO RHODANESE JOURNAL OF BACTERIOLOGY Laudenbach, D. E., Ehrhardt, D., Green, L., Grossman, A. 1991; 173 (9): 2751-2760

    Abstract

    During sulfur-limited growth, the cyanobacterium Synechococcus sp. strain PCC 7942 loses most of its photosynthetic pigments and develops an increased capacity to acquire sulfate. Sulfur deprivation also triggers the synthesis of several soluble polypeptides. We have isolated a prominent polypeptide of 33 kDa that accumulates specifically under sulfur-limiting conditions. This polypeptide was localized to the periplasmic space. The gene for this protein (designated rhdA) was isolated and discovered to lie within a region of the Synechococcus sp. strain PCC 7942 genome that encodes components of the sulfate permease system. The mRNA for the 33-kDa protein accumulates to high levels within an hour after the cells are deprived of sulfur and drops rapidly when sulfur is added back to the cultures. The amino acid sequence of the protein has similarity to bovine liver rhodanese, an enzyme that transfers the thiol group of thiosulfate to a thiophilic acceptor molecule, and a rhodaneselike protein of Saccharopolyspora erythraea. A strain in which rhdA was interrupted by a drug resistance marker exhibited marginally lower levels of rhodanese activity but was still capable of efficiently utilizing a variety of inorganic sulfur sources. The possible role of this protein in the transport of specific sulfur compounds is discussed.

    View details for Web of Science ID A1991FK03800003

    View details for PubMedID 1708376

  • LIGHT-HARVESTING PROTEINS OF DIATOMS - THEIR RELATIONSHIP TO THE CHLOROPHYLL A/B BINDING-PROTEINS OF HIGHER-PLANTS AND THEIR MODE OF TRANSPORT INTO PLASTIDS MOLECULAR & GENERAL GENETICS Grossman, A., MANODORI, A., Snyder, D. 1990; 224 (1): 91-100

    Abstract

    We have cloned and characterized members of a gene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex, a light-harvesting complex associated with photosystem II of diatoms and brown algae. Three cDNA clones encoding proteins associated with this complex in the diatom Phaeodactylum tricornutum have been isolated. As deduced from the nucleotide sequences, these light-harvesting proteins show homology to the chlorophyll a/b binding polypeptides of higher plants. Specifically, the N-terminal regions of the fucoxanthin, chlorophyll a/c-binding proteins are homologous to the chlorophyll a/b binding proteins in both the third membrane-spanning domain and the stroma-exposed region between membrane-spanning domains 2 and 3. Like the chlorophyll a/b-binding proteins, the mature fucoxanthin, chlorophyll a/c polypeptides have three hydrophobic alpha-helical domains which could span the membrane bilayer. The similarities between the two light-harvesting proteins might reflect the fact that both bind chlorophyll molecules and/or might be important for maintaining certain structural features of the complex. There is little similarity between the N-terminal sequences of the primary translation products of the fucoxanthin, chlorophyll a/c proteins and any transit sequences that have been characterized. Instead, the N-terminal sequences have features resembling those of signal sequences. Thus either transit peptides used in P. tricornutum show little resemblance to those of higher plants and green algae or the nuclear-encoded plastid proteins enter the organelle via a mechanism different from that used in higher plants.

    View details for Web of Science ID A1990EG01300012

    View details for PubMedID 2277634

  • CYTOCHROME-C-553 IS NOT REQUIRED FOR PHOTOSYNTHETIC ACTIVITY IN THE CYANOBACTERIUM SYNECHOCOCCUS PLANT CELL Laudenbach, D. E., Herbert, S. K., McDowell, C., FORK, D. C., Grossman, A. R., Straus, N. A. 1990; 2 (9): 913-924

    Abstract

    In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942.

    View details for Web of Science ID A1990EB41800010

    View details for PubMedID 1967057

  • CHROMATIC ADAPTATION AND THE EVENTS INVOLVED IN PHYCOBILISOME BIOSYNTHESIS PLANT CELL AND ENVIRONMENT Grossman, A. R. 1990; 13 (7): 651-666
  • CHARACTERIZATION OF THE LIGHT-REGULATED OPERON ENCODING THE PHYCOERYTHRIN-ASSOCIATED LINKER PROTEINS FROM THE CYANOBACTERIUM FREMYELLA-DIPLOSIPHON JOURNAL OF BACTERIOLOGY Federspiel, N. A., Grossman, A. R. 1990; 172 (7): 4072-4081

    Abstract

    Many biological processes in photosynthetic organisms can be regulated by light quantity or light quality or both. A unique example of the effect of specific wavelengths of light on the composition of the photosynthetic apparatus occurs in cyanobacteria that undergo complementary chromatic adaptation. These organisms alter the composition of their light-harvesting organelle, the phycobilisome, and exhibit distinct morphological features as a function of the wavelength of incident light. Fremyella diplosiphon, a filamentous cyanobacterium, responds to green light by activating transcription of the cpeBA operon, which encodes the pigmented light-harvesting component phycoerythrin. We have isolated and determined the complete nucleotide sequence of another operon, cpeCD, that encodes the linker proteins associated with phycoerythrin hexamers in the phycobilisome. The cpeCD operon is activated in green light and expressed as two major transcripts with the same 5' start site but differing 3' ends. Analysis of the kinetics of transcript accumulation in cultures of F. diplosiphon shifted from red light to green light and vice versa shows that the cpeBA and cpeCD operons are regulated coordinately. A common 17-base-pair sequence is found upstream of the transcription start sites of both operons. A comparison of the predicted amino acid sequences of the phycoerythrin-associated linker proteins CpeC and CpeD with sequences of other previously characterized rod linker proteins shows 49 invariant residues, most of which are in the amino-terminal half of the proteins.

