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Nitrogen fixation in the surface ocean impacts global marine nitrogen bioavailability and thus microbial primary productivity. Until now, cyanobacterial populations have been viewed as the main suppliers of bioavailable nitrogen in this habitat. Although PCR amplicon surveys targeting the nitrogenase reductase gene have revealed the existence of diverse non-cyanobacterial diazotrophic populations, subsequent quantitative PCR surveys suggest that they generally occur in low abundance. Here, we use state-of-the-art metagenomic assembly and binning strategies to recover nearly one thousand non-redundant microbial population genomes from the TARA Oceans metagenomes. Among these, we provide the first genomic evidence for non-cyanobacterial diazotrophs inhabiting surface waters of the open ocean, which correspond to lineages within the Proteobacteria and, most strikingly, the Planctomycetes. Members of the latter phylum are prevalent in aquatic systems, but have never been linked to nitrogen fixation previously. Moreover, using genome-wide quantitative read recruitment, we demonstrate that the discovered diazotrophs were not only widespread but also remarkably abundant (up to 0.3% of metagenomic reads for a single population) in both the Pacific Ocean and the Atlantic Ocean northwest. Our results extend decades of PCR-based gene surveys, and substantiate the importance of heterotrophic bacteria in the fixation of nitrogen in the surface ocean.

Our goal is to strengthen the foundations of metaproteomics as a microbial community analysis tool that links the functional identity of actively expressed gene products with host phylogeny. We used shotgun metaproteomics to survey waters in six disparate aquatic habitats (Cayuga Lake, NY; Oneida Lake, NY; Gulf of Maine; Chesapeake Bay, MD; Gulf of Mexico; and the South Pacific). Peptide pools prepared from filter-gathered microbial biomass, analyzed by nano-liquid chromatography-mass spectrometry (MS/MS) generating 9,693 ± 1,073 mass spectra identified 326 ± 107 bacterial proteins per sample. Distribution of proteobacterial (Alpha and Beta) and cyanobacterial (Prochlorococcus and Synechococcus spp.) protein hosts across all six samples was consistent with the previously published biogeography for these microorganisms. Marine samples were enriched in transport proteins (TRAP-type for dicarboxylates and ATP binding cassette (ABC)-type for amino acids and carbohydrates) compared with the freshwater samples. We were able to match in situ expression of many key proteins catalyzing C-, N-, and S-cycle processes with their bacterial hosts across all six habitats. Pelagibacter was identified as the host of ABC-type sugar-, organic polyanion-, and glycine betaine-transport proteins; this extends previously published studies of Pelagibacter's in situ biogeochemical role in marine C- and N-metabolism. Proteins matched to Ruegeria confirmed these organism's role in marine waters oxidizing both carbon monoxide and sulfide. By documenting both processes expressed in situ and the identity of host cells, metaproteomics tested several existing hypotheses about ecophysiological processes and provided fodder for new ones.

Members of the neutrophilic iron-oxidizing candidate class Zetaproteobacteria have predominantly been found at sites of microbially mediated iron oxidation in marine environments around the Pacific Ocean. Eighty-four full-length (>1,400-bp) and 48 partial-length Zetaproteobacteria small-subunit (SSU) rRNA gene sequences from five novel clone libraries, one novel Zetaproteobacteria isolate, and the GenBank database were analyzed to assess the biodiversity of this burgeoning class of the Proteobacteria and to investigate its biogeography between three major sampling regions in the Pacific Ocean: Loihi Seamount, the Southern Mariana Trough, and the Tonga Arc. Sequences were grouped into operational taxonomic units (OTUs) on the basis of a 97% minimum similarity. Of the 28 OTUs detected, 13 were found to be endemic to one of the three main sampling regions and 2 were ubiquitous throughout the Pacific Ocean. Additionally, two deeply rooted OTUs that potentially dominate communities of iron oxidizers originating in the deep subsurface were identified. Spatial autocorrelation analysis and analysis of molecular variance (AMOVA) showed that geographic distance played a significant role in the distribution of Zetaproteobacteria biodiversity, whereas environmental parameters, such as temperature, pH, or total Fe concentration, did not have a significant effect. These results, detected using the coarse resolution of the SSU rRNA gene, indicate that the Zetaproteobacteria have a strong biogeographic signal.

The abundance and phylogenetic diversity of the microbial community in the hydrogenetic ferromanganese crust, sandy sediment and overlying seawater were investigated using a culture-independent molecular analysis based on the 16S rRNA gene. These samples were carefully collected from the Takuyo-Daigo Seamount, located in the northwest Pacific Ocean, by a remotely operated vehicle. Based on quantitative PCR analysis, Archaea occupy a significant portion of the prokaryotic communities in the ferromanganese crust and the sediment samples, while Bacteria dominated in the seawater samples. Phylotypes belonging to Gammaproteobacteria and to Marine group I (MGI) Crenarchaeota were abundant in clone libraries constructed from the ferromanganese crust and sediment samples, while those belonging to Alphaproteobacteria were abundant in that from the seawater sample. Comparative analysis indicates that over 80% of the total phylotype richness estimates for the crust community were unique as compared with the sediment and seawater communities. Phylotypes related to Nitrosospira belonging to the Betaproteobacteria and those related to Nitrosopumilus belonging to MGI Crenarchaeota were detected in the ferromanganese crust, suggesting that these ammonia-oxidizing chemolithoautotrophs play a role as primary producers in the microbial ecosystem of hydrogenetic ferromanganese crusts that was formed as precipitates from seawater., (2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.

