Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea.
ABSTRACT: Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.
Project description:Spatial patterns of marine Synechococcus diversity across ocean domains have been reported on extensively. However, much less is known of seasonal and multiannual patterns of change in Synechococcus community composition. Here we report on the genotypic diversity of Synechococcus populations in the Gulf of Aqaba, Northern Red Sea, over seven annual cycles of deep mixing and stabile stratification, using ntcA as a phylogenetic marker. Synechococcus clone libraries were dominated by clade II and XII genotypes and a total of eight different clades were identified. Inclusion of ntcA sequences from the Global Ocean Sampling database in our analyses identified members of clade XII from beyond the Gulf of Aqaba, extending its known distribution. Most of the Synechococcus diversity was attributed to members of clade II during the spring bloom, while clade III contributed significantly to diversity during summer stratification. Clade XII diversity was most prevalent in fall and winter. Clade abundances were estimated from pyrosequencing of the V6 hypervariable region of 16S rRNA. Members of clade II dominated Synechococcus communities throughout the year, whereas the less frequent genotypes showed a pattern of seasonal succession. Based on the prevailing nutritional conditions we observed that clade I members thrive at higher nutrient concentrations during winter mixing. Clades V, VI and X became apparent during the transition periods between mixing and stratification. Clade III became prominent during sumeer stratification. We propose that members of clades V, VI, and X, and clade III are Synechococcus ecotypes that are adapted to intermediate and low nutrient levels respectively. This is the first time that molecular analyses have correlated population dynamics of Synechococcus genotypes with temporal fluctuations in nutrient regimes. Since these Synechococcus genotypes are routinely observed in the Gulf of Aqaba we suggest that seasonal fluctuations in nutrient levels create temporal niches that sustain their coexistence.
Project description:Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.
Project description: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.
Project description: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.
Project description:A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.
Project description:BACKGROUND: Aerobic gammaproteobacteria affiliated to the OM60/NOR5 clade are widespread in saline environments and of ecological importance in several marine ecosystems, especially the euphotic zone of coastal areas. Within this group a close relationship between aerobic anoxygenic photoheterotrophs and non-phototrophic members has been found. RESULTS: Several strains of aerobic red-pigmented bacteria affiliated to the OM60/NOR5 clade were obtained from tidal flat sediment samples at the island of Sylt (North Sea, Germany). Two of the novel isolates, Rap1red and Ivo14(T), were chosen for an analysis in detail. Strain Rap1red shared a 16S rRNA sequence identity of 99% with the type strain of Congregibacter litoralis and was genome-sequenced to reveal the extent of genetic microheterogeneity among closely related strains within this clade. In addition, a draft genome sequence was obtained from the isolate Ivo14(T), which belongs to the environmental important NOR5-1 lineage that contains so far no cultured representative with a comprehensive description. Strain Ivo14(T) was characterized using a polyphasic approach and compared with other red-pigmented members of the OM60/NOR5 clade, including Congregibacter litoralis DSM 17192(T), Haliea rubra DSM 19751(T) and Chromatocurvus halotolerans DSM 23344(T). All analyzed strains contained bacteriochlorophyll a and spirilloxanthin as photosynthetic pigments. Besides a detailed phenotypic characterization including physiological and chemotaxonomic traits, sequence information based on protein-coding genes and a comparison of draft genome data sets were used to identify possible features characteristic for distinct taxa within this clade. CONCLUSIONS: Comparative sequence analyses of the pufLM genes of genome-sequenced representatives of the OM60/NOR5 clade indicated that the photosynthetic apparatus of these species was derived from a common ancestor and not acquired by multiple horizontal gene transfer from phylogenetically distant species. An affiliation of the characterized bacteriochlorophyll a-containing strains to different genera was indicated by significant phenotypic differences and pufLM nucleotide sequence identity values below 82%. The revealed high genotypic and phenotypic diversity of closely related strains within this phylogenetic group reflects a rapid evolution and frequent niche separation in the OM60/NOR5 clade, which is possibly driven by the necessities of an adaptation to oligotrophic marine habitats.
Project description:The marine Roseobacter clade comprises several genera of marine bacteria related to the uncultured SAR83 cluster, the second most abundant marine picoplankton lineage. Cultivated representatives of this clade are physiologically heterogeneous, and only some have the capability for aerobic anoxygenic photosynthesis, a process of potentially great ecological importance in the world's oceans. In an attempt to correlate phylogeny with ecology, we investigated the diversity of Roseobacter clade strains from various marine habitats (water samples, biofilms, laminariae, diatoms, and dinoflagellate cultures) by using the 16S rRNA gene as a phylogenetic marker gene. The potential for aerobic anoxygenic photosynthesis was determined on the genetic level by PCR amplification and sequencing of the pufLM genes of the bacterial photosynthesis reaction center and on the physiological level by detection of bacteriochlorophyll (Bchl) a. A collection of ca. 1,000 marine isolates was screened for members of the marine Roseobacter clade by 16S rRNA gene-directed multiplex PCR and sequencing. The 42 Roseobacter clade isolates found tended to form habitat-specific subclusters. The pufLM genes were detected in two groups of strains from dinoflagellate cultures but in none of the other Roseobacter clade isolates. Strains within the first group (the DFL-12 cluster) also synthesized Bchl a. Strains within the second group (the DFL-35 cluster) formed a new species of Roseovarius and did not produce Bchl a under the conditions investigated here, thus demonstrating the importance of genetic methods for screening of cultivation-dependent metabolic traits. The pufL genes of the dinoflagellate isolates were phylogenetically closely related to pufL genes from Betaproteobacteria, confirming similar previous observations which have been interpreted as indications of gene transfer events.
