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Ma, AT, Beld J, Brahamsha B.  2017.  An amoebal grazer of cyanobacteria requires cobalamin produced by heterotrophic bacteria. Applied and Environmental Microbiology. 83   10.1128/aem.00035-17   AbstractWebsite

Amoebae are unicellular eukaryotes that consume microbial prey through phagocytosis, playing a role in shaping microbial food webs. Many amoebal species can be cultivated axenically in rich media or monoxenically with a single bacterial prey species. Here, we characterize heterolobosean amoeba LPG3, a recent natural isolate, which is unable to grow on unicellular cyanobacteria, its primary food source, in the absence of a heterotrophic bacterium, a Pseudomonas species coisolate. To investigate the molecular basis of this requirement for heterotrophic bacteria, we performed a screen using the defined nonredundant transposon library of Vibrio cholerae, which implicated genes in corrinoid uptake and biosynthesis. Furthermore, cobalamin synthase deletion mutations in V. cholerae and the Pseudomonas species coisolate do not support the growth of amoeba LPG3 on cyanobacteria. While cyanobacteria are robust producers of a corrinoid variant called pseudocobalamin, this variant does not support the growth of amoeba LPG3. Instead, we show that it requires cobalamin that is produced by the Pseudomonas species coisolate. The diversity of eukaryotes utilizing corrinoids is poorly understood, and this amoebal corrinoid auxotroph serves as a model for examining predator-prey interactions and micronutrient transfer in bacterivores underpinning microbial food webs. IMPORTANCE Cyanobacteria are important primary producers in aquatic environments, where they are grazed upon by a variety of phagotrophic protists and, hence, have an impact on nutrient flux at the base of microbial food webs. Here, we characterize amoebal isolate LPG3, which consumes cyanobacteria as its primary food source but also requires heterotrophic bacteria as a source of corrinoid vitamins. Amoeba LPG3 specifically requires the corrinoid variant produced by heterotrophic bacteria and cannot grow on cyanobacteria alone, as they produce a different corrinoid variant. This same corrinoid specificity is also exhibited by other eukaryotes, including humans and algae. This amoebal model system allows us to dissect predator-prey interactions to uncover factors that may shape microbial food webs while also providing insight into corrinoid specificity in eukaryotes.

Strom, S, Bright K, Fredrickson K, Brahamsha B.  2017.  The Synechococcus cell surface protein SwmA increases vulnerability to predation by flagellates and ciliates. Limnology and Oceanography. 62:784-794.   10.1002/lno.10460   AbstractWebsite

The genus Synechococcus, a major contributor to ocean productivity, exhibits wide genetic variability and a distinct biogeography of genetic subgroups. Synechococcus sp. strain WH8102 is a motile Sargasso Sea isolate belonging to Clade III in subcluster 5.1. Non-flagellar motility in WH8102 depends upon two large cell surface proteins, SwmA and SwmB. In the marine plankton, Synechococcus can experience high rates of mortality from protist predators, suggesting strong selective pressure for the maintenance of anti-predation defenses. We used knockout mutants deficient in large cell surface proteins (SwmA- and SwmB-) to test the hypothesis that these proteins defend WH8102 against predation by ciliates and flagellates, important consumers of Synechococcus in aquatic ecosystems. Contrary to our hypothesis, we found that wild-type WH8102 were preyed upon at higher rates than SwmA- by ciliates and by two of three tested flagellate species. Thus the SwmA protein, constitutively produced by WH8102, made this strain more rather than less vulnerable to predation. SwmA proteins link to form the S layer, a cell coating known to promote adhesion in other microbial systems, suggesting the predation vulnerability arises from increased attachment between consumers and prey. Given the SwmA-related enhancement of predation rates, our findings suggest two possibilities for WH8102 and perhaps other Clade III Synechococcus: (1) factors other than protist predation select for production of the S layer; (2) ingestion actually increases fitness, by exposing digestion-resistant cells to beneficial conditions in the food vacuole.

