Search

Marine Synechococcus is a principal component of the picophytoplankton and makes an important contribution to primary productivity in the ocean. Synechophages, infecting Synechococcus, are believed to have significant influences on the distribution and abundance of their hosts. Extensive previous ecological studies on cyanobacteria and viruses have been carried out in the East China Sea (ECS). Here we investigate the diversity and divergence of Synechococcus and their myoviruses (Synechomyoviruses) based on their shared photosynthesis psbA gene. Synechococcus is dominated by subclades 5.1A I, 5.1A II and 5.1A IV in the ECS, and clades I and II are the dominant groups in the Synechomyoviruses. As two phylogenetically independent clades, there is much higher diversity of the Synechomyoviruses than Synechococcus. Obvious partitioning characteristics of GC and GC3 (the GC content at the third codon position) contents are obtained among different picophytoplankton populations and their phages. The GC3 content causes the psbA gene in Synechococcus to have a higher GC content, while the opposite is true in the Synechomyoviruses. Analyzing more than one-time difference of the codon usage frequency of psbA sequences, the third position nucleotides of preferred codons for Synechococcus are all G and C, while most Synechomyoviral sequences (72.7%) have A and T at the third position of their preferred codons. This work shed light on the ecology and evolution of phage-host interactions in the environment.

It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. "Universal proteins" have been used to infer the organismal phylogeny, but have been criticized as being only the "tree of one percent." Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees - a "treecloud" - for each, as well as for a concatenated dataset based on putative "universal proteins." We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS). We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the "tree of one percent" is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.

Synechococcus are distributed throughout the world's oceans and are composed of diverse genetic lineages. However, as they are much less abundant than Prochlorococcus in oligotrophic open oceans, their in-depth genetic diversity cannot be investigated using commonly used primers targeting both Prochlorococcus and Synechococcus. Thus, in this study, we designed a primer specific to the 16S-23S rRNA internal transcribed spacer (ITS) of the Synechococcus subcluster 5.1. Using the primer, we could selectively amplify Synechococcus sequences in oligotrophic seawater samples. Further, we showed that a barcoded amplicon pyrosequencing method could be applicable to investigate Synechococcus diversity using sequences retrieved in GenBank and obtained from environmental samples. Allowing sequence analyses of a large number of samples, this high-throughput method would be useful to study global biodiversity and biogeographic patterns of Synechococcus in marine environments.

Hot springs and saline-alkaline lakes of East Africa are extreme habitats regarding temperature, or salinity and pH, respectively. This study examines whether divergent habitats of Lake Bogoria, Kenya, impacts cyanobacterial community structure. Samples from the hot springs, pelagic zone and sediment were analysed by light microscopy, multilocus 454-amplicons sequencing and metagenomics to compare the cyanobacterial diversity. Most of the phylogenetic lineages of Cyanobacteria occurred exclusively in the Bogoria hot springs suggesting a high degree of endemism. The prevalent phylotypes were mainly members of the Oscillatoriales (Leptolyngbya, Spirulina, Oscillatoria-like and Planktothricoides). The Chroococcales were represented by different clades of Synechococcus but not a single phylotype clustered with any of the lineages described earlier from different continents. In contrast, we found that the pelagic zone and the sediments were inhabited by only a few taxa, dominated by Arthrospira and Anabaenopsis. Arthrospira, the main food base of Lesser Flamingo, was detected in all three habitats by amplicons pyrosequencing, indicating its resilience and key role as a primary producer. Despite the close connection between the three habitats studied, the cyanobacterial communities in the hot springs and lake differed considerably, suggesting that they are unable to adapt to the extreme conditions of the neighbouring habitat., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & sons Ltd. All rights reserved.)

We studied the phylogenetic diversity of nonmarine picocyanobacteria broadening the sequence data set with 43 new sequences of the 16S rRNA gene. The sequences were derived from monoclonal strains isolated from four volcanic high-altitude athalassohaline lakes in Mexico, five glacial ultraoligotrophic North Patagonian lakes and six Italian lakes of glacial, volcanic and morenic origin. The new sequences fall into a number of both novel and previously described clades within the phylogenetic tree of 16S rRNA gene. The new cluster of Lake Nahuel Huapi (North Patagonia) forms a sister clade to the subalpine cluster II and the marine Synechococcus subcluster 5.2. Our finding of the novel clade of 'halotolerants' close to the marine subcluster 5.3 (Synechococcus RCC307) constitutes an important demonstration that euryhaline and marine strains affiliate closely. The intriguing results obtained shed new light on the importance of the nonmarine halotolerants in the phylogenesis of picocyanobacteria., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Phytoplankton are single-celled, photosynthetic algae and cyanobacteria found in all aquatic environments. Differential pigmentation between phytoplankton taxa allows use of fluorescence excitation spectroscopy for discrimination and classification. For this work, we applied multivariate optical computing (MOC) to emulate linear discriminant vectors of phytoplankton fluorescence excitation spectra by using a simple filter-fluorometer arrangement. We grew nutrient-replete cultures of three differently pigmented species: the coccolithophore Emiliania huxleyi, the diatom Thalassiosira pseudonana, and the cyanobacterium Synechococcus sp. Linear discriminant analysis (LDA) was used to determine a suitable set of linear discriminant functions for classification of these species over an optimal wavelength range. Multivariate optical elements (MOEs) were then designed to predict the linear discriminant scores for the same calibration spectra. The theoretical performance specifications of these MOEs are described.

Three new cyanobacterial strains, that have been previously purified from the hydroid Dynamena pumila (L., 1758), isolated from the White Sea, were studied using scanning and transmission electron microscopy methods and were characterized by using almost complete sequence of the 16S rRNA gene, internal transcribed spacer 16S-23S rRNA, and part of the gene for 23S rRNA. The full nucleotide sequences of the rRNA gene clusters were deposited to GenBank (HM064496.1, GU265558.1, JQ259187.1). Comparison of rRNA gene cluster sequences of Synechococcus cyanobacterium 1Dp66E-1, Oscillatoriales cyanobacterium 2Dp86E, and Nostoc sp. 10Dp66E with all sequences present at the GenBank shows that these cyanobacterial strains do not have 100% identity with any organisms investigated previously. Furthermore, for the first time heterotrophic bacterium, associated with Nostoc sp. 10Dp66E, was identified as a member of the new phylum Gemmatimonadetes, genus of Gemmatimonas (GenBank accession number is JX437625.1). Phylogenetic analysis showed that cyanobacterium Synechococcus sp. 1Dp66E-1 forms the unique branch and belongs to a cluster of Synechococcus, including freshwater and sea strains. Oscillatoriales cyanobacterium 2Dp86E belongs to a cluster of Leptolyngbya strains. Isolate Nostoc sp. 10Dp66E forms unique branch and belongs to a cluster of the genus Nostoc, with the closest relative of Nostoc commune isolates.