The Pacific Nodule Province is a unique ocean area containing an abundance of polymetallic nodules. To explore more genetic information and discover potentially industrial useful genes of the microbial community from this particular area, a cosmid library with an average insert of about 35 kb was constructed from the deep-sea sediment. The bacteria in the cosmid library were composed mainly of Proteobacteria including Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. The end sequences of some cosmid clones were determined and the complete insert sequences of two cosmid clones, 10D02 and 17H9, are presented. 10D02 has a length of 40.8 kb and contains 40 predicted encoding genes. It contains a partial 16S rRNA gene of Alphaproteobacteria. 17H9 is 36.8 kb and predicted to have 31 encoding genes and a 16S-23S-5S rRNA gene operon. Phylogenetic analysis of 16S and 23S rRNA gene sequence on the 17H9 both reveals that the inserted DNA from 17H9 came from a novel Alphaproteobacteria and is closely related to Magnetospirillum species. The predicted proteins of ORF 1-11 also have high identity to those of Magnetospirillum species, and the organization of these genes is highly conserved among known Magnetospirillum species. The data suggest that the retrieved DNA in 17H9 might be derived from a novel Magnetospirillum species.

A systematic investigation of marine pigmented heterotrophic bacteria (PHB) based on the cultivation method and sequencing analysis of 16S rRNA genes was conducted in Chinese coastal and shelf waters and the Pacific Ocean. Both the abundance of PHB and the ratio of PHB to CFU decreased along trophic gradients from coastal to oceanic waters, with the highest values of 9.9 x 10(3) cell mL(-1) and 39.6%, respectively, in the Yangtze River Estuary. In contrast to the total heterotrophic bacteria (TB) and CFU, which were present in the whole water column, PHB were primarily confined to the euphotic zone, with the highest abundance of PHB and ratio of PHB to CFU occurring in surface water. In total, 247 pigmented isolates were obtained during this study, and the phylogenetic analysis showed a wide genetic diversity covering 25 genera of six phylogenetic classes: Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria. PHB belonging to Alphaproteobacteria, Flavobacteria and Sphingobacteria were obtained mainly from the South China Sea and East China Sea; PHB from the Pacific Ocean water were predominantly affiliated with Gammaproteobacteria, and most isolates from the Yangtze River Estuary fell into the classes Actinobacteria and Bacilli. The isolates exhibited various colours (e.g. golden, yellow, red, pink and orange), with genus or species specificity. Furthermore, the pigment of PHB cells absorbed light mainly in the wavelength range between 450 and 550 nm. In conclusion, our work has revealed that PHB with broad genetic diversity are widely distributed in the marine environment, and may account for up to 39.6% of culturable bacteria, equivalent to 1.4% of the total microbial community. This value might even be underestimated because it is probable that not all pigmented bacteria were isolated. Their abundance and genetic distribution are heavily influenced by environmental properties, such as light and nutrition, suggesting that they have important roles in the marine ecosystem, especially in the absorption of visible light.

Olavius crassitunicatus is a small symbiont-bearing worm that occurs at high abundance in oxygen-deficient sediments in the East Pacific Ocean. Using comparative 16S rRNA sequence analysis and fluorescence in situ hybridization, we examined the diversity and phylogeny of bacterial symbionts in two geographically distant O. crassitunicatus populations (separated by 385 km) on the Peru margin (water depth, approximately 300 m). Five distinct bacterial phylotypes co-occurred in all specimens from both sites: two members of the gamma-Proteobacteria (Gamma 1 and 2 symbionts), two members of the delta-Proteobacteria (Delta 1 and 2 symbionts), and one spirochete. A sixth phylotype belonging to the delta-Proteobacteria (Delta 3 symbiont) was found in only one of the two host populations. Three of the O. crassitunicatus bacterial phylotypes are closely related to symbionts of other gutless oligochaete species; the Gamma 1 phylotype is closely related to sulfide-oxidizing symbionts of Olavius algarvensis, Olavius loisae, and Inanidrilus leukodermatus, the Delta 1 phylotype is closely related to sulfate-reducing symbionts of O. algarvensis, and the spirochete is closely related to spirochetal symbionts of O. loisae. In contrast, the Gamma 2 phylotype and the Delta 2 and 3 phylotypes belong to novel lineages that are not related to other bacterial symbionts. Such a phylogenetically diverse yet highly specific and stable association in which multiple bacterial phylotypes coexist within a single host has not been described previously for marine invertebrates.

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5 degrees C and natural vent fluids at 7 degrees C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and gamma- and epsilon-Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with "Nanoarchaeota." The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300 degrees C were affiliated with the delta-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4 degrees C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.

A depth profile of naturally occurring bacterial community structures associated with the deep-sea cold seep push-core sediment in the Japan Trench at a depth of 5343 m were evaluated using molecular phylogenetic analyses of RNA reverse transcription-PCR (RT-PCR) amplified 16S crDNA fragments. A total of 137 clones of bacterial crDNA (complimentary rDNA) phylotypes (phylogenetic types) obtained at three different depths (2-4, 8-10 and 14-16 cm) were identified in partial crDNA sequencings. crDNA phylotypes from the cold seep sediment were dominantly composed of delta- and epsilon-Proteobacteria (36% and 42% respectively). Phylotype analysis of crDNA clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the majority of bacterial components shifted from delta- Proteobacteria to epsilon-Proteobacteria with increasing depth. Among the delta-proteobacterial crDNA clones, the sequences related to the genus Desulfosarcina were dominant. Almost all sequences of crDNA belonging to epsilon-Proteobacteria were affiliated with the same cluster (epsilon-CSG: epsilon-proteobacterial cold seep group), and were closely related with rDNA sequences from deep-sea hydrothermal vent environments.