Hogle, SL, Brahamsha B, Barbeau KA.  2017.  Direct heme uptake by phytoplankton-associated Roseobacter bacteria. mSystems. 2( Weitz J, Ed.).   10.1128/mSystems.00124-16   Abstract

Iron is an essential micronutrient and can limit the growth of both marine phytoplankton and heterotrophic bacterioplankton. In this study, we investigated the molecular basis of heme transport, an organic iron acquisition pathway, in phytoplankton-associated Roseobacter bacteria and explored the potential role of bacterial heme uptake in the marine environment. We searched 153 Roseobacter genomes and found that nearly half contained putative complete heme transport systems with nearly the same synteny. We also examined a publicly available coculture transcriptome and found that Roseobacter strain Sulfitobacter sp. strain SA11 strongly downregulated a putative heme transport gene cluster during mutualistic growth with a marine diatom, suggesting that the regulation of heme transport might be influenced by host cues. We generated a mutant of phytoplankton-associated Roseobacter strain Ruegeria sp. strain TM1040 by insertionally inactivating its homolog of the TonB-dependent heme transporter hmuR and confirmed the role of this gene in the uptake of heme and hemoproteins. We performed competition experiments between iron-limited wild-type and mutant TM1040 strains and found that the wild type maintains a growth advantage when competing with the mutant for iron compounds derived solely from lysed diatom cells. Heme transport systems were largely absent from public marine metagenomes and metatranscriptomes, suggesting that marine bacteria with the potential for heme transport likely have small standing populations in the free-living bacterioplankton. Heme transport is likely a useful strategy for phytoplankton-associated bacteria because it provides direct access to components of the host intracellular iron pool after lysis.IMPORTANCE Ecosystem productivity in large regions of the surface ocean is fueled by iron that has been microbially regenerated from biomass. Currently, the specific microbes and molecules that mediate the transfer of recycled iron between microbial trophic levels remain largely unknown. We characterized a marine bacterial heme transporter and verified its role in acquiring heme, an abundant iron-containing enzyme cofactor. We present evidence that after host cell lysis, phytoplankton-associated bacteria directly extract heme and hemoproteins from algal cellular debris in order to fulfill their iron requirements and that the regulation of this process may be modulated by host cues. Direct heme transport, in contrast to multistep extracellular processing of hemoproteins, may allow certain phytoplankton-associated bacteria to rapidly extract iron from decaying phytoplankton, thus efficiently recycling cellular iron into the wider microbial loop.

Ma, AT, Daniels EF, Gulizia N, Brahamsha B.  2016.  Isolation of diverse amoebal grazers of freshwater cyanobacteria for the development of model systems to study predator-prey interactions. Algal Research-Biomass Biofuels and Bioproducts. 13:85-93.   10.1016/j.algal.2015.11.010   AbstractWebsite

A common method for large-scale production of algal crops is growth in outdoor open-air ponds. While this approach is more cost-effective, outdoor open-air ponds are prone to contamination by competing algae, pathogens, and eukaryotic grazers, including ciliates, flagellates, and amoebae. To characterize grazers of cyanobacteria, or blue-green algae, we have performed enrichments and isolations from water samples obtained from environmental sites and from an experimental production pond. We obtained a set of amoebal isolates that show diversity in phylogeny, morphology, and locomotion. After examination of grazing on solid medium and in liquid medium, we found that some amoebal isolates can graze on a range of cyanobacterial species, while other amoebal isolates appear to have a more limited prey range. These prey ranges correlate with observed growth rates and cyst formation, suggesting differing growth and survival strategies for amoebae in the environment. Taken together, this work provides a glimpse into the range of natural amoebal predators of cyanobacteria and establishes model systems of predator-prey interactions. Further characterization of these systems will facilitate development of strategies for crop protection of open-air algal production ponds. (C) 2015 Elsevier B.V. All rights reserved.