Background: In the last decade, a considerable amount of research has been devoted to investigating the phylogenetic properties of organisms from a systems-level perspective. Most studies have focused on the classification of organisms based on structural comparison and local alignment of metabolic pathways. In contrast, global alignment of multiple metabolic networks complements sequence-based phylogenetic analyses and provides more comprehensive information., Results: We explored the phylogenetic relationships between microorganisms through global alignment of multiple metabolic networks. The proposed approach integrates sequence homology data with topological information of metabolic networks. In general, compared to recent studies, the resulting trees reflect the living style of organisms as well as classical taxa. Moreover, for phylogenetically closely related organisms, the classification results are consistent with specific metabolic characteristics, such as the light-harvesting systems, fermentation types, and sources of electrons in photosynthesis., Conclusions: We demonstrate the usefulness of global alignment of multiple metabolic networks to infer phylogenetic relationships between species. In addition, our exhaustive analysis of microbial metabolic pathways reveals differences in metabolic features between phylogenetically closely related organisms. With the ongoing increase in the number of genomic sequences and metabolic annotations, the proposed approach will help identify phenotypic variations that may not be apparent based solely on sequence-based classification.

Marine sponges often harbor photosynthetic symbionts that may enhance host metabolism and ecological success, yet little is known about the factors that structure the diversity, specificity, and nature of these relationships. Here, we characterized the cyanobacterial symbionts in two congeneric and sympatric host sponges that exhibit distinct habitat preferences correlated with irradiance: Ircinia fasciculata (higher irradiance) and Ircinia variabilis (lower irradiance). Symbiont composition was similar among hosts and dominated by the sponge-specific cyanobacterium Synechococcus spongiarum. Phylogenetic analyses of 16S-23S rRNA internal transcribed spacer (ITS) gene sequences revealed that Mediterranean Ircinia spp. host a specific, novel symbiont clade ("M") within the S. spongiarum species complex. A second, rare cyanobacterium related to the ascidian symbiont Synechocystis trididemni was observed in low abundance in I. fasciculata and likewise corresponded to a new symbiont clade. Symbiont communities in I. fasciculata exhibited nearly twice the chlorophyll a concentrations of I. variabilis. Further, S. spongiarum clade M symbionts in I. fasciculata exhibited dense intracellular aggregations of glycogen granules, a storage product of photosynthetic carbon assimilation rarely observed in I. variabilis symbionts. In both host sponges, S. spongiarum cells were observed interacting with host archeocytes, although the lower photosynthetic activity of Cyanobacteria in I. variabilis suggests less symbiont-derived nutritional benefit. The observed differences in clade M symbionts among sponge hosts suggest that ambient irradiance conditions dictate symbiont photosynthetic activity and consequently may mediate the nature of host-symbiont relationships. In addition, the plasticity exhibited by clade M symbionts may be an adaptive attribute that allows for flexibility in host-symbiont interactions across the seasonal fluctuations in light and temperature characteristic of temperate environments.

In marine Synechococcus there is evidence for the adaptive evolution of spectrally distinct forms of the major light harvesting pigment phycoerythrin (PE). Recent research has suggested that these spectral forms of PE have a different evolutionary history than the core genome. However, a lack of explicit statistical testing of alternative hypotheses or for selection on these genes has made it difficult to evaluate the evolutionary relationships between spectral forms of PE or the role horizontal gene transfer (HGT) may have had in the adaptive phenotypic evolution of the pigment system in marine Synechococcus. In this work, PE phylogenies of picocyanobacteria with known spectral phenotypes, including newly co-isolated strains of marine Synechococcus from the Gulf of Mexico, were constructed to explore the diversification of spectral phenotype and PE evolution in this group more completely. For the first time, statistical evaluation of competing evolutionary hypotheses and tests for positive selection on the PE locus in picocyanobacteria were performed. Genes for PEs associated with specific PE spectral phenotypes formed strongly supported monophyletic clades within the PE tree with positive directional selection driving evolution towards higher phycourobilin (PUB) content. The presence of the PUB-lacking phenotype in PE-containing marine picocyanobacteria from cyanobacterial lineages identified as Cyanobium is best explained by HGT into this group from marine Synechococcus. Taken together, these data provide strong examples of adaptive evolution of a single phenotypic trait in bacteria via mutation, positive directional selection and horizontal gene transfer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have identified a homologue of 4-deoxygadusol (core of mycosporine-like amino acids) synthesizing gene (ZP_05036788) from Synechococcus sp. PCC 7335 that was found to have additional functionally unknown N-terminal domain similar to homologues from dinoflagellates based on the ClustalW analysis. Phylogenetic analysis revealed that Synechococcus sp. (ZP_05036788) makes a clade together with dinoflagellates and was closest to the Oxyrrhis marina. This study shows for the first time that N-terminal additional sequences that possess upstream plastid targeting sequence in Heterocapsa triquetra and Karlodinium micrum were already evolved in cyanobacteria, and plastid targeting sequence were evolved later in dinoflagellates after divergence from chloroplast lacking Oxyrrhis marina. Thus, MAAs synthesizing genes were transferred from cyanobacteria to dinoflagellates and possibly Synechococcus sp. PCC 7335 acted as a donor during lateral gene transfer event. In addition, we also tried to mutate 4-deoxygadusol synthesizing gene (YP_324358) of Anabaena variabilis PCC 7937 by homologous recombination, however, all approaches to get complete segregation of the mutants from the wild-type were unsuccessful, showing the essentiality of YP_324358 for A. variabilis PCC 7937., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We found that the clade-specific abundance dynamics of Synechococcus type picocyanobacteria in the pelagic and littoral zone macro-habitats of Lake Constance (Germany) challenge the hypothesis of a regular annual succession of picocyanobacteria genotypes in temperate zone lakes. Methods used in this study were quantitative Taq nuclease assays (TNA), denaturing gradient gel electrophoresis (DGGE), a 19-month time series analysis (with two isothermal and two stratified periods) and genotyping of a new littoral phycocyanin (PC)-rich Synechococcus strain collection. The recorded differences between the two macro-habitats and between seasons or years, and the observed effect of water column mixis in winter on the inversion of clade-specific dominance ratios in Lake Constance might explain the known inter-annual differences in abundance and dynamics of the autotrophic picoplankton (APP) in lakes. The APP in Lake Constance shows a high genetic diversity with a low overall abundance, similar to the APP in the Baltic Sea, but different from Lake Biwa in Japan or lakes in the UK. Our results indicate that APP bloom events in both macro-habitats of Lake Constance are driven by phycoerythrin-rich Synechococcus genotypes of the Subalpine Cluster I. DGGE revealed the presence of a diverse periphyton (biofilm) community of the PC-rich Synechococcus pigment type in the littoral zone in early spring, when no such community was detectable in the pelagic habitat. A more sensitive and quantitative approach with TNA, however, revealed an intermittent presence of one PC-rich genotype in the plankton. We discuss the seasonal development of the pelagic and littoral PC-rich community, and while we cannot rule out a strain isolation bias, we found that isolated PC-rich strains from the pelagic habitat have different genotypes when compared to new littoral strains. We also observed littoral substrates colonized by specific PC-rich Synechococcus genotypes., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Synechococcus is a cosmopolitan marine cyanobacterial genus, and is often the most abundant picocyanobacterial genus in coastal waters. Little is known about Synechococcus seasonal dynamics in coastal zones highly impacted by upwelling. This was investigated by collecting seasonal samples from an upwelling-impacted Monterey Bay (MB) monitoring station M0, in parallel with measurements of oceanographic conditions during 2006-2008. Synechococcus abundances were determined using quantitative PCR (qPCR) assays and flow cytometry (FCM). A new qPCR assay was designed to target dominant Synechococcus in MB using the rbcL gene, while previously designed assays targeted distinct phylotypes (called narB subgroups) with the narB gene. The rbcL qPCR assay successfully tracked abundant Synechococcus in MB, accounting for on average 89% (± 57%) of FCM-based counts. Annual spring upwelling caused decreases in Synechococcus and narB subgroup abundances. Differences in narB subgroup abundance maxima and abundance patterns support the view that subgroups differ in their ecologies, including subgroup D_C1, which seems to specifically thrive in coastal waters. Correlations between narB subgroup abundances and measured environmental variables were similar among the subgroups. Therefore, non-measured environmental factors (e.g. metals, mortality) likely had different influences on subgroups, which led to their distinct abundance patterns at M0., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Picocyanobacteria represented by Prochlorococcus and Synechococcus have an important role in oceanic carbon fixation and nutrient cycling. In this study, we compared the community composition of picocyanobacteria from diverse marine ecosystems ranging from estuary to open oceans, tropical to polar oceans and surface to deep water, based on the sequences of 16S-23S rRNA internal transcribed spacer (ITS). A total of 1339 ITS sequences recovered from 20 samples unveiled diverse and several previously unknown clades of Prochlorococcus and Synechococcus. Six high-light (HL)-adapted Prochlorococcus clades were identified, among which clade HLVI had not been described previously. Prochlorococcus clades HLIII, HLIV and HLV, detected in the Equatorial Pacific samples, could be related to the HNLC clades recently found in the high-nutrient, low-chlorophyll (HNLC), iron-depleted tropical oceans. At least four novel Synechococcus clades (out of six clades in total) in subcluster 5.3 were found in subtropical open oceans and the South China Sea. A niche partitioning with depth was observed in the Synechococcus subcluster 5.3. Members of Synechococcus subcluster 5.2 were dominant in the high-latitude waters (northern Bering Sea and Chukchi Sea), suggesting a possible cold-adaptation of some marine Synechococcus in this subcluster. A distinct shift of the picocyanobacterial community was observed from the Bering Sea to the Chukchi Sea, which reflected the change of water temperature. Our study demonstrates that oceanic systems contain a large pool of diverse picocyanobacteria, and further suggest that new genotypes or ecotypes of picocyanobacteria will continue to emerge, as microbial consortia are explored with advanced sequencing technology.