Project description:The OM43 clade within the family Methylophilaceae of Betaproteobacteria represents a group of methylotrophs that play important roles in the metabolism of C1 compounds in marine environments and other aquatic environments around the globe. Using dilution-to-extinction cultivation techniques, we successfully isolated a novel species of this clade (here designated MBRS-H7) from the ultraoligotrophic open ocean waters of the central Red Sea. Phylogenomic analyses indicate that MBRS-H7 is a novel species that forms a distinct cluster together with isolate KB13 from Hawaii (Hawaii-Red Sea [H-RS] cluster) that is separate from the cluster represented by strain HTCC2181 (from the Oregon coast). Phylogenetic analyses using the robust 16S-23S internal transcribed spacer revealed a potential ecotype separation of the marine OM43 clade members, which was further confirmed by metagenomic fragment recruitment analyses that showed trends of higher abundance in low-chlorophyll and/or high-temperature provinces for the H-RS cluster but a preference for colder, highly productive waters for the HTCC2181 cluster. This potential environmentally driven niche differentiation is also reflected in the metabolic gene inventories, which in the case of the H-RS cluster include those conferring resistance to high levels of UV irradiation, temperature, and salinity. Interestingly, we also found different energy conservation modules between these OM43 subclades, namely, the existence of the NADH:quinone oxidoreductase complex I (NUO) system in the H-RS cluster and the nonhomologous NADH:quinone oxidoreductase (NQR) system in the HTCC2181 cluster, which might have implications for their overall energetic yields.
Project description:Marine picocyanobacteria, Prochlorococcus and Synechococcus, substantially contribute to marine primary production and have been the subject of extensive ecological and genomic studies. Little is known about their close relatives from freshwater and non-marine environments. Phylogenomic analyses (using 136 proteins) provide strong support for the monophyly of a clade of non-marine picocyanobacteria consisting of Cyanobium, Synechococcus and marine Sub-cluster 5.2; this clade itself is sister to marine Synechococcus and Prochlorococcus. The most basal lineage within the Syn/Pro clade, Sub-Cluster 5.3, includes marine and freshwater strains. Relaxed molecular clock (SSU, LSU) analyses show that while ancestors of the Syn/Pro clade date as far back as the end of the Pre-Cambrian, modern crown groups evolved during the Carboniferous and Triassic. Comparative genomic analyses reveal novel gene cluster arrangements involved in phycobilisome (PBS) metabolism in freshwater strains. Whilst PBS genes in marine Synechococcus are mostly found in one type of phycoerythrin (PE) rich gene cluster (Type III), strains from non-marine habitats, so far, appear to be more diverse both in terms of pigment content and gene arrangement, likely reflecting a wider range of habitats. Our phylogenetic analyses show that the PE genes (mpeBA) evolved via a duplication of the cpeBA genes in an ancestor of the marine and non-marine picocyanobacteria and of the symbiotic strains Synechococcus spongiarum. A 'primitive' Type III-like ancestor containing cpeBA and mpeBA had thus evolved prior to the divergence of the Syn/Pro clade and S. spongiarum. During the diversification of Synechococcus lineages, losses of mpeBA genes may explain the emergence of pigment cluster Types I, II, IIB, and III in both marine and non-marine habitats, with few lateral gene transfer events in specific taxa.
Project description:Prasinophytes clade VII is a group of pico/nano-planktonic green algae (division Chlorophyta) for which numerous ribosomal RNA (rRNA) sequences have been retrieved from the marine environment in the last 15 years. A large number of strains have also been isolated but have not yet received a formal taxonomic description. A phylogenetic analysis of available strains using both the nuclear 18S and plastidial 16S rRNA genes demonstrates that this group composes at least 10 different clades: A1-A7 and B1-B3. Analysis of sequences from the variable V9 region of the 18S rRNA gene collected during the Tara Oceans expedition and in the frame of the Ocean Sampling Day consortium reveal that clade VII is the dominant Chlorophyta group in oceanic waters, replacing Mamiellophyceae, which have this role in coastal waters. At some location, prasinophytes clade VII can even be the dominant photosynthetic eukaryote representing up to 80% of photosynthetic metabarcodes overall. B1 and A4 are the overall dominant clades and different clades seem to occupy distinct niches, for example, A6 is dominant in surface Mediterranean Sea waters, whereas A4 extend to high temperate latitudes. Our work demonstrates that prasinophytes clade VII constitute a highly diversified group, which is a key component of phytoplankton in open oceanic waters but has been neglected in the conceptualization of marine microbial diversity and carbon cycle.