Lake Superior supports novel clusters of cyanobacterial picoplankton

Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the "picocyanobacterial clade" consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 10(5) cells ml-1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.     

Distribution patterns of marine planktonic cyanobacterial assemblages in transitional marine habitats using 16S rRNA phylogeny

The community composition of marine planktonic cyanobacteria in transitional marine habitats can influence its overall contribution to aquatic primary production. To understand distribution patterns of marine planktonic cyanobacterial assemblages, phylogenetic and statistical analyses were undertaken on planktonic cyanobacterial 16S rRNA gene sequences from four transitional marine habitats [Baltic Sea (BL), Monterey Bay (MB), South China Sea (SCS) and Sundarbans (SB)]. Out of 3255 sequences analyzed, only 546 sequences were found to be planktonic cyanobacteria and were considered in this study. Among these, 338 sequences representative of Sundarbans, the world's largest mangrove were generated based on Sanger and Illumina sequencing approaches. Based on 16S rRNA phylogeny, four major taxonomic orders of marine planktonic cyanobacteria were recovered in varying proportions with several novel 16S rRNA sequences in each of the four targeted sites. Members of the order Synechococcales were dominant in all the sites (?94% sequences) while the orders Chroococcales and Oscillatoriales were only detected in SB and SCS sites, respectively. In the phylogenetic tree, sequences representing the major marine picocyanobacterial genus Synechococcus showed overwhelming dominance in SB and they were found in three other sites. Prochlorococcus ‐like sequences were found in sizeable number in MB and SCS but were absent in SB and coastal BL. Synechococcus ‐like sequences were represented by three major marine clusters (5.1, 5.2, and 5.3). Three novel clades as part of Synechococcus cluster were detected only in SB and one novel clade in BL. The majority of OTUs were found to be exclusive to each site, whereas some were shared by two or more sites as revealed by beta‐diversity analysis.     

Prokaryotic abundance and 16S rRNA gene sequences detected in marine aerosols on the East Sea (Korea)

Modern studies on marine airborne prokaryotes over open seas have been rare. Here, to understand their distribution and composition, aerosol as well as surface seawater samples were collected at three sites in the East Sea. The abundance of airborne prokaryotes was 0.7-1.2 × 10(5) cells m(-3) . Partial sequences of the 16S rRNA gene from the aerosols were dominated by Gammaproteobacteria and Bacteroidetes. Interestingly, the numbers of phylotypes of airborne bacteria were comparable to those of seawater bacteria in coastal and remote offshore sites. Over half of the bacterial operational taxonomic units (OTUs) detected in these aerosols (12 out of 20) were of marine origin. Eight of the 19 bacterial OTUs found in the surface waters occurred in the aerosols. Thus, surface seawater seems to be a main source of marine airborne bacteria. However, in the intermediate offshore site, airborne bacterial OTUs (n=4) were all terrestrial, indicating that variations of dominant airborne bacterial phylotypes occurred over the sites (each ～160 km apart) sampled at 1-4-day intervals. Euryarchaeota sequences belonging to the MSP8 clade reported from geographically distant regions were detected in marine aerosols. Long-distance aerial transport of the marine prokaryotes seems to be quite possible.     

Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments

The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.     

Microbes in high arctic snow and implications for the cold biosphere

We applied molecular, microscopic, and culture techniques to characterize the microbial communities in snow and air at remote sites in the Canadian High Arctic (Ward Hunt Island, Ellesmere Island, and Cornwallis Island, latitudes 74 to 83(o)N). Members of the Bacteria and Eukarya were prevalent in the snow, and their small subunit (SSU) rRNA gene signatures indicated strong local aerial transport within the region over the preceding 8 months of winter snowpack accumulation. Many of the operational taxonomic units (OTUs) were similar to previously reported SSU rRNA gene sequences from the Arctic Ocean, suggesting the importance of local aerial transport processes for marine microbiota. More than 47% of the cyanobacterial OTUs in the snow have been previously found in microbial mats in the region, indicating that this group was also substantially derived from local sources. Viable cyanobacteria isolated from the snow indicated free exchange between the snow and adjacent mat communities. Other sequences were most similar to those found outside the Canadian Arctic but were from snow, lake and sea ice, glaciers and permafrost, alpine regions, Antarctica, and other regions of the Arctic, supporting the concept of global distribution of microbial ecotypes throughout the cold biosphere.     

Detection and expression of the phosphonate transporter gene phnD in marine and freshwater picocyanobacteria

We describe a PCR-based assay designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high-molecular-weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. Last, RT-PCR of environmental RNA samples from the Sargasso Sea and Pacific Ocean detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. The method described herein will allow future studies aimed at understanding the utilization of naturally occurring phoshonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in freshwaters.     