Conserved markers such as the 16S rRNA gene do not provide sufficient molecular resolution to identify spatially structured populations of marine Synechococcus, or 'ecotypes' adapted to distinct ecological niches. Multi-locus sequence analysis targeting seven 'core' genes was employed to taxonomically resolve Synechococcus isolates and correlate previous phylogenetic analyses encompassing a range of markers. Despite the recognized importance of lateral gene transfer in shaping the genomes of marine cyanobacteria, multi-locus sequence analysis of more than 120 isolates reflects a clonal population structure of major lineages and subgroups. A single core genome locus, petB, encoding the cytochrome b(6) subunit of the cytochrome b(6) f complex, was selected to expand our understanding of the diversity and ecology of marine Synechococcus populations. Environmental petB sequences cloned from contrasting sites highlight numerous genetically and ecologically distinct clusters, some of which represent novel, environmentally abundant clades without cultured representatives. With a view to scaling ecological analyses, the short sequence, taxonomic resolution and accurate automated alignment of petB is ideally suited to high-throughput and high-resolution sequencing projects to explore links between the ecology, evolution and biology of marine Synechococcus., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The distributed genome hypothesis states that the gene pool of a bacterial taxon is much more complex than that found in a single individual genome. However, the possible fitness advantage, why such genomic diversity is maintained, whether this variation is largely adaptive or neutral, and why these distinct individuals can coexist, remains poorly understood. Here, we present the infinitely many genes (IMG) model, which is a quantitative, evolutionary model for the distributed genome. It is based on a genealogy of individual genomes and the possibility of gene gain (from an unbounded reservoir of novel genes, e.g., by horizontal gene transfer from distant taxa) and gene loss, for example, by pseudogenization and deletion of genes, during reproduction. By implementing these mechanisms, the IMG model differs from existing concepts for the distributed genome, which cannot differentiate between neutral evolution and adaptation as drivers of the observed genomic diversity. Using the IMG model, we tested whether the distributed genome of 22 full genomes of picocyanobacteria (Prochlorococcus and Synechococcus) shows signs of adaptation or neutrality. We calculated the effective population size of Prochlorococcus at 1.01 × 10(11) and predicted 18 distinct clades for this population, only six of which have been isolated and cultured thus far. We predicted that the Prochlorococcus pangenome contains 57,792 genes and found that the evolution of the distributed genome of Prochlorococcus was possibly neutral, whereas that of Synechococcus and the combined sample shows a clear deviation from neutrality.

Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-μm vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O₂, pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O₂ concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.

UV radiation (UVR) has different effects on prokaryotic cells, such as, for instance, filamentation and aggregation in bacteria. Here we studied the effect of UVR on microcolony formation in two freshwater Synechococcus strains of different ribotypes (group B and group I) and phycobiliprotein compositions (phycoerythrin [PE] and phycocyanin [PC]). Each strain was photoacclimated at two light intensities, low light (LL) (10 μmol m⁻² s⁻¹) and moderate light (ML) (100 μmol m⁻² s⁻¹). The cultures were exposed for 6 days to treatments with UVR or without UVR. PE-rich Synechococcus acclimated to LL had a low carotenoid/chlorophyll a (car/chl) ratio but responded faster to UVR treatment, producing the highest percentages of microcolonies and of cells in microcolonies. Conversely, the same strain acclimated to ML, with a higher car/chl ratio, did not aggregate significantly. These results suggest that microcolony formation by PE-rich Synechococcus is induced by UVR if carotenoid levels are low. PC-rich Synechococcus formed a very low percentage of microcolonies in both acclimations even with low car/chl ratio. The different responses of the two Synechococcus strains to UVR depend on their pigment compositions. On the other hand, this study does not exclude that UVR-induced microcolony formation could also be related to specific ribotypes.