Johnson, TL, Brahamsha B, Palenik B, Muhle J.  2015.  Halomethane production by vanadium-dependent bromoperoxidase in marine Synechococcus. Limnology and Oceanography. 60:1823-1835.   10.1002/lno.10135   AbstractWebsite

To investigate the role of vanadium-dependent bromoperoxidase (VBPO) for the production of halogenated methanes in marine prokaryotes, we measured VBPO activity and halomethane production in two strains of Synechococcus; one with VBPO (strain CC9311) and one without VBPO (strain WH8102). A mutant strain of CC9311, VMUT2, in which the gene for VBPO is disrupted, was also tested. A suite of halomethanes was measured in the headspace above cultures as well as in the culture medium with a purge-and-trap method. Monohalomethanes were the most consistently produced molecules among the three strains tested. Additionally, CC9311 produced 301 +/- 109 molecules cell(-1) d(-1) of bromoform (CHBr3) when VBPO activity was detected, while production was not significantly different from zero when VBPO activity was not detected. VBPO activity and CHBr3 production were only detected when cultures of CC9311 were stirred, which may contribute to the often moderate to weak correlations between CHBr3 concentration and biological markers in the ocean. No production was seen by VMUT2 or WH8102. These data show that CHBr3 production rates are dramatically increased with or exclusive to the presence of VBPO, supporting its involvement in CHBr3 synthesis. This study thus provides genetic evidence that certain strains of marine Synechococcus, under particular conditions, can be a natural source of marine CHBr3, which contributes to ozone depletion in the stratosphere.

Taton, A, Unglaub F, Wright NE, Zeng WY, Paz-Yepes J, Brahamsha B, Palenik B, Peterson TC, Haerizadeh F, Golden SS, Golden JW.  2014.  Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria. Nucleic Acids Research. 42   10.1093/nar/gku673   AbstractWebsite

Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bio-products, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.

Paz-Yepes, J, Brahamsha B, Palenik B.  2013.  Role of a Microcin-C-like biosynthetic gene cluster in allelopathic interactions in marine Synechococcus. Proceedings of the National Academy of Sciences of the United States of America. 110:12030-12035.   10.1073/pnas.1306260110   AbstractWebsite

Competition between phytoplankton species for nutrients and light has been studied for many years, but allelopathic interactions between them have been more difficult to characterize. We used liquid and plate assays to determine whether these interactions occur between marine unicellular cyanobacteria of the genus Synechococcus. We have found a clear growth impairment of Synechococcus sp. CC9311 and Synechococcus sp. WH8102 when they are cultured in the presence of Synechococcus sp. CC9605. The genome of CC9605 contains a region showing homology to genes of the Escherichia coli Microcin C (McC) biosynthetic pathway. McC is a ribosome-synthesized peptide that inhibits translation in susceptible strains. We show that the CC9605 McC gene cluster is expressed and that three genes (mccD, mccA, and mccB) are further induced by coculture with CC9311. CC9605 was resistant to McC purified from E. coli, whereas strains CC9311 and WH8102 were sensitive. Cloning the CC9605 McC biosynthetic gene cluster into sensitive CC9311 led this strain to become resistant to both purified E. coli McC and Synechococcus sp. CC9605. A CC9605 mutant lacking mccA1, mccA2, and the N-terminal domain of mccB did not inhibit CC9311 growth, whereas the inhibition of WH8102 was reduced. Our results suggest that an McC-like molecule is involved in the allelopathic interactions with CC9605.

Stuart, RK, Brahamsha B, Busby K, Palenik B.  2013.  Genomic island genes in a coastal marine Synechococcus strain confer enhanced tolerance to copper and oxidative stress. Isme Journal. 7:1139-1149.   10.1038/ismej.2012.175   AbstractWebsite

Highly variable regions called genomic islands are found in the genomes of marine picocyano-bacteria, and have been predicted to be involved in niche adaptation and the ecological success of these microbes. These picocyanobacteria are typically highly sensitive to copper stress and thus, increased copper tolerance could confer a selective advantage under some conditions seen in the marine environment. Through targeted gene inactivation of genomic island genes that were known to be upregulated in response to copper stress in Synechococcus sp. strain CC9311, we found two genes (sync_1495 and sync_1217) conferred tolerance to both methyl viologen and copper stress in culture. The prevalence of one gene, sync_1495, was then investigated in natural samples, and had a predictable temporal variability in abundance at a coastal monitoring site with higher abundance in winter months. Together, this shows that genomic island genes can confer an adaptive advantage to specific stresses in marine Synechococcus, and may help structure their population diversity.