    View details for Web of Science ID A1990DM73700070

    View details for PubMedID 1694529

  • STRUCTURE AND LIGHT-REGULATED EXPRESSION OF PHYCOERYTHRIN GENES IN WILD-TYPE AND PHYCOBILISOME ASSEMBLY MUTANTS OF SYNECHOCYSTIS SP STRAIN PCC-6701 JOURNAL OF BACTERIOLOGY Anderson, L. K., Grossman, A. R. 1990; 172 (3): 1297-1305

    Abstract

    Phycoerythrin is a major pigmented component of the phycobilisome, a cyanobacterial light-harvesting complex. It contains bilin-type chromophores that absorb and transfer light energy to chlorophyll protein complexes of the photosynthetic membranes. In many cyanobacteria, phycoerythrin expression is regulated by light wavelength in a response known as chromatic adaptation. Green light-grown cells contain higher levels of this biliprotein than do cells grown in red light. The phycoerythrin gene set from the unicellular cyanobacterium Synechocystis sp. strain PCC 6701 was cloned and sequenced, and the 5' end of the phycoerythrin mRNA was localized. The amino acid sequences of the phycoerythrin subunits from Synechocystis strain 6701 and Fremyella diplosiphon were 90% identical. As observed in F. diplosiphon, the Synechocystis strain 6701 phycoerythrin transcript accumulated to high levels in green light-grown cells and low levels in red light-grown cells. Similar nucleotide sequences, which might control gene expression, occurred upstream of the transcription initiation sites of the phycoerythrin genes in both organisms. While the phycoerythrin structure and light-regulated transcript accumulation were similar in Synechocystis strain 6701 and F. diplosiphon, the steady-state levels of phycoerythrin subunits during growth in red light were quite different for the two organisms. This observation suggests that control of phycoerythrin levels in Synechocystis strain 6701 is complex and may involve posttranscriptional processes. We also characterized the phycoerythrin genes and mRNA levels in two phycobilisome assembly mutants, UV16-40 and UV16.

    View details for Web of Science ID A1990CQ75200020

    View details for PubMedID 2106507

  • GENES FOR PHYCOCYANIN SUBUNITS IN SYNECHOCYSTIS SP STRAIN PCC-6701 AND ASSEMBLY MUTANT UV16 JOURNAL OF BACTERIOLOGY Anderson, L. K., Grossman, A. R. 1990; 172 (3): 1289-1296

    Abstract

    The cyanobacterial phycobilisome is a large protein complex located on the photosynthetic membrane. It harvests light energy and transfers it to chlorophyll for use in photosynthesis. Phycobilisome assembly mutants in the unicellular cyanobacterium Synechocystis sp. strain 6701 have been characterized. One such mutant, UV16, contains a defect in the assembly of the biliprotein phycocyanin. We report the cloning and sequencing of the phycocyanin genes from wild-type Synechocystis strain 6701 and demonstrate an alteration in the gene for the phycocyanin alpha subunit in UV16. Possible consequences of the lesion on phycobilisome assembly were assessed from its position in the phycocyanin tertiary and quaternary structures. The UV16 phenotype is complex and includes a reduced level of phycocyanin relative to that in the wild type. To determine whether the lower phycocyanin content results from lower transcript levels, a fragment of cpcBA was used as a probe for quantitating phycocyanin mRNA. Both the wild type and UV16 contained two phycocyanin transcripts of approximately 1.4 and 1.5 kilobases that were equal in abundance and that did not vary with light quality during cell growth. Equal levels of these transcripts in the wild type and UV16 suggest that the lower phycocyanin content in the mutant may be due to posttranscriptional events. The 5' ends of the two phycocyanin mRNAs were mapped at 100 and 223 base pairs upstream of the cpcB initiation codon. Homologous regions upstream of the putative transcription initiation sites may be important for maintaining high levels of transcription from the Synechocystis strain 6701 phycocyanin gene set.