Several isolates of the marine cyanobacterial genus Prochlorococcus have smaller genome sizes than those of the closely related genus Synechococcus. In order to test whether loss of protein-coding genes has contributed to genome size reduction in Prochlorococcus, we reconstructed events of gene family evolution over a strongly supported phylogeny of 12 Prochlorococcus genomes and 9 Synechococcus genomes. Significantly, more events both of loss of paralogs within gene families and of loss of entire gene families occurred in Prochlorococcus than in Synechococcus. The number of nonancestral gene families in genomes of both genera was positively correlated with the extent of genomic islands (GIs), consistent with the hypothesis that horizontal gene transfer (HGT) is associated with GIs. However, even when only isolates with comparable extents of GIs were compared, significantly more events of gene family loss and of paralog loss were seen in Prochlorococcus than in Synechococcus, implying that HGT is not the primary reason for the genome size difference between the two genera.

The cyanobacterial CO(2)-concentrating mechanism (CCM) is an effective adaptation that increases the carbon dioxide (CO(2)) concentration around the primary photosynthetic enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO). α-Cyanobacteria (those containing Form1-A RuBisCO within cso-type α-carboxysomes) have a limited CCM composed of a small number of Ci-transporters whereas β-cyanobacteria (those species containing Form-1B RuBisCO within ccm-type β-carboxysomes) exhibit a more diverse CCM with a greater variety in Ci-transporter complement and regulation. In the coastal species Synechococcus sp. WH5701 (α-cyanobacteria), the minimal α-cyanobacterial CCM has been supplemented with β-cyanobacterial Ci transporters through the process of horizontal gene transfer (HGT). These transporters are transcriptionally regulated in response to external Ci-depletion however this change in transcript abundance is not correlated with a physiological induction. WH5701 exhibits identical physiological responses grown at 4% CO(2) (K (1/2) ≈ 31 μM Ci) and after induction with 0.04% CO(2) (K (1/2) ≈ 29 μM Ci). Insensitivity to external Ci concentration is an unusual characteristic of the WH5701 CCM which is a result of evolution by HGT. Our bioinformatic and physiological data support the hypothesis that WH5701 represents a clade of α-cyanobacterial species in transition from the marine/oligotrophic environment to a coastal/freshwater environment.

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.

The main goal of this study was to determine the distribution of potentially toxic cyanobacteria in 39 selected Polish water bodies. From the water bodies with blooms and also from those in which blooms were not visible 87 samples were investigated. For the first time samples from ponds localized in villages with high agricultural activities were included. Lakes for which microcystin concentrations had been determined before were included as a reference for the research. The detection of cyanobacteria was conducted by microscopic observation as well as by PCR amplification of the rpoC1 gene fragment. Cyanobacteria were present in 75 out of 87 samples. The presence of potentially toxic cyanobacteria was detected by amplification of the mcyB and mcyE genes, which are involved in the biosynthesis of microcystins. Both genes were detected in 7 out of 9 blooms investigated. In the case of samples collected from water bodies in which blooms were not observed, the mcyB and mcyE genes were detected in 20 out of 36. In order to identify the cyanobacteria occurring in selected reservoirs, 16S plus ITS clone libraries were constructed. The method allowed distinguishing 18 different genotypes. After sequence analysis, cyanobacteria belonging to genera Microcystis, Planktothrix, Anabaena, Pseudanabaena, Synechocystis, Synechococcus and Woronichinia were identified. Results confirmed the usefulness of the rpoC1 and mcy genes for monitoring water bodies and detection of potentially toxic cyanobacteria. Application of molecular markers allowed detecting potentially toxic cyanobacteria before the bloom was visible. This is the first comprehensive study concerning cyanobacteria present in different types of Polish water bodies performed using molecular markers.

A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shotgun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Global estimates indicate the oceans are responsible for approximately half of the carbon dioxide fixed on Earth. Organisms < or =5 microm in size dominate open ocean phytoplankton communities in terms of abundance and CO(2) fixation, with the cyanobacterial genera Prochlorococcus and Synechococcus numerically the most abundant and more extensively studied compared with small eukaryotes. However, the contribution of specific taxonomic groups to marine CO(2) fixation is still poorly known. In this study, we show that among the phytoplankton, small eukaryotes contribute significantly to CO(2) fixation (44%) because of their larger cell volume and thereby higher cell-specific CO(2) fixation rates. Within the eukaryotes, two groups, herein called Euk-A and Euk-B, were distinguished based on their flow cytometric signature. Euk-A, the most abundant group, contained cells 1.8+/-0.1 microm in size while Euk-B was the least abundant but cells were larger (2.8+/-0.2 microm). The Euk-B group comprising prymnesiophytes (73+/-13%) belonging largely to lineages with no close cultured counterparts accounted for up to 38% of the total primary production in the subtropical and tropical northeast Atlantic Ocean, suggesting a key role of this group in oceanic CO(2) fixation.

To investigate the energy utilization efficiency of Synechococcus sp. PCC7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and acetic acid respectively and discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could better enhance the growth of Synechococcus sp. PCC7942, and the latter was more effective. The metabolic flux through glycolytic pathway in mixotrophic cultures was stimulated by glucose whereas depressed by acetate, while the flux through the tricarboxylic acid cycle increased in both cases. Under mixotrophic conditions, glucose makes more significant impact on the diminishment of photochemical efficiency of Synechococcus sp. PCC7942. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher comparing with photoautotrophic culture. The energy conversion efficiencies based on ATP synthesis in photoautotrophic culture, mixotrophic cultures with glucose and with acetate were evaluated to be 6.81%, 7.43% and 8.77%, respectively.

In the Chesapeake Bay, picocyanobacteria were usually 100-fold less abundant in winter than in summer. However, little is known about how picocyanobacterial populations shift between winter and summer in the bay. This is due mainly to undetectable winter picocyanobacterial populations in bacterial 16S rRNA clone libraries. In this study, the winter and summer picocyanobacterial populations in the bay were detected using picocyanobacterium-specific primers and were compared based on the analysis of rRNA internal transcribed spacer sequences. Temperature was found to be the dominant environmental factor controlling picocyanobacterial populations in the Chesapeake Bay. In the summer, marine cluster B Synechococcus dominated the upper bay, while a unique cluster, CB1 (marine cluster A [MC-A] Synechococcus), made up the vast majority in the middle and lower bay. In the winter, the picocyanobacteria shifted to completely different populations. Subclades CB6 and CB7, which belong to MC-A Synechococcus and Cyanobium, respectively, made up the entire winter picocyanobacterial populations in the bay. Interestingly, the winter members in subclade CB6 clustered closely with Synechococcus CC9311, a coastal strain known to have a greater capacity to sense and respond to changing environments than oceanic strains.

