Prevalence of the Chloroflexi-Related SAR202 Bacterioplankton Cluster throughout the Mesopelagic Zone and Deep Ocean

Since their initial discovery in samples from the north Atlantic Ocean, 16S rRNA genes related to the environmental gene clone cluster known as SAR202 have been recovered from pelagic freshwater, marine sediment, soil, and deep subsurface terrestrial environments. Together, these clones form a major, monophyletic subgroup of the phylum Chloroflexi. While members of this diverse group are consistently identified in the marine environment, there are currently no cultured representatives, and very little is known about their distribution or abundance in the world's oceans. In this study, published and newly identified SAR202-related 16S rRNA gene sequences were used to further resolve the phylogeny of this cluster and to design taxon-specific oligonucleotide probes for fluorescence in situ hybridization. Direct cell counts from the Bermuda Atlantic time series study site in the north Atlantic Ocean, the Hawaii ocean time series site in the central Pacific Ocean, and along the Newport hydroline in eastern Pacific coastal waters showed that SAR202 cluster cells were most abundant below the deep chlorophyll maximum and that they persisted to 3,600 m in the Atlantic Ocean and to 4,000 m in the Pacific Ocean, the deepest samples used in this study. On average, members of the SAR202 group accounted for 10.2% (±5.7%) of all DNA-containing bacterioplankton between 500 and 4,000 m.     

Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans.

A gene lineage (SAR406) related to Chlorobium and Fibrobacter species was found in 16S rRNA gene clone libraries prepared from samples from two oceans. The clone libraries were constructed from total picoplankton genomic DNA to assess bacterial diversity in the lower surface layer. The samples were collected by filtration from a depth of 80 m at a site in the western Sargasso Sea and from a depth of 120 m at a site in the Pacific Ocean, approximately 70 km from the Oregon coast. The PCR and primers which amplified nearly full-length 16S rRNA genes were used to prepare the clone libraries. Among the diverse gene clones in these libraries were two related clones (SAR406 and OCS307) which could not be assigned to any of the major bacterial phyla. Phylogenetic analyses demonstrated that these genes were distant relatives of the genus Fibrobacter and the green sulfur bacterial phylum, which includes the genus Chlorobium. The inclusion of SAR406 in phylogenetic trees inferred by several methods resulted in support from bootstrap replicates for the conclusion that Fibrobacter and Chlorobium species and SAR406 are a monophyletic group. An oligonucleotide probe that selectively hybridized to clone SAR406 was used to examine the distribution of this gene lineage in vertical profiles from the Atlantic and Pacific Oceans and in monthly time series at 0 and 200 m in the Atlantic Ocean. During stratified periods, the genes were most abundant slightly below the deep chlorophyll layer. Seasonal changes in the surface abundance of SAR406 rDNA were highly correlated with chlorophyll a levels (r = 0.75).     

Dynamics of the SAR11 bacterioplankton lineage in relation to environmental conditions in the oligotrophic North Pacific subtropical gyre

A quantitative PCR assay for the SAR11 clade of marine Alphaproteobacteria was applied to nucleic acids extracted from monthly depth profiles sampled over a 3-year period (2004–2007) at the open-ocean Station ALOHA (A Long-term Oligotrophic Habitat Assessment; 22°45'N, 158°00'W) in the oligotrophic North Pacific Ocean. This analysis revealed a high contribution (averaging 36% of 16S rRNA gene copies) of SAR11 to the total detected 16S rRNA gene copies over depths ranging from the surface layer to 4000 m, and revealed consistent spatial and temporal variation in the relative abundance of SAR11 16S rRNA gene copies. On average, a higher proportion of SAR11 rRNA gene copies were detected in the photic zone (< 175 m depth; mean = 38%) compared with aphotic (> 175 m depth; mean = 30%), and in the winter months compared with the summer (mean =  44% versus 33%, integrated over 175 m depth). Partial least square to latent structure projections identified environmental variables that correlate with variation in the absolute abundance of SAR11, and provided tools for developing a predictive model to explain time and depth-dependent variations in SAR11. Moreover, this information was used to hindcast temporal dynamics of the SAR11 clade between 1997 and 2006 using the existing HOT data set, which suggested that interannual variations in upper ocean SAR11 abundances were related to ocean-climate variability such as the El Niño Southern Oscillation.     