Genome sequences of siphoviruses infecting marine Synechococcus unveil a diverse cyanophage group and extensive phage-host genetic exchanges

Investigating the interactions between marine cyanobacteria and their viruses (phages) is important towards understanding the dynamic of ocean's primary productivity. Genome sequencing of marine cyanophages has greatly advanced our understanding about their ecology and evolution. Among 24 reported genomes of cyanophages that infect marine picocyanobacteria, 17 are from cyanomyoviruses and six from cyanopodoviruses, and only one from cyanosiphovirus (Prochlorococcus phage P-SS2). Here we present four complete genome sequences of siphoviruses (S-CBS1, S-CBS2, S-CBS3 and S-CBS4) that infect four different marine Synechococcus strains. Three distinct subtypes were recognized among the five known marine siphoviruses (including P-SS2) in terms of morphology, genome architecture, gene content and sequence similarity. Our study revealed that cyanosiphoviruses are genetically diverse with polyphyletic origin. No core genes were found across these five cyanosiphovirus genomes, and this is in contrast to the fact that many core genes have been found in cyanomyovirus or cyanopodovirus genomes. Interestingly, genes encoding three structural proteins and a lysozyme of S-CBS1 and S-CBS3 showed homology to a prophage-like genetic element in two freshwater Synechococcus elongatus genomes. Re-annotation of the prophage-like genomic region suggests that S.?elongatus may contain an intact prophage. Cyanosiphovirus genes involved in DNA metabolism and replication share high sequence homology with those in cyanobacteria, and further phylogenetic analysis based on these genes suggests that ancient and selective genetic exchanges occurred, possibly due to past prophage integration. Metagenomic analysis based on the Global Ocean Sampling database showed that cyanosiphoviruses are present in relatively low abundance in the ocean surface water compared to cyanomyoviruses and cyanopodoviruses.     

Cyanobacterial assimilatory nitrate reductase gene diversity in coastal and oligotrophic marine environments

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.     

Diversity and phylogeny of Baltic Sea picocyanobacteria inferred from their ITS and phycobiliprotein operons

Picocyanobacteria of the genus Synechococcus span a range of different colours, from red strains rich in phycoerythrin (PE) to green strains rich in phycocyanin (PC). Here, we show that coexistence of red and green picocyanobacteria in the Baltic Sea is widespread. The diversity and phylogeny of red and green picocyanobacteria was analysed using three different genes: 16S rRNA-ITS, the cpeBA operon of the red PE pigment and the cpcBA operon of the green PC pigment. Sequencing of 209 clones showed that Baltic Sea picocyanobacteria exhibit high levels of microdiversity. The partial nucleotide sequences of the cpcBA and cpeBA operons from the clone libraries of the Baltic Sea revealed two distinct phylogenetic clades: one clade containing mainly sequences from cultured PC-rich picocyanobacteria, while the other contains only sequences from cultivated PE-rich strains. A third clade of phycourobilin (PUB) containing strains of PE-rich Synechococcus spp. did not contain sequences from the Baltic Sea clone libraries. These findings differ from previously published phylogenies based on 16S rRNA gene analysis. Our data suggest that, in terms of their pigmentation, Synechococcus spp. represent three different lineages occupying different ecological niches in the underwater light spectrum. Strains from different lineages can coexist in light environments that overlap with their light absorption spectra.     

Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.     

Cyanophycin production in a phycoerythrin-containing marine synechococcus strain of unusual phylogenetic affinity

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.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge <Emphasis Type="Italic">Gelliodes carnosa</Emphasis> Collected from the Hainan Island Coastal Waters of the South China Sea

Several molecular techniques were employed to document the bacterial diversity associated with the marine sponge Gelliodes carnosa. Cultivation-dependent and cultivation-independent methods were used to obtain the 16S rRNA gene sequences of the bacteria. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the bacterial community structure was highly diverse with representatives of the high G + C Gram-positive bacteria, cyanobacteria, low G + C Gram-positive bacteria, and proteobacteria (α-, β-, and γ-), most of which were also found in other marine environments, including in association with other sponges. Overall, 300 bacterial isolates were cultivated, and a total of 62 operational taxonomic units (OTUs) were identified from these isolates by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of the 16S rRNA genes. Approximately 1,000 16S rRNA gene clones were obtained by the cultivation-independent method. A total of 310 clones were randomly selected for RFLP analysis, from which 33 OTUs were acquired by further DNA sequencing and chimera checking. A total of 12 cultured OTUs (19.4% of the total cultured OTUs) and 13 uncultured OTUs (39.4% of the total uncultured OTUs) had low sequence identity (≤97%) with their closest matches in GenBank and were probably new species. Our data provide strong evidence for the presence of a diverse variety of unidentified bacteria in the marine sponge G. carnosa. A relatively high proportion of the isolates exhibited antimicrobial activity, and the deferred antagonism assay showed that over half of the active isolates exhibited a much stronger bioactivity when grown on medium containing seawater. In addition to demonstrating that the sponge-associated bacteria could be a rich source of new biologically active natural products, the results may have ecological implications. This study expands our knowledge of the diversity of sponge-associated bacteria and contributes to the growing database of the bacterial communities within sponges.     