Simkovsky, R, Daniels EF, Tang K, Huynh SC, Golden SS, Brahamsha B.  2012.  Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proceedings of the National Academy of Sciences of the United States of America. 109:16678-16683.   10.1073/pnas.1214904109   AbstractWebsite

The grazing activity of predators on photosynthetic organisms is a major mechanism of mortality and population restructuring in natural environments. Grazing is also one of the primary difficulties in growing cyanobacteria and other microalgae in large, open ponds for the production of biofuels, as contaminants destroy valuable biomass and prevent stable, continuous production of biofuel crops. To address this problem, we have isolated a heterolobosean amoeba, HGG1, that grazes upon unicellular and filamentous freshwater cyanobacterial species. We have established a model predator-prey system using this amoeba and Synechococcus elongatus PCC 7942. Application of amoebae to a library of mutants of S. elongatus led to the identification of a grazer-resistant knockout mutant of the wzm ABC O-antigen transporter gene, SynPCC7942_1126. Mutations in three other genes involved in O-antigen synthesis and transport also prevented the expression of O-antigen and conferred resistance to HGG1. Complementation of these rough mutants returned O-antigen expression and susceptibility to amoebae. Rough mutants are easily identifiable by appearance, are capable of autoflocculation, and do not display growth defects under standard laboratory growth conditions, all of which are desired traits for a biofuel production strain. Thus, preventing the production of O-antigen is a pathway for producing resistance to grazing by certain amoebae.

Dupont, CL, Johnson DA, Phillippy K, Paulsen IT, Brahamsha B, Palenik B.  2012.  Genetic identification of a high-affinity Ni transporter and the transcriptional response to Ni deprivation in Synechococcus sp. strain WH8102. Applied and Environmental Microbiology. 78:7822-7832.   10.1128/aem.01739-12   AbstractWebsite

One biological need for Ni in marine cyanobacteria stems from the utilization of the Ni metalloenzyme urease for the assimilation of urea as a nitrogen source. In many of the same cyanobacteria, including Synechococcus sp. strain WH8102, an additional and obligate nutrient requirement for Ni results from usage of a Ni superoxide dismutase (Ni-SOD), which is encoded by sodN. To better understand the effects of Ni deprivation on WH8102, parallel microarray-based analysis of gene expression and gene knockout experiments were conducted. The global transcriptional response to Ni deprivation depends upon the nitrogen source provided for growth; fewer than 1% of differentially expressed genes for Ni deprivation on ammonium or urea were concordantly expressed. Surprisingly, genes for putative Ni transporters, including one colocalized on the genome with sodN, sodT, were not induced despite an increase in Ni transport. Knockouts of the putative Ni transporter gene sodT appeared to be lethal in WH8102, so the genes for sodT and sodN in WH8102 were interrupted with the gene for Fe-SOD, sodB, and its promoter from Synechococcus sp. strain WH7803. The sodT:: sodB exconjugants were unable to grow at low Ni concentrations, confirming that SodT is a Ni transporter. The sodN::sodB exconjugants displayed higher growth rates at low Ni concentrations than did the wild type, presumably due to a relaxed competition between urease and Ni-SOD for Ni. Both sodT::sodB and sodN::sodB lines exhibited an impaired ability to grow at low Fe concentrations. We propose a posttranslational allosteric SodT regulation involving the binding of Ni to a histidine-rich intracellular protein loop.