Distribution of Roseobacter RCA and SAR11 lineages and distinct bacterial communities from the subtropics to the Southern Ocean

We assessed the composition of the bacterioplankton in the Atlantic sector of the Southern Ocean in austral fall and winter and in New Zealand coastal waters in summer. The various water masses between the subtropics/Agulhas–Benguela boundary region and the Antarctic coastal current exhibited distinct bacterioplankton communities with the highest richness in the polar frontal region, as shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. The SAR11 clade and the Roseobacter clade‐affiliated (RCA) cluster were quantified by real‐time quantitative PCR. SAR11 was detected in all samples analysed from subtropical waters to the coastal current and to depths of > 1000 m. In fall and winter, this clade constituted < 3% to 48% and 4–28% of total bacterial 16S rRNA genes respectively, with highest fractions in subtropical to polar frontal regions. The RCA cluster was only present in New Zealand coastal surface waters not exceeding 17°C, in the Agulhas–Benguela boundary region (visited only during the winter cruise), in subantarctic waters and in the Southern Ocean. In fall, this cluster constituted up to 36% of total bacterial 16S rRNA genes with highest fractions in the Antarctic coastal current and outnumbered the SAR11 clade at most stations in the polar frontal region and further south. In winter, the RCA cluster constituted lower proportions than the SAR11 clade and did not exceed 8% of total bacterial 16S rRNA genes. In fall, the RCA cluster exhibited significant positive correlations with latitude and ammonium concentrations and negative correlations with concentrations of nitrate, phosphate, and for near‐surface samples also with chlorophyll a, biomass production of heterotrophic prokaryotes and glucose turnover rates. The findings show that the various water masses between the subtropics and the Antarctic coastal current harbour distinct bacterioplankton communities. They further indicate that the RCA cluster, despite the narrow sequence similarity of > 98% of its 16S rRNA gene, is an abundant component of the heterotrophic bacterioplankton in the Southern Ocean, in particular in its coldest regions.     

Abundance and activity of Chloroflexi-type SAR202 bacterioplankton in the meso- and bathypelagic waters of the (sub)tropical Atlantic

The contribution of Chloroflexi-type SAR202 cells to total picoplankton and bacterial abundance and uptake of D- and L-aspartic acids (Asp) was determined in the different meso- and bathypelagic water masses of the (sub)tropical Atlantic (from 35 degrees N to 5 degrees S). Fluorescence in situ hybridization (FISH) revealed that the overall abundance of SAR202 was < or = 1 x 10(3) cells ml(-1) in subsurface waters (100 m layer), increasing in the mesopelagic zone to 3 x 10(3) cells ml(-1) and remaining fairly constant down to 4000 m depth. Overall, the percentage of total picoplankton identified as SAR202 increased from < 1% in subsurface waters to 10-20% in the bathypelagic waters. On average, members of the SAR202 cluster accounted for about 30% of the Bacteria in the bathypelagic waters, whereas in the mesopelagic and subsurface waters, SAR202 cells contributed < 5% to total bacterial abundance. The ratio of D-Asp : L-Asp uptake by the bulk picoplankton community increased from the subsurface layer (D-Asp : L-Asp uptake ratio approximately 0.03) to the deeper layers reaching a ratio of approximately 1 at 4000 m depth. Combining FISH with microautoradiography to determine the proportion of SAR202 cells taking up D-Asp versus L-Asp, we found that approximately 30% of the SAR202 cells were taking up L-Asp throughout the water column while D-Asp was essentially not taken up by SAR202. This D-Asp : L-Asp uptake pattern of SAR202 cells is in contrast to that of the bulk bacterial and crenarchaeal community in the bathypelagic ocean, both sustaining a higher fraction of D-Asp-positive cells than L-Asp-positive cells. Thus, although the Chloroflexi-type SAR202 constitutes a major bathypelagic bacterial cluster, it does not contribute to the large fraction of d-Asp utilizing prokaryotic community in the meso- and bathypelagic waters of the North Atlantic, but rather utilizes preferentially L-amino acids.     