    View details for Web of Science ID A1990CQ75200019

    View details for PubMedID 2106506

  • SEQUENCE HOMOLOGY BETWEEN LIGHT HARVESTING POLYPEPTIDES OF PLANTS AND THE DIATOM PHAEODACTYLUM-TRICORNUTUM 8TH INTERNATIONAL CONGRESS ON PHOTOSYNTHESIS MANODORI, A., Grossman, A. R. KLUWER ACADEMIC PUBL. 1990: C541–C544
  • STRUCTURE AND EXPRESSION OF THE GENE ENCODING THE PERIPLASMIC ARYLSULFATASE OF CHLAMYDOMONAS-REINHARDTII MOLECULAR AND GENERAL GENETICS deHostos, E. L., Schilling, J., Grossman, A. R. 1989; 218 (2): 229-239

    Abstract

    Chlamydomonas reinhardtii produces a periplasmic arylsulfatase in response to sulfur deprivation. We have isolated and sequenced arylsulfatase cDNAs from a lambda gt11 expression library. The amino acid sequence of the protein, as deduced from the nucleotide sequence, has features characteristic of secreted proteins, including a signal sequence and putative glycosylation sites. The gene has a broad codon usage with seven codons, all having A residues in the third position, not previously observed in C. reinhardtii genes. Arylsulfatase transcription is tightly regulated by sulfur availability. The approximately 2.7 kb arylsulfatase transcript is very susceptible to degradation, disappearing in less than an hour after sulfur starved cells are administered either sulfate or alpha-amanitin. The accumulation of the arylsulfatase transcript is also suppressed by the addition of cycloheximide. Transcription initiation from the arylsulfatase gene occurs approximately 100 bp upstream of the initiation codon, in a region that is 5' to a 43 bp imperfect inverted repeat. Preceding the transcription start site are sequences similar to those present in promoter regions of other genes from C. reinhardtii.

    View details for Web of Science ID A1989AJ18300007

    View details for PubMedID 2476654

  • ROLE OF PROTEIN-SYNTHESIS IN REGULATION OF PHYCOBILIPROTEIN MESSENGER-RNA ABUNDANCE BY LIGHT QUALITY IN FREMYELLA-DIPLOSIPHON PLANT PHYSIOLOGY Oelmuller, R., Grossman, A. R., Briggs, W. R. 1989; 90 (4): 1486-1491

    Abstract

    If green light-acclimated Fremyella diplosiphon cultures are transferred to red light, the transcription from the inducible phycocyanin gene set increases at least 30-fold within 60 minutes. This effect is inhibited completely by the protein synthesis inhibitors chloramphenicol and spectinomycin. Application of chloramphenicol 30 minutes after transfer of cultures to inductive red light prevents further phycocyanin mRNA accumulation within 10 minutes. If red light-acclimated cells are transferred to green light, the phycocyanin transcript level declines by about 70% within 1 hour. Most of the green light-dependent decline results from the rapid cessation of transcription from the PC gene set. Chloramphenicol slows the decline to some extent by decreasing the rate of mRNA degradation in a light-independent manner. The accumulation of phycoerythrin mRNA after transfer of red light-acclimated cells to green light is also inhibited by chloramphenicol. However, there is no red light-dependent mechanism that rapidly halts phycoerythin mRNA synthesis after transfer of cultures from green to red light. Therefore, at least three light-dependent processes are involved in regulating phycobiliproteingene expression: chloramphenicol-sensitive processes required for the activation of phycocyanin and phycoerythrin gene sets and a chloramphenicol-insensitive process which blocks phycocyanin mRNA synthesis after transfer of cells from red to green light.

    View details for Web of Science ID A1989AL17500044

    View details for PubMedID 16666955

  • A REGION OF A CYANOBACTERIAL GENOME REQUIRED FOR SULFATE TRANSPORT PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Green, L. S., Laudenbach, D. E., Grossman, A. R. 1989; 86 (6): 1949-1953

    Abstract

    Using the cysA locus of Salmonella typhimurium as a heterologous probe, we have cloned a region of the Anacystis nidulans R2 (Synechococcus PCC 7942) genome involved in sulfate assimilation. The 8.3-kilobase-pair region encodes at least five transcripts that cannot be detected unless the cells are deprived of sulfur. One of the genes in this region has been sequenced, and the protein that it encodes is homologous to a polypeptide component of other permease systems of Escherichia coli and Salmonella. Insertional inactivation of the putative sulfate permease gene, designated cysA, as well as of other genes within this region, results in cysteine auxotrophy, reduced sulfate uptake, and altered expression of soluble and cytoplasmic-membrane polypeptides associated with sulfur starvation.

    View details for Web of Science ID A1989T788900043

    View details for PubMedID 2538823

  • MOLECULAR CHARACTERIZATION OF PHYCOBILISOME REGULATORY MUTANTS OF FREMYELLA-DIPLOSIPHON JOURNAL OF BACTERIOLOGY BRUNS, B. U., Briggs, W. R., Grossman, A. R. 1989; 171 (2): 901-908

    Abstract

    Three classes of pigment mutants were generated in Fremyella diplosiphon in the course of electroporation experiments. The red mutant class had high levels of phycoerythrin in both red and green light and no inducible phycocyanin in red light. Thus, this mutant behaved as if it were always in green light, regardless of light conditions. Blue mutants exhibited normal phycoerythrin photoregulation, whereas the inducible phycocyanin was present at high levels in both red- and green-light-grown cells. Furthermore, the absolute amount of allophycocyanin was increased threefold in comparison with our wild-type strain. Green mutants lost the capacity to accumulate phycoerythrin in green light but showed normal photoregulation of phycocyanin. Analyses of transcript abundance in these mutants demonstrated that changes in the levels of the different phycobilisome components correlated with changes in the levels of mRNAs encoding those components. The characterization of these mutants supports hypotheses previously discussed concerning molecular mechanisms involved in the regulation of the phycobiliprotein gene sets during chromatic adaptation.