Strom, SL, Brahamsha B, Fredrickson KA, Apple JK, Rodriguez AG.  2012.  A giant cell surface protein in Synechococcus WH8102 inhibits feeding by a dinoflagellate predator. Environmental Microbiology. 14:807-816.   10.1111/j.1462-2920.2011.02640.x   AbstractWebsite

Diverse strains of the marine planktonic cyanobacterium Synechococcus sp. show consistent differences in their susceptibility to predation. We used mutants of Sargasso Sea strain WH8102 (clade III) to test the hypothesis that cell surface proteins play a role in defence against predation by protists. Predation rates by the heterotrophic dinoflagellate Oxyrrhis marina on mutants lacking the giant SwmB protein were always higher (by 1.6 to 3.9x) than those on wild-type WH8102 cells, and equalled predation rates on a clade I strain (CC9311). In contrast, absence of the SwmA protein, which comprises the S-layer (surface layer of the cell envelope that is external to the outer membrane), had no effect on predation by O. marina. Reductions in predation rate were not due to dissolved substances in Synechococcus cultures, and could not be accounted for by variations in cell hydrophobicity. We hypothesize that SwmB defends Synechococcus WH8102 by interfering with attachment of dinoflagellate prey capture organelles or cell surface receptors. Giant proteins are predicted in the genomes of multiple Synechococcus isolates, suggesting that this defence strategy may be more general. Strategies for resisting predation will contribute to the differential competitive success of different Synechococcus groups, and to the diversity of natural picophytoplankton assemblages.

Apple, JK, Strom SL, Palenik B, Brahamsha B.  2011.  Variability in protist grazing and growth on different marine Synechococcus isolates. Applied and Environmental Microbiology. 77:3074-3084.   10.1128/aem.02241-10   AbstractWebsite

Grazing mortality of the marine phytoplankton Synechococcus is dominated by planktonic protists, yet rates of consumption and factors regulating grazer-Synechococcus interactions are poorly understood. One aspect of predator-prey interactions for which little is known are the mechanisms by which Synechococcus avoids or resists predation and, in turn, how this relates to the ability of Synechococcus to support growth of protist grazer populations. Grazing experiments conducted with the raptorial dinoflagellate Oxyrrhis marina and phylogenetically diverse Synechococcus isolates (strains WH8102, CC9605, CC9311, and CC9902) revealed marked differences in grazing rates-specifically that WH8102 was grazed at significantly lower rates than all other isolates. Additional experiments using the heterotrophic nanoflagellate Goniomonas pacifica and the filter-feeding tintinnid ciliate Eutintinnis sp. revealed that this pattern in grazing susceptibility among the isolates transcended feeding guilds and grazer taxon. Synechococcus cell size, elemental ratios, and motility were not able to explain differences in grazing rates, indicating that other features play a primary role in grazing resistance. Growth of heterotrophic protists was poorly coupled to prey ingestion and was influenced by the strain of Synechococcus being consumed. Although Synechococcus was generally a poor-quality food source, it tended to support higher growth and survival of G. pacifica and O. marina relative to Eutintinnis sp., indicating that suitability of Synechococcus varies among grazer taxa and may be a more suitable food source for the smaller protist grazers. This work has developed tractable model systems for further studies of grazer-Synechococcus interactions in marine microbial food webs.

Johnson, TL, Palenik B, Brahamsha B.  2011.  Characterization of a functional vanadium-dependent bromoperoxidase in the marine cyanobacterium Synechococcus sp. CC9311. Journal of Phycology. 47:792-801.   10.1111/j.1529-8817.2011.01007.x   AbstractWebsite

Vanadium-dependent bromoperoxidases (VBPOs) are characterized by the ability to oxidize halides using hydrogen peroxide. These enzymes are well-studied in eukaryotic macroalgae and are known to produce a variety of brominated secondary metabolites. Though genes have been annotated as VBPO in multiple prokaryotic genomes, they remain un-characterized. The genome of the coastal marine cyanobacterium Synechococcus sp. CC9311 encodes a predicted VBPO (YP_731869.1, sync_2681), and in this study, we show that protein extracts from axenic cultures of Synechococcus possess bromoperoxidase activity, oxidizing bromide and iodide, but not chloride. In-gel activity assays of Synechococcus proteins separated using PAGE reveal a single band having VBPO activity. When sequenced via liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), peptides from the band aligned to the VBPO sequence predicted by the open reading frame (ORF) sync_2681. We show that a VBPO gene is present in a closely related strain, Synechococcus sp. WH8020, but not other clade I Synechococcus strains, consistent with recent horizontal transfer of the gene into Synechococcus. Diverse cyanobacterial-like VBPO genes were detected in a pelagic environment off the California coast using PCR. Investigation of functional VBPOs in unicellular cyanobacteria may lead to discovery of novel halogenated molecules and a better understanding of these organisms' chemical ecology and physiology.