Widespread distribution in polar oceans of a 16S rRNA gene sequence with affinity to Nitrosospira-like ammonia-oxidizing bacteria

We analyzed the phylogenetic compositions of ammonia-oxidizing bacteria of the beta subclass of Proteobacteria from 42 Southern Ocean samples. We found a Nitrosospira-like 16S rRNA gene sequence in all 20 samples that yielded PCR products (8 of 30 samples from the Ross Sea and 12 of 12 samples from the Palmer Peninsula). We also found this sequence in Arctic Ocean samples, indicating a transpolar, if not global, distribution; however, slight differences between Arctic and Antarctic sequences may be evidence of polar endemism.     

第六次北极科学考察海洋沉积物可培养细菌的多样性分析

【目的】研究北极海洋沉积物可培养细菌的菌种资源多样性。【方法】采用海水Zobell2216E培养基和涂布平板法对第六次北极科学考察获得的海洋沉积物开展细菌分离培养,通过16S rRNA基因系统发育分析了解可培养细菌的多样性。【结果】根据菌落形态特征,从40个站位的北极海洋沉积物样品中共分离并获得16S rRNA基因有效序列的细菌达445株;基于16S rRNA基因的相似性分析与系统发育研究结果表明,分离获得的细菌分属于细菌域的4个门、6个纲、13个目、28个科、49个属、91个种,其中γ-Proteobacteria占大多数;有12株与模式菌株的16S rRNA基因序列相似性小于97%,可能代表了6个潜在的细菌新物种;此次获得的细菌种类组成与以往第五次北极科考获得的相比,在属水平上差异较大。【结论】北极海洋沉积物中存在着丰富的微生物菌种资源,具有很多新型微生物仍未被发现,是亟待开发的微生物资源宝库。     

Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters

Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ , and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.     

Diversity and distribution of DNA sequences with affinity to ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Arctic Ocean

The spatial distribution and diversity of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria (hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to Escherichia coli 16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine Nitrosospira-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marine Nitrosomonas-like sequences. Our results suggest that the Arctic Ocean beta-proteobacterial ammonia oxidizers have low diversity and are dominated by marine Nitrosospira-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the Pacific Ocean through the Bering and Chukchi seas.     

Influence of salinity on bacterioplankton communities from the Brazilian rain forest to the coastal Atlantic Ocean

Background

Planktonic bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems, however, the taxa that make up these communities are poorly known. The aim of this study was to investigate bacterial communities in aquatic ecosystems at Ilha Grande, Rio de Janeiro, Brazil, a preserved insular environment of the Atlantic rain forest and how they correlate with a salinity gradient going from terrestrial aquatic habitats to the coastal Atlantic Ocean.

Methodology/Principal Findings

We analyzed chemical and microbiological parameters of water samples and constructed 16S rRNA gene libraries of free living bacteria obtained at three marine (two coastal and one offshore) and three freshwater (water spring, river, and mangrove) environments. A total of 836 sequences were analyzed by MOTHUR, yielding 269 freshwater and 219 marine operational taxonomic units (OTUs) grouped at 97% stringency. Richness and diversity indexes indicated that freshwater environments were the most diverse, especially the water spring. The main bacterial group in freshwater environments was Betaproteobacteria (43.5%), whereas Cyanobacteria (30.5%), Alphaproteobacteria (25.5%), and Gammaproteobacteria (26.3%) dominated the marine ones. Venn diagram showed no overlap between marine and freshwater OTUs at 97% stringency. LIBSHUFF statistics and PCA analysis revealed marked differences between the freshwater and marine libraries suggesting the importance of salinity as a driver of community composition in this habitat. The phylogenetic analysis of marine and freshwater libraries showed that the differences in community composition are consistent.

Conclusions/Significance

Our data supports the notion that a divergent evolutionary scenario is driving community composition in the studied habitats. This work also improves the comprehension of microbial community dynamics in tropical waters and how they are structured in relation to physicochemical parameters. Furthermore, this paper reveals for the first time the pristine bacterioplankton communities in a tropical island at the South Atlantic Ocean.