    View details for Web of Science ID A1989T041700039

    View details for PubMedID 2464582

  • PHOTOREVERSIBILITY OF THE EFFECT OF RED AND GREEN LIGHT-PULSES ON THE ACCUMULATION IN DARKNESS OF MESSENGER-RNAS CODING FOR PHYCOCYANIN AND PHYCOERYTHRIN IN FREMYELLA-DIPLOSIPHON PLANT PHYSIOLOGY Oelmuller, R., Grossman, A. R., Briggs, W. R. 1988; 88 (4): 1084-1091

    Abstract

    DNA fragments encoding a red light-inducible phycocyanin gene and a green light-inducible phycoerythrin gene have been used to investigate the effect of red and green pulses on the accumulation of phycocyanin and phycoerythrin mRNA in subsequent darkness. A red pulse promotes phycocyanin and suppresses phycoerythrin mRNA accumulation while a green pulse has an opposite effect on both transcript levels. The effect of a saturating light pulse is canceled by a subsequently given pulse of the other light quality. For a given mRNA, the positive and negative effects require the same fluence for saturation, whereas to saturate the phycoerythrin mRNA response requires at least twice as much light as to saturate the phycocyanin mRNA response. Calculations of the apparent extinction coefficients for the pigments mediating the light-regulated mRNA increase and decrease are of the order of 2 x 10(4) for phycocyanin mRNA and less than 10(4) for phycoerythrin mRNA. The data are consistent with the hypothesis that the light-induced increase and decrease of a particular phycobiliprotein mRNA is controlled by a single red/green photoreversible photosystem, but that phycoerythrin and phycocyanin mRNA levels are either controlled by two distinct photoreversible systems or that marked differences occur in the chain of events leading from photoperception to gene activation. These system(s) differ from most phytochrome systems in several ways: First, they remain fully on or off depending upon the light quality of the terminal irradiation. Second, they can be completely reversed by light of the appropriate wavelength after several hours of darkness without diminution of the effectiveness of the reversing light pulse. These two features argue against the existence of dark reversion or dark destruction of the biologically active moiety. Third, signal transduction is rapid-measurable mRNA changes occur even during a 10 minute irradiation.

    View details for Web of Science ID A1988R574400029

    View details for PubMedID 16666426

  • CHANGES IN ACCUMULATION AND SYNTHESIS OF TRANSCRIPTS ENCODING PHYCOBILISOME COMPONENTS DURING ACCLIMATION OF FREMYELLA-DIPLOSIPHON TO DIFFERENT LIGHT QUALITIES PLANT PHYSIOLOGY Oelmuller, R., Conley, P. B., Federspiel, N., Briggs, W. R., Grossman, A. R. 1988; 88 (4): 1077-1083

    Abstract

    We have used gene-specific DNA fragments as hybridization probes to quantitate the levels of transcripts encoding several phycobilisome polypeptides in the cyanobacterium Fremyella diplosiphon in response to changes in the light environment. While the levels of transcripts encoding allophycocyanin, the core linker polypeptide, and the constitutive phycocyanin subunits are similar in F. diplosiphon grown either in red or green light, the levels of other transcripts change dramatically. Transcripts encoding the inducible phycocyanin subunits are barely detected in green light-grown cells and very abundant in red light-grown cells, while the level of phycoerythrin mRNA is approximately 10-fold more in green than red light-grown cells. Quantitation of the phycoerythrin and inducible phycocyanin transcripts after transfer of cultures from green to red light and red to green light demonstrate that both increase rapidly upon exposure of cells to inductive illumination. The decrease in the phycoerythrin mRNA level in red light is much slower than the decline in the levels of the inducible phycocyanin transcripts in green light. Since the half-lives of the inducible phycocyanin and phycoerythrin transcripts do not change when F. diplosiphon is exposed to red or green illumination, the steady state levels of these mRNAs are primarily controlled by the rate of transcription. Therefore, the high level of phycoerythrin mRNA maintained for several hours after cultures are transferred from green to red illumination must result from continued transcription of the phycoerythrin gene set. Differences in expression from the phycoerythrin and inducible phycocyanin gene sets in response to light quality are discussed in terms of possible mechanisms involved in their regulation.

    View details for Web of Science ID A1988R574400028

    View details for PubMedID 16666425

  • CHARACTERIZATION OF PHYCOBILIPROTEIN AND LINKER POLYPEPTIDE GENES IN FREMYELLA-DIPLOSIPHON AND THEIR REGULATED EXPRESSION DURING COMPLEMENTARY CHROMATIC ADAPTATION PHOTOSYNTHESIS RESEARCH Grossman, A. R., Lemaux, P. G., Conley, P. B., BRUNS, B. U., Anderson, L. K. 1988; 17 (1-2): 23-56
  • IDENTIFICATION AND PURIFICATION OF A DEREPRESSIBLE ALKALINE-PHOSPHATASE FROM ANACYSTIS-NIDULANS R2 PLANT PHYSIOLOGY Block, M. A., Grossman, A. R. 1988; 86 (4): 1179-1184