Background: The phylogeny and taxonomy of cyanobacteria is currently poorly understood due to paucity of reliable markers for identification and circumscription of its major clades., Results: A combination of phylogenomic and protein signature based approaches was used to characterize the major clades of cyanobacteria. Phylogenetic trees were constructed for 44 cyanobacteria based on 44 conserved proteins. In parallel, Blastp searches were carried out on each ORF in the genomes of Synechococcus WH8102, Synechocystis PCC6803, Nostoc PCC7120, Synechococcus JA-3-3Ab, Prochlorococcus MIT9215 and Prochlor. marinus subsp. marinus CCMP1375 to identify proteins that are specific for various main clades of cyanobacteria. These studies have identified 39 proteins that are specific for all (or most) cyanobacteria and large numbers of proteins for other cyanobacterial clades. The identified signature proteins include: (i) 14 proteins for a deep branching clade (Clade A) of Gloebacter violaceus and two diazotrophic Synechococcus strains (JA-3-3Ab and JA2-3-B'a); (ii) 5 proteins that are present in all other cyanobacteria except those from Clade A; (iii) 60 proteins that are specific for a clade (Clade C) consisting of various marine unicellular cyanobacteria (viz. Synechococcus and Prochlorococcus); (iv) 14 and 19 signature proteins that are specific for the Clade C Synechococcus and Prochlorococcus strains, respectively; (v) 67 proteins that are specific for the Low B/A ecotype Prochlorococcus strains, containing lower ratio of chl b/a2 and adapted to growth at high light intensities; (vi) 65 and 8 proteins that are specific for the Nostocales and Chroococcales orders, respectively; and (vii) 22 and 9 proteins that are uniquely shared by various Nostocales and Oscillatoriales orders, or by these two orders and the Chroococcales, respectively. We also describe 3 conserved indels in flavoprotein, heme oxygenase and protochlorophyllide oxidoreductase proteins that are specific for either Clade C cyanobacteria or for various subclades of Prochlorococcus. Many other conserved indels for cyanobacterial clades have been described recently., Conclusions: These signature proteins and indels provide novel means for circumscription of various cyanobacterial clades in clear molecular terms. Their functional studies should lead to discovery of novel properties that are unique to these groups of cyanobacteria.

Phylogenetic relationships among 33 Synechococcus strains isolated from the East China Sea (ECS) and the East Sea (ES) were studied based on 16S rRNA gene sequences and 16S-23S rRNA gene internal transcribed spacer (ITS) sequences. Pigment patterns of the culture strains were also examined. Based on 16S rRNA gene and ITS sequence phylogenies, the Synechococcus isolates were clustered into 10 clades, among which eight were previously identified and two were novel. Half of the culture strains belonged to clade V or VI. All strains that clustered into novel clades exhibited both phycoerythrobilin and phycourobilin. Interestingly, the pigment compositions of isolates belonging to clades V and VI differed from those reported for other oceanic regions. None of the isolates in clade V showed phycourobilin, whereas strains in clade VI exhibited both phycourobilin and phycoerythrobilin, which is in contrast to previous studies. The presence of novel lineages and the different pigment patterns in the ECS and the ES suggests the possibility that some Synechococcus lineages are distributed only in geographically restricted areas and have evolved in these regions. Therefore, further elucidation of the physiological, ecological, and genetic characteristics of the diverse Synechococcus strains is required to understand their spatial and geographical distribution.

Marine cyanobacteria from the genus Synechococcus are found throughout the world's oceans and are important contributors to global primary productivity and carbon cycling. Cultured isolates and environmental DNA clone libraries of Synechococcus have demonstrated the diversity of these microbes. However, the natural distribution of this diversity through space and time and the ecological significance of their distribution are still poorly understood. To understand the seasonal dynamics of Synechococcus diversity, we have developed a quantitative PCR strategy using the gene encoding as a subunit of DNA-dependent RNA polymerase (rpoC1) and applied it to a 3-year time series of surface samples from the Scripps Institution of Oceanography pier (La Jolla, CA, USA), a coastal site in the northeastern Pacific Ocean. Synechococcus from clades I and IV were dominant throughout the time series and correlated with total Synechococcus abundance. The relative abundance of these two dominant clades showed evidence of a seasonal cycle. Synechococcus from clade IV were typically more abundant, but those from clade I dominated during periods just before the annual spring bloom of Synechococcus. Synechococcus from clades II and III were absent during spring and early summer, but appeared at low abundances in late summer and winter possibly due to changes in circulation in the Southern California Bight. As the first long-term time series describing Synechococcus population diversity, these temporal dynamics were used to interpret the genetic/genomic diversity observed in the environment and the potential factors regulating their distribution.

Background: Cyanobacteria of the genera Synechococcus and Prochlorococcus play a key role in marine photosynthesis, which contributes to the global carbon cycle and to the world oxygen supply. Recently, genes encoding the photosystem II reaction center (psbA and psbD) were found in cyanophage genomes. This phenomenon suggested that the horizontal transfer of these genes may be involved in increasing phage fitness. To date, a very small percentage of marine bacteria and phages has been cultured. Thus, mapping genomic data extracted directly from the environment to its taxonomic origin is necessary for a better understanding of phage-host relationships and dynamics., Results: To achieve an accurate and rapid taxonomic classification, we employed a computational approach combining a multi-class Support Vector Machine (SVM) with a codon usage position specific scoring matrix (cuPSSM). Our method has been applied successfully to classify core-photosystem-II gene fragments, including partial sequences coming directly from the ocean, to seven different taxonomic classes. Applying the method on a large set of DNA and RNA psbA clones from the Mediterranean Sea, we studied the distribution of cyanobacterial psbA genes and transcripts in their natural environment. Using our approach, we were able to simultaneously examine taxonomic and ecological distributions in the marine environment., Conclusion: The ability to accurately classify the origin of individual genes and transcripts coming directly from the environment is of great importance in studying marine ecology. The classification method presented in this paper could be applied further to classify other genes amplified from the environment, for which training data is available.

Synechococcus is a cosmopolitan genus of picocyanobacteria living in the photic zone of freshwater and marine ecosystems. Here, we describe the isolation of 46 closely related picocyanobacterial strains from the Baltic Sea. The isolates showed considerable variation in their cell size and pigmentation phenotypes, yielding a colorful variety of red, pink and blue-green strains. These pigmentation phenotypes could not be differentiated on the basis of their 16S rRNA-internal transcribed spacer (ITS) sequences. Thirty-nine strains, designated BSea, possessed 16S rRNA-ITS sequences almost identical with Synechococcus strain WH5701. Despite their similar 16S rRNA-ITS sequences, the BSea strains separated into several different clusters when comparing the phycocyanin (cpcBA) operon. This separation was largely consistent with the phycobiliprotein composition of the different BSea strains. The majority of phycocyanin (PC)-rich Bsea strains clustered with WH5701. Remarkably, the phycoerythrin (PE)-rich strains of BSea formed an as yet unidentified cluster within the cpcBA phylogeny, distantly related to other PE-rich groups. Detailed analysis of the cpcBA operon using neighbour-net analysis indicated that the PE-rich BSea strains are probably endemic for the Baltic Sea. Comparison of the phylogenies obtained by the 16S rRNA-ITS, the cpcBA, and the concatenated 16S rRNA-ITS and cpcBA operon sequences revealed possible events of horizontal gene transfer among different Synechococcus lineages. Our results show that microdiversity is important in Synechococcus populations and that it can reflect extensive diversification of different pigmentation phenotypes into different ecological niches.