Snyder, DS, Brahamsha B, Azadi P, Palenik B.  2009.  Structure of compositionally simple lipopolysaccharide from marine Synechococcus. Journal of Bacteriology. 191:5499-5509.   10.1128/jb.00121-09   AbstractWebsite

Lipopolysaccharide (LPS) is the first defense against changing environmental factors for many bacteria. Here, we report the first structure of the LPS from cyanobacteria based on two strains of marine Synechococcus, WH8102 and CC9311. While enteric LPS contains some of the most complex carbohydrate residues in nature, the full-length versions of these cyanobacterial LPSs have neither heptose nor 3-deoxy-D-manno-octulosonic acid (Kdo) but instead 4-linked glucose as their main saccharide component, with low levels of glucosamine and galacturonic acid also present. Matrix-assisted laser desorption ionization mass spectrometry of the intact minimal core LPS reveals triacylated and tetraacylated structures having a heterogeneous mix of both hydroxylated and nonhydroxylated fatty acids connected to the diglucosamine backbone and a predominantly glucose outer core-like region for both strains. WH8102 incorporated rhamnose in this region as well, contributing to differences in sugar composition and possibly nutritional differences between the strains. In contrast to enteric lipid A, which can be liberated from LPS by mild acid hydrolysis, lipid A from these organisms could be produced by only two novel procedures: triethylamine-assisted periodate oxidation and acetolysis. The lipid A contains odd-chain hydroxylated fatty acids, lacks phosphate, and contains a single galacturonic acid. The LPS lacks any limulus amoebocyte lysate gelation activity. The highly simplified nature of LPSs from these organisms leads us to believe that they may represent either a primordial structure or an adaptation to the relatively higher salt and potentially growth-limiting phosphate levels in marine environments.

Tetu, SG, Brahamsha B, Johnson DA, Tai V, Phillippy K, Palenik B, Paulsen IT.  2009.  Microarray analysis of phosphate regulation in the marine cyanobacterium Synechococcus sp WH8102. ISME Journal. 3:835-849.   10.1038/ismej.2009.31   AbstractWebsite

Primary productivity of open ocean environments, such as those inhabited by marine picocyanobacteria, is often limited by low inorganic phosphate (P). To observe how these organisms cope with P starvation, we constructed a full genome microarray for Synechococcus sp. WH8102 and compared differences in gene expression under P-replete and P-limited growth conditions, including both early P stress, during extracellular alkaline phosphatase induction, and late P stress. A total of 36 genes showed significant upregulation (>log(2) fold) whereas 23 genes were highly downregulated at the early time point; however, these changes in expression were maintained during late P stress for only 5 of the upregulated genes. Knockout mutants were constructed for genes SYNW0947 and SYNW0948, comprising a two-component regulator hypothesized to have a key function in regulating P metabolism. A high degree of overlap in the sets of genes affected by P stress conditions and in the knockout mutants supports this hypothesis; however, there is some indication that other regulators may be involved in this response in Synechococcus sp. WH8102. Consistent with what has been observed in many other cyanobacteria, the Pho regulon of this strain is comprised largely of genes for alkaline phosphatases, P transport or P metabolism. Interestingly, however, the exact composition and arrangement of the Pho regulon appears highly variable in marine cyanobacteria. The ISME Journal (2009) 3, 835-849; doi: 10.1038/ismej.2009.31; published online 2 April 2009

McCarren, J, Brahamsha B.  2009.  Swimming motility mutants of marine Synechococcus Affected in production and localization of the S-layer protein SwmA. Journal of Bacteriology. 191:1111-1114.   10.1128/jb.01401-08   AbstractWebsite

The S-layer protein SwmA is required for nonflagellar swimming in marine Synechococcus. An analysis of mutations in seven genes at two loci in the Synechococcus sp. strain WH8102 genome indicates that a multicomponent transporter and glycosyltransferases are required for the production and proper localization of SwmA.