    Abstract

    We have examined the increase in alkaline phosphatase activity in the cyanobacterium Anacystis nidulans R2 upon phosphate deprivation. Much of the activity is released into the medium when A. nidulans is osmotically shocked, indicating that the enzyme is located either in the periplasmic space or is loosely bound to the cell wall. The polypeptide associated with phosphatase activity has been identified as a single species of M(r) 160,000. Several lines of evidence demonstrate that this polypeptide is responsible for alkaline phosphatase activity: (a) It is absent when cells are grown in the presence of phosphate and specifically accumulates during phosphate deprivation. (b) It is the major periplasmic polypeptide extracted by osmotic shock. (c) It represents over 90% of the protein in a fraction of periplasmic polypeptides enriched for phosphatase activity. (d) Antibodies raised against the purified species of M(r) 160,000 inhibit phosphatase activity by approximately 70%.

    View details for Web of Science ID A1988N059700038

    View details for PubMedID 16666051

  • MOLECULAR CHARACTERIZATION AND EVOLUTION OF SEQUENCES ENCODING LIGHT-HARVESTING COMPONENTS IN THE CHROMATICALLY ADAPTING CYANOBACTERIUM FREMYELLA-DIPLOSIPHON JOURNAL OF MOLECULAR BIOLOGY Conley, P. B., Lemaux, P. G., Grossman, A. 1988; 199 (3): 447-465

    Abstract

    The major light-harvesting complex in eukaryotic red algae and prokaryotic cyanobacteria is the phycobilisome, a water-soluble complex located on the outer surface of the photosynthetic membranes and composed of both pigmented phycobiliproteins (85%) and non-pigmented linker (15%) polypeptides. The phycobiliproteins are encoded by a gene family and exhibit varying degrees of sequence homology (25 to 55%). Some cyanobacteria can maximize the absorption of prevalent wavelengths of light by adjusting the phycobiliprotein composition of the phycobilisome, a process called complementary chromatic adaptation. In the chromatically adapting species Fremyella displosiphon, there are at least two sets of phycocyanin genes; one is transcribed as two red light-induced transcripts and the other is encoded on a single transcript present in both red and green light. We have determined the complete nucleotide sequences of both sets of phycocyanin subunit genes and their associated 5' and 3' regulatory regions. Based on S1 nuclease protection experiments, the transcripts (1600 and 3800 bases) encoding the inducible phycocyanin subunits have the same 5' end, and possible mechanisms for their synthesis are presented. The 5' end of the 1500-base transcript encoding the constitutive phycocyanin subunits was determined and revealed an Escherichia coli-like "-10" and "-35" region, and sequences near the transcription initiation site homologous to the analogous region of the phycocyanin gene set of Anabaena sp. 7120. Determination of the 3' ends of the transcripts encoding both F. diplosiphon phycocyanin gene sets revealed regions of potential secondary structure that may be important for transcription termination and/or transcript stability. In addition, the sequence of an open reading frame (encoding a 30 kDa polypeptide), located 3' to the constitutive phycocyanin gene set in F. diplosiphon and highly conserved in at least three cyanobacterial species, is presented. The same high degree of sequence homology between the two F. diplosiphon PC alpha and PC beta sequences (85 and 77%, respectively) was found at both the nucleotide and amino acid levels, and similar results were obtained for interspecies comparisons. Implications of these homologies with regard to the evolution of phycobiliprotein subunits are discussed.

    View details for Web of Science ID A1988M072200005

    View details for PubMedID 3127591

  • CHANGES IN SULFATE TRANSPORT CHARACTERISTICS AND PROTEIN-COMPOSITION OF ANACYSTIS-NIDULANS R2 DURING SULFUR DEPRIVATION JOURNAL OF BACTERIOLOGY Green, L. S., Grossman, A. R. 1988; 170 (2): 583-587

    Abstract

    Sulfur-starved cells of Anacystis nidulans have an increased capacity to take up sulfate. The apparent Vmax for sulfate uptake increased at least 10-fold after 24 h of sulfur deprivation, whereas the K1/2 remained unchanged at approximately 1.35 microM. The initial rate of sulfate uptake increased between 2 and 6 h after transfer of the cells to sulfur-free medium, in concert with elevated levels of three cytoplasmic membrane polypeptides with molecular masses of 43, 42, and 36 kilodaltons (kDa). The amounts of these polypeptides did not increase in response to nitrogen or phosphorus deprivation. A fourth cytoplasmic membrane polypeptide of 17 kDa did not appear until 24 h after transfer to sulfur-deficient medium. In the total soluble fraction, three polypeptides with masses of 36.5, 33.5, and 28.5 kDa increased dramatically in response to sulfur deprivation, but not in response to nitrogen or phosphorus deprivation. The specificity and abundance of these polypeptides indicate that they could play an important role in the response of A. nidulans to sulfur deprivation.