The phylogenetic diversity of the marine Synechococcus community in the subtropical coastal waters of Hong Kong, China, was examined through intergenic transcribed spacer clone libraries. All the sequences obtained fell within both marine cluster A (MC-A) and B (MC-B), with MC-A phylotypes dominating throughout the year. Distinct phylogenetic lineages specific to Hong Kong waters were detected from both MC-A and MC-B. The highest Synechococcus community diversity occurred in December, but the highest Synechococcus abundance occurred in August. On the other hand, both the abundance and diversity of Synechococcus showed a minimum in February. The remarkable seasonal variations of Synechococcus diversity observed were likely the result of the changes of hydrographic condition modulated by monsoons. Principal component analysis revealed that the in situ abiotic water characteristics, especially salinity and water turbidity, explained much of the variability of the marine Synechococcus population diversity in Hong Kong coastal waters. In addition, the temporal changes of Synechococcus abundance were largely driven by water temperature.

We investigated the dark-to-light transition in Synechococcus sp. PCC 7942 cells by a detailed analysis of fluorescence transients induced by strong red light. The transients, recorded with high data-acquisition, revealed all the steps of the fast (OJIP; 10(-5)-1 s) and slow phase (PSM(T); 1-10(3) s), kinetically distinguished with precision. Focusing on the OJIP-rise, we show, for the first time, how the variable to initial fluorescence ratio and the relative height of J-level can serve as indexes of the plastoquinone redox poise and the established state in the dark; hence, differences among cyanobacteria can be recognised in a simple way. Applying intermittent illumination (20-s light pulses separated by 10-s dark intervals) to induce dark-to-light transition and analysing the individual transients, we establish a method by which we determine the fluorescence component not originating from photosystem (PS) II and we assess PSII dynamics during state 2 to state 1 transition. The development of photochemical and non-photochemical quenching is also discussed, as well as evidences favouring the mobile antenna model.

The extent to which cultured strains represent the genetic diversity of a population of microorganisms is poorly understood. Because they do not require culturing, metagenomic approaches have the potential to reveal the genetic diversity of the microbes actually present in an environment. From coastal California seawater, a complex and diverse environment, the marine cyanobacteria of the genus Synechococcus were enriched by flow cytometry-based sorting and the population metagenome was analysed with 454 sequencing technology. The sequence data were compared with model Synechococcus genomes, including those of two coastal strains, one isolated from the same and one from a very similar environment. The natural population metagenome had high sequence identity to most genes from the coastal model strains but diverged greatly from these genomes in multiple regions of atypical trinucleotide content that encoded diverse functions. These results can be explained by extensive horizontal gene transfer presumably with large differences in horizontally transferred genetic material between different strains. Some assembled contigs showed the presence of novel open reading frames not found in the model genomes, but these could not yet be unambiguously assigned to a Synechococcus clade. At least three distinct mobile DNA elements (plasmids) not found in model strain genomes were detected in the assembled contigs, suggesting for the first time their likely importance in marine cyanobacterial populations and possible role in horizontal gene transfer.

The marine cyanobacterium Prochlorococcus MED4 has the smallest sequenced genome of any photosynthetic organism. Prochlorococcus MED4 shares many genomic characteristics with chloroplasts and bacterial endosymbionts, including a reduced coding capacity, missing DNA repair genes, a minimal transcriptional regulatory network, a marked AT% bias, and an accelerated rate of amino acid changes. In chloroplasts and endosymbionts, these molecular phenotypes appear to be symptomatic of a relative increase in genetic drift due to restrictions on effective population size in the host environment. As a free-living bacterium, Prochlorococcus MED4 is not known to be subject to similar ecological constraints. To test whether the high-light-adapted Prochlorococcus MED4 is experiencing a reduction in selection efficiency resulting from genetic drift, we examine two data sets, namely, the environmental genome shotgun sequencing data from the Sargasso Sea and a set of cyanobacterial genome sequences. After integrating these data sets, we compare the evolutionary profile of a high-light Prochlorococcus group to that of a group of Synechococcus (a closely related group of marine cyanobacteria) that does not exhibit a similar small-genome syndrome. The average pairwise dN/dS ratios in the high-light-adapted Prochlorococcus group are significantly lower than those in the Synechococcus group, leading us to reject the hypothesis that the Prochlorococcus group is currently experiencing higher levels of genetic drift.

Cyanobacteria are common members of sponge-associated bacterial communities and are particularly abundant symbionts of coral reef sponges. The unicellular cyanobacterium Synechococcus spongiarum is the most prevalent photosynthetic symbiont in marine sponges and inhabits taxonomically diverse hosts from tropical and temperate reefs worldwide. Despite the global distribution of S. spongiarum, molecular analyses report low levels of genetic divergence among 16S ribosomal RNA (rRNA) gene sequences from diverse sponge hosts, resulting either from the widespread dispersal ability of these symbionts or the low phylogenetic resolution of a conserved molecular marker. Partial 16S rRNA and entire 16S-23S rRNA internal transcribed spacer (ITS) genes were sequenced from cyanobacteria inhabiting 32 sponges (representing 18 species, six families and four orders) from six geographical regions. ITS phylogenies revealed 12 distinct clades of S. spongiarum that displayed 9% mean sequence divergence among clades and less than 1% sequence divergence within clades. Symbiont clades ranged in specificity from generalists to specialists, with most (10 of 12) clades detected in one or several closely related hosts. Although multiple symbiont clades inhabited some host sponges, symbiont communities appear to be structured by both geography and host phylogeny. In contrast, 16S rRNA sequences were highly conserved, exhibiting less than 1% sequence divergence among symbiont clades. ITS gene sequences displayed much higher variability than 16S rRNA sequences, highlighting the utility of ITS sequences in determining the genetic diversity and host specificity of S. spongiarum populations among reef sponges. The genetic diversity of S. spongiarum revealed by ITS sequences may be correlated with different physiological capabilities and environmental preferences that may generate variable host-symbiont interactions.