Thomas, EV, Phillippy KH, Brahamsha B, Haaland DM, Timlin JA, Elbourne LDH, Palenik B, Paulsen IT.  2009.  Statistical analysis of microarray data with replicated spots: a case study with Synechococcus WH8102. Comparative and Functional Genomics.   10.1155/2009/950171   AbstractWebsite

Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in part to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition. Copyright (C) 2009 E. V. Thomas et al.

McCarren, J, Brahamsha B.  2007.  SwmB, a 1.12-megadalton protein that is required for nonflagellar swimming motility in Synechococcus. Journal of Bacteriology. 189:1158-1162.   10.1128/jb.01500-06   AbstractWebsite

SwmB is required for swimming motility in Synechococcus sp. strain WH8102. This highly repetitive 1.12-MDa polypeptide is associated with the cell surface, where it is arranged in a punctate manner. Inactivation of swmB does not affect the localization of SwmA, an S-layer protein also required for swimming.

McCarren, J, Brahamsha B.  2005.  Transposon mutagenesis in a marine synechococcus strain: Isolation of swimming motility mutants. Journal of Bacteriology. 187:4457-4462.   10.1128/jb.187.13.4457-4462.2005   AbstractWebsite

Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique type of swimming motility characterized by the absence of flagella or any other obvious organelles of motility. While the abundant cell surface-associated 130-kDa glycoprotein SwmA is known to be required for the generation of thrust, identification of other components of the motility apparatus has, until recently, been unsuccessful. Here we report on the development of a transposon mutagenesis system for use with marine Synechococcus sp. strain WH8102, a model organism for which the genome has been sequenced. Utilizing this mutagenesis technique, we have isolated 17 independent mutants impaired in swimming motility. These 17 transposon insertions are located in nine open reading frames, which cluster in three separate regions of the genome. Included within these clusters are several multicomponent transport systems as well as a number of glycosyltransferases.

McCarren, J, Heuser J, Roth R, Yamada N, Martone M, Brahamsha B.  2005.  Inactivation of swmA results in the loss of an outer cell layer in a swimming Synechococcus strain. Journal of Bacteriology. 187:224-230.   10.1128/jb.187.1.224-230.2005   AbstractWebsite

The mechanism of nonflagellar swimming of marine unicellular cyanobacteria remains poorly understood. SwmA is an abundant cell surface-associated 130-kDa glycoprotein that is required for the generation of thrust in Synechococcus sp. strain WH8102. Ultrastructural comparisons of wild-type cells to a mutant strain in which the gene encoding SwmA has been insertionally inactivated reveal that the mutant lacks a layer external to the outer membrane. Cryofixation and freeze-substitution are required for the preservation of this external layer. Freeze fracturing and etching reveal that this additional layer is an S-layer. How the S-layer might function in motility remains elusive; however, this work describes an ultrastructural component required for this unique type of swimming. In addition, the work presented here describes the envelope structure of a model swimming cyanobacterium.

Six, C, Thomas JC, Brahamsha B, Lemoine Y, Partensky F.  2004.  Photophysiology of the marine cyanobacterium Synechococcus sp. WH8102, a new model organism. Aquatic Microbial Ecology. 35:17-29.   10.3354/ame035017   AbstractWebsite