    View details for Web of Science ID A1988L890200015

    View details for PubMedID 3123460

  • PURIFICATION AND BIOSYNTHESIS OF A DEREPRESSIBLE PERIPLASMIC ARYLSULFATASE FROM CHLAMYDOMONAS-REINHARDTII JOURNAL OF CELL BIOLOGY deHostos, E. L., Togasaki, R. K., Grossman, A. 1988; 106 (1): 29-37

    Abstract

    The unicellular green alga Chlamydomonas reinhardtii responds to sulfate deprivation by producing an arylsulfatase (Lien, T., and O. Schreiner. 1975. Biochim. Biophys. Acta. 384:168-179; Schreiner, O., 1975. Biochim. Biophys. Acta. 384:180-193) and by developing the capacity to transport sulfate more rapidly (our unpublished data). The arylsulfatase activity, detectable 3 h after the transfer of the cells to low sulfate medium (less than or equal to 10 microM sulfate), is a periplasmic protein released into the culture medium by cw15, a cell wall-less mutant of C. reinhardtii. We have purified the derepressible arylsulfatase to homogeneity and have raised monospecific antibodies to it. The protein monomer (67.6 kD) associates into a dimer, and the enzyme activity shows an alkaline pH optimum and a Km of 0.3 mM for p-nitrophenylsulfate. Studies focused on arylsulfatase biosynthesis demonstrate that it is glycosylated and synthesized as a higher molecular mass precursor. The mature protein contains complex N-linked oligosaccharides and the primary translation product has an apparent molecular mass approximately 5 kD larger than the deglycosylated monomer. Since translatable RNA encoding the arylsulfatase can only be detected in cells after sulfate starvation, it is likely that accumulation of the enzyme is regulated at the level of transcription, although posttranscriptional processes may also be involved.

    View details for Web of Science ID A1988L864400004

    View details for PubMedID 3339089

  • ISOLATION AND CHARACTERIZATION OF LIGHT-REGULATED PHYCOBILISOME LINKER POLYPEPTIDE GENES AND THEIR TRANSCRIPTION AS A POLYCISTRONIC MESSENGER-RNA JOURNAL OF BACTERIOLOGY Lomax, T. L., Conley, P. B., Schilling, J., Grossman, A. R. 1987; 169 (6): 2675-2684

    Abstract

    Several cyanobacteria adjust both the phycobiliprotein and linker protein composition of the phycobilisome, a light-harvesting complex in cyanobacteria and some eucaryotic algae, to maximize absorption of prevalent wavelengths of light. This process is called complementary chromatic adaptation. We sequenced the amino terminus of a linker polypeptide which is associated with phycocyanin and accumulates to high levels during growth of the cyanobacterium Fremyella diplosiphon in red light. A mixed oligonucleotide encoding a region of this amino terminus was synthesized and used to identify a fragment of F. diplosiphon genomic DNA encoding the linker polypeptide. This linker gene was located between two other linker genes and contiguous to the red-light-induced phycocyanin gene set. Sequences of all three linker genes are presented. These genes were transcribed together onto a large polycistronic mRNA which also encoded the red-light-induced phycocyanin subunits. The relationship of this transcript to the biogenesis of the phycobilisome when F. diplosiphon is grown under different conditions of illumination is discussed.

    View details for Web of Science ID A1987H492300050

    View details for PubMedID 3108238

  • REGULATED SYNTHESIS OF PHYCOBILISOME COMPONENTS PHOTOCHEMISTRY AND PHOTOBIOLOGY Grossman, A. R., Lemaux, P. G., Conley, P. B. 1986; 44 (6): 829-837
  • Regulated synthesis of phycobilisome components. Photochemistry and photobiology Grossman, A. R., Lemaux, P. G., Conley, P. B. 1986; 44 (6): 827-837

    View details for PubMedID 3104939

  • GENES ENCODING MAJOR LIGHT-HARVESTING POLYPEPTIDES ARE CLUSTERED ON THE GENOME OF THE CYANOBACTERIUM FREMYELLA-DIPLOSIPHON PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Conley, P. B., Lemaux, P. G., Lomax, T. L., Grossman, A. R. 1986; 83 (11): 3924-3928

    Abstract

    The polypeptide composition of the phycobilisome, the major light-harvesting complex of prokaryotic cyanobacteria and certain eukaryotic algae, can be modulated by different light qualities in cyanobacteria exhibiting chromatic adaptation. We have identified genomic fragments encoding a cluster of phycobilisome polypeptides (phycobiliproteins) from the chromatically adapting cyanobacterium Fremyella diplosiphon using previously characterized DNA fragments of phycobiliprotein genes from the eukaryotic alga Cyanophora paradoxa and from F. diplosiphon. Characterization of two lambda-EMBL3 clones containing overlapping genomic fragments indicates that three sets of phycobiliprotein genes--the alpha- and beta-allophycocyanin genes plus two sets of alpha- and beta-phycocyanin genes--are clustered within 13 kilobases on the cyanobacterial genome and transcribed off the same strand. The gene order (alpha-allophycocyanin followed by beta-allophycocyanin and beta-phycocyanin followed by alpha-phycocyanin) appears to be a conserved arrangement found previously in a eukaryotic alga and another cyanobacterium. We have reported that one set of phycocyanin genes is transcribed as two abundant red light-induced mRNAs (1600 and 3800 bases). We now present data showing that the allophycocyanin genes and a second set of phycocyanin genes are transcribed into major mRNAs of 1400 and 1600 bases, respectively. These transcripts are present in RNA isolated from cultures grown in red and green light, although lower levels of the 1600-base phycocyanin transcript are present in cells grown in green light. Furthermore, a larger transcript of 1750 bases hybridizes to the allophycocyanin genes and may be a precursor to the 1400-base species.