Two cyanobacterial strains, Pseudanabaena sp. 0411 and Synechococcus sp. 0431, were isolated from a sample collected in the Kotel'nikovskii hot spring of the Baikal rift. According to the results of light and transmission electron microscopy, as well as of the phylogenetic analysis of the 16S rRNA gene, these cyanobacteria were classified as Pseudanabaena sp. nov. and Synechococcus bigranulatus Skuja. The constructed phylogenetic tree shows that the studied strains are positioned in the clades of cyanobacteria isolated from hydrothermal vents of Asia and New Zealand, separately from marine and freshwater members of these genera, including those isolated from Lake Baikal.

Uroporphyrinogen decarboxylase (HemE) is important due to its location at the first branch-point in tetrapyrrole biosynthesis. We detected a complex formation between full-length polypeptides of HtpG and HemE by biochemical studies in vivo and in vitro. The interaction suppressed the enzyme activity, suggesting a regulatory role of HtpG in tetrapyrrole biosynthesis.

Background: The picocyanobacterial genus Synechococcus occurs over wide oceanic expanses, having colonized most available niches in the photic zone. Large scale distribution patterns of the different Synechococcus clades (based on 16S rRNA gene markers) suggest the occurrence of two major lifestyles ('opportunists'/'specialists'), corresponding to two distinct broad habitats ('coastal'/'open ocean'). Yet, the genetic basis of niche partitioning is still poorly understood in this ecologically important group., Results: Here, we compare the genomes of 11 marine Synechococcus isolates, representing 10 distinct lineages. Phylogenies inferred from the core genome allowed us to refine the taxonomic relationships between clades by revealing a clear dichotomy within the main subcluster, reminiscent of the two aforementioned lifestyles. Genome size is strongly correlated with the cumulative lengths of hypervariable regions (or 'islands'). One of these, encompassing most genes encoding the light-harvesting phycobilisome rod complexes, is involved in adaptation to changes in light quality and has clearly been transferred between members of different Synechococcus lineages. Furthermore, we observed that two strains (RS9917 and WH5701) that have similar pigmentation and physiology have an unusually high number of genes in common, given their phylogenetic distance., Conclusion: We propose that while members of a given marine Synechococcus lineage may have the same broad geographical distribution, local niche occupancy is facilitated by lateral gene transfers, a process in which genomic islands play a key role as a repository for transferred genes. Our work also highlights the need for developing picocyanobacterial systematics based on genome-derived parameters combined with ecological and physiological data.

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the world's oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the "leaves of the tree," between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The Amt proteins constitute a ubiquitous family of transmembrane ammonia channels that permit the net uptake of ammonium by cells. In many organisms, there is more than one amt gene, and these genes are subjected to nitrogen control. The mature Amt protein is a homo- or heterooligomer of three Amt subunits. We previously characterized an amt1 gene in the unicellular cyanobacterium Synechococcus elongatus strain PCC 7942. In this work, we describe the presence in this organism of a second amt gene, amtB, which encodes a protein more similar to the bacterial AmtB proteins than to any other characterized cyanobacterial Amt protein. The expression of amtB took place in response to nitrogen step-down, required the NtcA transcription factor, and occurred parallel to the expression of amt1. However, the transcript levels of amtB measured after 2 h of nitrogen deprivation were about 100-fold lower than those of amt1. An S. elongatus amtB insertional mutant exhibited an activity for uptake of [14C]methylammonium that was about 55% of that observed in the wild type, but inactivation of amtB had no noticeable effect on the uptake of ammonium when it was supplied at a concentration of 100 microM or more. Because an S. elongatus amt1 mutant is essentially devoid of [14C]methylammonium uptake activity, the mature Amt transporter is functional in the absence of AmtB subunits but not in the absence of Amt1 subunits. However, the S. elongatus amtB mutant could not concentrate [14C]methylammonium within the cells to the same extent as the wild type. Therefore, AmtB is necessary for full methylammonium uptake activity in S. elongatus.

The abundance and distribution of Synechococcus spp. in the autotrophic picoplankton of Lake Constance, were followed in the pelagic and littoral habitat by qPCR over 2 years. One genotype, represented by isolated phycoerythrin-rich strain BO 8807, showed a seasonal distribution pattern in both habitats. Before a stable thermal stratification, the maximum of both the Synechococcus population and genotype BO 8807 occurred at 15 or 20 m water depth in the pelagic habitat. During the summer stratification, when the absolute abundance of all Synechococcus spp. was highest above 15 m, the absolute and relative abundance of genotype BO 8807 was maximal at 20 m. These results indicate that Synechococcus spp. or single genotypes are present in deep maxima in Lake Constance. The in situ dynamics of genotype BO 8807 is consistent with the observation that isolated strain BO 8807 requires higher phosphate concentrations for maximum growth rates than a strain from the same phylogenetic cluster that dominates the pelagic summer population. In contrast to these findings, low genome numbers of phycocyanin-rich genotype BO 8805 were found temporarily only in both the littoral and pelagic plankton. Microscopy revealed that PC-rich cells in general occurred preferentially in the littoral habitat. We discuss our results with respect to the versatility of picocyanobacteria of the evolutionary lineage VI of cyanobacteria, and a habitat-related distribution pattern of Synechococcus genotypes.

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.

Two thermophilic cyanobacterial strains, Ts and Bs, collected from Asian geothermal springs were identified morphologically and phylogenetically as Synechococcus in the order Chroococcales and were isolated into axenic cultures. In addition to the high similarities between their full 16S rRNA gene sequences, both strains also shared similar pigment profiles and fatty acid compositions but with varied ratios. Strain Ts had elevated levels of photoprotective pigments such as carotenoid and scytonemin even after prolonged culture under identical laboratory conditions, whereas strain Bs produced more chlorophyll a per unit cell volume, perhaps resulting from UV adaptation in the natural habitats. In addition, strain Ts had more content than strain Bs in terms of the total fatty acids and the proportion of unsaturated fatty acids. Neither isolate was able to fix nitrogen, and they had zero susceptibility to ampicillin and streptomycin.

Background: Marine Synechococcus owe their specific vivid color (ranging from blue-green to orange) to their large extrinsic antenna complexes called phycobilisomes, comprising a central allophycocyanin core and rods of variable phycobiliprotein composition. Three major pigment types can be defined depending on the major phycobiliprotein found in the rods (phycocyanin, phycoerythrin I or phycoerythrin II). Among strains containing both phycoerythrins I and II, four subtypes can be distinguished based on the ratio of the two chromophores bound to these phycobiliproteins. Genomes of eleven marine Synechococcus strains recently became available with one to four strains per pigment type or subtype, allowing an unprecedented comparative genomics study of genes involved in phycobilisome metabolism., Results: By carefully comparing the Synechococcus genomes, we have retrieved candidate genes potentially required for the synthesis of phycobiliproteins in each pigment type. This includes linker polypeptides, phycobilin lyases and a number of novel genes of uncharacterized function. Interestingly, strains belonging to a given pigment type have similar phycobilisome gene complements and organization, independent of the core genome phylogeny (as assessed using concatenated ribosomal proteins). While phylogenetic trees based on concatenated allophycocyanin protein sequences are congruent with the latter, those based on phycocyanin and phycoerythrin notably differ and match the Synechococcus pigment types., Conclusion: We conclude that the phycobilisome core has likely evolved together with the core genome, while rods must have evolved independently, possibly by lateral transfer of phycobilisome rod genes or gene clusters between Synechococcus strains, either via viruses or by natural transformation, allowing rapid adaptation to a variety of light niches.