Synechococcus spp. constitute a major and ubiquitous component of marine ecosystems. The genome of one strain of this genus, WH8102, has recently been completely sequenced. Since it can also be genetically manipulated, this clone has the potential to become a new model organism however, to date, it remains poorly characterised in terms of pigment composition, optical properties and photophysiology. It has a very high phycourobilin to phycoerythrobilin (PUB:PEB) ratio (ca. 1,95 at low light), and is therefore representative of Synechococcus populations found in oligotrophic areas of the ocean. We show here that this strain has a very wide growth irradiance range from < 15 to > 650 mumol photons m(-2) s(-1) continuous white light, with a maximum growth rate (mu(max) = 1.13 +/- 0.02 d(-1)) at 207 mumol quanta m(-2) s(-1) (I-max). As cells acclimated to high light, drastic variations in the chlorophyll a (chl a), beta-carotene and phycoerythrin (PE) contents were observed, reaching a quasi steady state around In contrast, the zeaxanthin content remained approximately constant whatever the light level. Similarly, the carbon and nitrogen contents did not significantly vary with irradiance, Red and orange fluorescences, as measured by flow cytometry, were found to correlate well with chl a and PE contents, respectively. Spectrometric analyses of phycobilisome (PBS)-containing fractions from cells grown under different photon fluxes suggest. a specific reduction of the PEII content relative to other phycobiliproteins (PBPs) during acclimation of the PBSs to high light.

Palenik, B, Brahamsha B, Larimer FW, Land M, Hauser L, Chain P, Lamerdin J, Regala W, Allen EE, McCarren J, Paulsen I, Dufresne A, Partensky F, Webb EA, Waterbury J.  2003.  The genome of a motile marine Synechococcus. Nature. 424:1037.: Macmillan Magazines Ltd.   10.1038/nature01943   Abstract

Marine unicellular cyanobacteria are responsible for an estimated 20–40% of chlorophyll biomass and carbon fixation in the oceans1. Here we have sequenced and analysed the 2.4-megabase genome of Synechococcus sp. strain WH8102, revealing some of the ways that these organisms have adapted to their largely oligotrophic environment. WH8102 uses organic nitrogen and phosphorus sources and more sodium-dependent transporters than a model freshwater cyanobacterium. Furthermore, it seems to have adopted strategies for conserving limited iron stores by using nickel and cobalt in some enzymes, has reduced its regulatory machinery (consistent with the fact that the open ocean constitutes a far more constant and buffered environment than fresh water), and has evolved a unique type of swimming motility. The genome of WH8102 seems to have been greatly influenced by horizontal gene transfer, partially through phages. The genetic material contributed by horizontal gene transfer includes genes involved in the modification of the cell surface and in swimming motility. On the basis of its genome, WH8102 is more of a generalist than two related marine cyanobacteria2.

Brahamsha, B.  2000.  Non-flagellar swimming in marine Synechococcus. Molecular marine microbiology. ( Bartlett D, Ed.).:59-62., Wymondham, Norfolk, England: Horizon Scientific Abstract
Collier, JL, Brahamsha B, Palenik B.  1999.  The marine cyanobacterium Synechococcus sp. WH7805 requires urease (urea amidohydrolase, EC to utilize urea as a nitrogen source: molecular-genetic and biochemical analysis of the enzyme. Microbiology-Sgm. 145:447-459.   10.1099/13500872-145-2-447   AbstractWebsite

Cyanobacteria assigned to the genus Synechococcus are an important component of oligotrophic marine ecosystems, where their growth may be constrained by low availability of fixed nitrogen. Urea appears to be a major nitrogen resource in the sea, but little molecular information exists about its utilization by marine organisms, including Synechococcus. Oligonucleotide primers were used to amplify a conserved fragment of the urease (urea amidohydrolase, EC coding region from cyanobacteria. A 5.7 kbp region of the genome of the unicellular marine cyanobacterium Synechococcus sp. strain WH7805 was then cloned, and genes encoding three urease structural subunits and four urease accessory proteins were sequenced and identified by homology. The WH7805 urease had a predicted subunit composition typical of bacterial ureases, but the organization of the WH7805 urease genes was unique. Biochemical characteristics of the WH7805 urease enzyme were consistent with the predictions of the sequence data. Physiological data and sequence analysis both suggested that the urease operon may be nitrogen-regulated by the ntcA system in WH7805. Inactivation of the large subunit of urease, ureC, prevented WH7805 and Synechococcus WH8102 from growing on urea, demonstrating that the urease genes cloned are essential to the ability of these cyanobacteria to utilize urea as a nitrogen source.

Toledo, G, Palenik B, Brahamsha B.  1999.  Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group. Applied and Environmental Microbiology. 65:5247-5251. AbstractWebsite

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim, Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/FEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.