    View details for Web of Science ID A1986C590700076

    View details for PubMedID 3086870

  • CYANOBACTERIAL LIGHT-HARVESTING COMPLEX SUBUNITS ENCODED IN 2 RED-LIGHT INDUCED TRANSCRIPTS SCIENCE Conley, P. B., Lemaux, P. G., Grossman, A. R. 1985; 230 (4725): 550-553

    Abstract

    The major light-harvesting complex in cyanobacteria and red algae, the phycobilisome, is composed of chromophoric and nonchromophoric polypeptides. Two linked genes encoding major chromophoric components, the polypeptide subunits of phycocyanin, were isolated from the cyanobacterium Fremyella diplosiphon. Transcripts from this phycocyanin subunit gene cluster were present as major species in the cyanobacterium grown in red light, but not in cultures maintained in green light. The genes for the subunits of the red light-induced phycocyanin were transcribed together (beta-phycocyanin followed by alpha-phycocyanin) on two messenger RNA species; one contained 1600 bases while the other had 3800 bases. The latter, which encompassed the smaller transcript, contained additional sequences extending from the 3' end of the coding region of the alpha-phycocyanin gene. It may encode other light-induced components of the phycobilisome. Since phycocyanin, which effectively absorbs red light, becomes a dominant constituent of the phycobilisome in red light, these different levels may reflect an important adaptive mechanism of these organisms to their environment.

    View details for Web of Science ID A1985ASW2500031

    View details for PubMedID 3931221

  • MAJOR LIGHT-HARVESTING POLYPEPTIDES ENCODED IN POLYCISTRONIC TRANSCRIPTS IN A EUKARYOTIC ALGA EMBO JOURNAL Lemaux, P. G., Grossman, A. R. 1985; 4 (8): 1911-1919

    Abstract

    By sequence analysis of previously identified fragments and low stringency hybridization of an identified gene for a phycobiliprotein subunit to total plastid DNA, we have identified four phycobiliprotein subunit genes in a eukaryotic alga, Cyanophora paradoxa. The four phycobiliprotein subunits, alpha and beta of phycocyanin (PC) and allophycocyanin (APC), comprise the bulk of the light-harvesting complex in this alga. The alpha and beta subunit genes encoding each phycobiliprotein (the products of which are required in a 1:1 ratio in the light-harvesting complex) are contiguous; however, the genes for different phycobiliproteins, PC and APC, are located in different regions of the genome. The two PC subunit genes are in the small single copy region of the plastid genome whereas the APC subunit genes are in the large single copy region and the two sets of phycobiliprotein genes are transcribed from opposite strands. The alpha and beta subunits of both PC and APC are encoded in dicistronic transcripts and this arrangement may provide a mechanism by which the two subunits can be synthesized in equimolar amounts. Levels of the PC transcript are approximately five times that of the APC transcript which may reflect the relative abundance of their gene products in the phycobilisome. The 5' ends of the transcripts for PC and APC were mapped and the regulatory regions identified. Several features of the promoter regions for these highly transcribed genes are described.

    View details for Web of Science ID A1985ANN7800002

    View details for PubMedID 2998775

  • ISOLATION AND CHARACTERIZATION OF A GENE FOR A MAJOR LIGHT-HARVESTING POLYPEPTIDE FROM CYANOPHORA-PARADOXA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Lemaux, P. G., Grossman, A. 1984; 81 (13): 4100-4104

    Abstract

    Antibodies raised against mixtures of phycobilisome polypeptides from the eukaryotic alga Cyanidium caldarium were used in an immunological screen to detect expression of phycobiliprotein genes in an Escherichia coli library containing segments of plastid (chloroplast, cyanelle) DNA from another eukaryotic alga, Cyanophora paradoxa. The four candidate clones obtained were mapped by restriction analysis and found to be overlapping. The clone with the smallest insert (1.4 kilobases) was partially sequenced and a coding region similar to the carboxyl terminus of the phycobiliprotein subunit beta-phycocyanin was found. The coding region for the beta-phycocyanin gene in C. paradoxa has been mapped to the small single copy region on the cyanelle genome, and its orientation has been determined. A short probe unique to a conserved chromophore binding site shared by at least two phycobiliprotein subunits has now been generated from the carboxyl terminus of the beta-phycocyanin gene. This probe may be useful in identifying specific phycobiliprotein subunit genes, beta-phycocyanin, beta-phycoerythrocyanin, and possibly beta-phycoerythrin, in other eukaryotic algae and in prokaryotic cyanobacteria.