Cyanobacteria are important primary producers in many marine ecosystems and their abundances and growth rates depend on their ability to assimilate various nitrogen sources. To examine the diversity of nitrate-utilizing marine cyanobacteria, we developed PCR primers specific for cyanobacterial assimilatory nitrate reductase (narB) genes. We obtained amplification products from diverse strains of cultivated cyanobacteria and from several marine environments. Phylogenetic trees constructed with the narB gene are congruent with those based on ribosomal RNA genes and RNA polymerase genes. Analysis of sequence library data from coastal and oligotrophic marine environments shows distinct groups of Synechococcus sp. in each environment; some of which are represented by sequences from cultivated organisms and others that are unrelated to known sequences and likely represent novel phylogenetic groups. We observed spatial differences in the distribution of sequences between two sites in Monterey Bay and differences in the vertical distribution of sequence types at the Hawai'i Ocean Time-series Station ALOHA, suggesting that nitrogen assimilation in Synechococcus living in different ecological niches can be followed with the nitrate reductase gene.

Marine microbial communities often contain multiple closely related phylogenetic clades, but in many cases, it is still unclear what physiological traits differentiate these putative ecotypes. The numerically abundant marine cyanobacterium Synechococcus can be divided into at least 14 clades. In order to better understand ecotype differentiation in this genus, we assessed the diversity of a Synechococcus community from a well-mixed water column in the Sargasso Sea during March 2002, a time of year when this genus typically reaches its annual peak in abundance. Diversity was estimated from water sampled at three depths (approximately 5, 70, and 170 m) using both culture isolation and construction of cyanobacterial 16S-23S rRNA internal transcribed sequence clone libraries. Clonal isolates were obtained by enrichment with ammonium, nitrite, or nitrate as the sole N source, followed by pour plating. Each method sampled the in situ diversity differently. The combined methods revealed a total of seven Synechococcus phylotypes including two new putative ecotypes, labeled XV and XVI. Although most other isolates grow on nitrate, clade XV exhibited a reduced efficiency in nitrate utilization, and both clade XV and XVI are capable of chromatic adaptation, demonstrating that this trait is more widely distributed among Synechococcus strains than previously known. Thus, as in its sister genus Prochlorococcus, light and nitrogen utilization are important factors in ecotype differentiation in the marine Synechococcus lineage.

Interactions with Gijs Kuenen and other Dutch scientists have led my lab to fundamental insights into the composition, structure and function of a hot spring cyanobacterial mat community that should influence our thinking about all microbial communities. By focusing on the distribution of molecular sequence variants of predominant mat phototrophs, we have discovered that small-scale sequence variation can be ecologically meaningful. By applying novel cultivation approaches, we have been able to obtain genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary radiation. By applying the analytical tools of organic geochemistry we have gained insight into the metabolisms of major phototrophic members of the mat community as well as interactions between phototrophic guilds. These observations challenge traditional paradigms about prokaryotic species and cause us to consider evolutionary ecology theory as we develop genome-based methods for high-resolution analysis of the species-like fundamental units comprising microbial communities, and for investigating how such units coordinate the physiological activities within guilds of the community.

Using 1128 protein-coding gene families from 11 completely sequenced cyanobacterial genomes, we attempt to quantify horizontal gene transfer events within cyanobacteria, as well as between cyanobacteria and other phyla. A novel method of detecting and enumerating potential horizontal gene transfer events within a group of organisms based on analyses of "embedded quartets" allows us to identify phylogenetic signal consistent with a plurality of gene families, as well as to delineate cases of conflict to the plurality signal, which include horizontally transferred genes. To infer horizontal gene transfer events between cyanobacteria and other phyla, we added homologs from 168 available genomes. We screened phylogenetic trees reconstructed for each of these extended gene families for highly supported monophyly of cyanobacteria (or lack of it). Cyanobacterial genomes reveal a complex evolutionary history, which cannot be represented by a single strictly bifurcating tree for all genes or even most genes, although a single completely resolved phylogeny was recovered from the quartets' plurality signals. We find more conflicts within cyanobacteria than between cyanobacteria and other phyla. We also find that genes from all functional categories are subject to transfer. However, in interphylum as compared to intraphylum transfers, the proportion of metabolic (operational) gene transfers increases, while the proportion of informational gene transfers decreases.

Cyanophages (cyanobacterial viruses) are important agents of horizontal gene transfer among marine cyanobacteria, the numerically dominant photosynthetic organisms in the oceans. Some cyanophage genomes carry and express host-like photosynthesis genes, presumably to augment the host photosynthetic machinery during infection. To study the prevalence and evolutionary dynamics of this phenomenon, 33 cultured cyanophages of known family and host range and viral DNA from field samples were screened for the presence of two core photosystem reaction center genes, psbA and psbD. Combining this expanded dataset with published data for nine other cyanophages, we found that 88% of the phage genomes contain psbA, and 50% contain both psbA and psbD. The psbA gene was found in all myoviruses and Prochlorococcus podoviruses, but could not be amplified from Prochlorococcus siphoviruses or Synechococcus podoviruses. Nearly all of the phages that encoded both psbA and psbD had broad host ranges. We speculate that the presence or absence of psbA in a phage genome may be determined by the length of the latent period of infection. Whether it also carries psbD may reflect constraints on coupling of viral- and host-encoded PsbA-PsbD in the photosynthetic reaction center across divergent hosts. Phylogenetic clustering patterns of these genes from cultured phages suggest that whole genes have been transferred from host to phage in a discrete number of events over the course of evolution (four for psbA, and two for psbD), followed by horizontal and vertical transfer between cyanophages. Clustering patterns of psbA and psbD from Synechococcus cells were inconsistent with other molecular phylogenetic markers, suggesting genetic exchanges involving Synechococcus lineages. Signatures of intragenic recombination, detected within the cyanophage gene pool as well as between hosts and phages in both directions, support this hypothesis. The analysis of cyanophage psbA and psbD genes from field populations revealed significant sequence diversity, much of which is represented in our cultured isolates. Collectively, these findings show that photosynthesis genes are common in cyanophages and that significant genetic exchanges occur from host to phage, phage to host, and within the phage gene pool. This generates genetic diversity among the phage, which serves as a reservoir for their hosts, and in turn influences photosystem evolution.