    View details for Web of Science ID A1984TA46100036

    View details for PubMedID 16593484

  • BIOSYNTHESIS OF CARBONIC-ANHYDRASE IN CHLAMYDOMONAS-REINHARDTII DURING ADAPTATION TO LOW CO2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Coleman, J. R., Grossman, A. R. 1984; 81 (19): 6049-6053

    Abstract

    The unicellular green alga Chlamydomonas reinhardtii synthesizes carbonic anhydrase in response to low levels of CO(2) (i.e., air levels of CO(2)). This enzyme, localized predominantly in the periplasmic space of the alga (or associated with the cell wall), is an important component of the machinery required for the active accumulation of inorganic carbon by C. reinhardtii and the saturation of ribulose-1,5-bisphosphate carboxylase at low extracellular carbon concentrations. We have begun to examine the synthesis and compartmentalization of carbonic anhydrase in C. reinhardtii. The monomeric species associated with carbonic anhydrase activity is synthesized as a precursor on 80S cytoplasmic ribosomes. This precursor can be detected immunologically in the profiles of translation products when a reticulocyte lysate, cell-free system is primed with poly(A)-RNA from either air-grown C. reinhardtii or cells shifted from growth on 5% CO(2) to air for 12 hr. It is not synthesized when the in vitro system is primed with poly(A)-RNA from CO(2)-grown algae. Since translatable RNA for the polypeptide responsible for carbonic anhydrase activity was only present in cells that experienced low levels of CO(2), the adaptation process either involves the regulation of transcription of the carbonic anhydrase gene (and perhaps other genes involved in adaptation) or the post-transcriptional processing of the messenger RNA. Furthermore, the appearance of the mature polypeptide associated with carbonic anhydrase activity in the periplasmic space of C. reinhardtii is inhibited by tunicamycin, an antibiotic that prevents core glycosylation of polypeptides on the endoplasmic reticulum. Together, these results suggest that the biosynthesis of this extracellular algal enzyme involves the translation of mRNA for the carbonic anhydrase monomer on ribosomes bound to the endoplasmic reticulum, the cleavage of a signal sequence during transport of the nascent polypeptide into the lumen of the endoplasmic reticulum, and subsequent glycosylation events prior to export across the plasmalemma.

    View details for Web of Science ID A1984TP31700029

    View details for PubMedID 16593518

  • IDENTIFICATION OF EXTRACELLULAR CARBONIC-ANHYDRASE OF CHLAMYDOMONAS-REINHARDTII PLANT PHYSIOLOGY Coleman, J. R., Berry, J. A., Togasaki, R. K., Grossman, A. R. 1984; 76 (2): 472-477

    Abstract

    We have examined the induction of carbonic anhydrase activity in Chlamydomonas reinhardtii and have identified the polypeptide responsible for this activity. This polypeptide was not synthesized when the alga was grown photoautotrophically on 5% CO(2), but its synthesis was induced under low concentrations of CO(2) (air levels of CO(2)). In CW-15, a mutant of C. reinhardtii which lacks a cell wall, between 80 and 90% of the carbonic anhydrase activity of air-adapted cells was present in the growth medium. Furthermore, between 80 and 90% of the carbonic anhydrase is released if wild type cells are treated with autolysin, a hydrolytic enzyme responsible for cell wall degradation during mating of C. reinhardtii. These data extend the work of Kimpel, Togasaki, Miyachi (1983 Plant Cell Physiol 24: 255-259) and indicate that the bulk of the carbonic anhydrase is located either in the periplasmic space or is loosely bound to the algal cell wall. The polypeptide associated with carbonic anhydrase activity has a molecular weight of approximately 37,000. Several lines of evidence indicate that this polypeptide is responsible for carbonic anhydrase activity: (a) it appears following the transfer of C. reinhardtii from growth on 5% CO(2) to growth on air levels of CO(2), (b) it is located in the periplasmic space or associated with the cell wall, like the bulk of the carbonic anhydrase activity, (c) it binds dansylamide, an inhibitor of the enzyme which fluoresces upon illumination with ultraviolet light, (d) antibodies which inhibit carbonic anhydrase activity only cross-react with this 37,000 dalton species.

    View details for Web of Science ID A1984TQ81300036

    View details for PubMedID 16663867

  • CYTOPLASMIC AND CHLOROPLAST SYNTHESIS OF PHYCOBILISOME POLYPEPTIDES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Egelhoff, T., Grossman, A. 1983; 80 (11): 3339-3343

    Abstract

    In vivo labeling of eukaryotic phycobilisomes in the presence of inhibitors of translation on 70S and 80S ribosomes demonstrates that some of the polypeptides of this light-harvesting complex are synthesized in the cytoplasm while others are synthesized in the chloroplast. The major pigmented polypeptides, the alpha and beta subunits of the biliproteins (molecular weights between 15,000 and 20,000) and the anchor protein (molecular weight about 90,000) are translated on 70S ribosomes. This suggests that these polypeptides are made within the algal chloroplast. Because the alpha and beta subunits comprise a group of closely related polypeptides, the genes encoding these polypeptides may reside in the plastid genome as a multigene family. Other prominent phycobilisome polypeptides, including a nonpigmented polypeptide that may be involved in maintaining the structural integrity of the complex, are synthesized on cytoplasmic ribosomes. Because the synthesis of phycobilisomes appears to require the expression of genes in two subcellular compartments, this system may be an excellent model for: (i) examining interaction between nuclear and plastid genomes: (ii) elucidating the molecular processes involved in the evolution of plastid genes: (iii) clarifying the events in the synthesis and assembly of macromolecular complexes in the chloroplast.

    View details for Web of Science ID A1983QT04900043

    View details for PubMedID 16593323