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria

Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.     

Phylogenetic comparisons of a coastal bacterioplankton community with its counterparts in open ocean and freshwater systems

In order to extend previous comparisons between coastal marine bacterioplankton communities and their open ocean and freshwater counterparts, here we summarize and provide new data on a clone library of 105 SSU rRNA genes recovered from seawater collected over the western continental shelf of the USA in the Pacific Ocean. Comparisons to previously published data revealed that this coastal bacterioplankton clone library was dominated by SSU rRNA gene phylotypes originally described from surface waters of the open ocean, but also revealed unique SSU rRNA gene lineages of beta Proteobacteria related to those found in clone libraries from freshwater habitats. beta Proteobacteria lineages common to coastal and freshwater samples included members of a clade of obligately methylotrophic bacteria, SSU rRNA genes affiliated with Xylophilus ampelinus, and a clade related to the genus Duganella. In addition, SSU rRNA genes were recovered from such previously recognized marine bacterioplankton SSU rRNA gene clone clusters as the SAR86, SAR11, and SAR116 clusters within the class Proteobacteria, the Roseobacter clade of the alpha subclass of the Proteobacteria, the marine group A/SAR406 cluster, and the marine Actinobacteria clade. Overall, these results support and extend previous observations concerning the global distribution of several marine planktonic prokaryote SSU rRNA gene phylotypes, but also show that coastal bacterioplankton communities contain SSU rRNA gene lineages (and presumably bacterioplankton) shown previously to be prevalent in freshwater habitats.     

Phylogenetic diversity of subsurface marine microbial communities from the Atlantic and Pacific Oceans.

The extent of the diversity of marine prokaryotes is not well known, primarily because of poor cultivability. However, new techniques permit the characterization of such organisms without culturing, via 16S rRNA sequences obtained directly from biomass. We performed such an analysis by polymerase chain reaction amplification with universal primers on five oligotrophic open-ocean samples: from 100-m (three samples) and 500-m depths in the western California Current (Pacific Ocean) and from a 10-m depth in the Atlantic Ocean near Bermuda. Of 61 clones, 90% were in clusters of two or more related marine clones obtained by ourselves or others. We report 15 clones related to clone SAR 11 found earlier near Bermuda (S. J. Giovannoni, T. B. Britschgi, C. L. Moyer, and K. G. Field, Nature [London] 345:60-63, 1990), 11 related to marine cyanobacteria, 9 clustered in a group affiliated with gram-positive bacteria, 9 in an archaeal cluster we recently described (mostly from the 500-m sample), 4 in a novel gamma-proteobacterial cluster, and 6 in three two-membered clusters (including other archaea). One clone was related to flavobacteria. Only the cyanobacteria plus one other clone, related to Roseobacter denitrificans (formerly Erythrobacter longus Och114), were within 10% sequence identity to any previously sequenced cultured organism in a major data base. We never found more than two occurrences of the same sequence in a sample, although four times we found identical sequences between samples, two of which were between oceans; one of these sequences was also identical to SAR 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM ¹³C-NaHCO₃, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.     

A new look at geographic and phylogenetic relationships within the species group surrounding Octopus vulgaris (Mollusca, Cephalopoda): indications of very wide distribution from mitochondrial DNA sequences

The distribution of Octopus vulgaris has not yet been completely clarified. For a long time, a cosmopolitan distribution with unknown distribution limits had been assumed. This assumption has recently been questioned and it has been postulated that the distribution is restricted to the Mediterranean and the northeastern Atlantic. However, as our previous studies show, the existence of O. vulgaris can be confirmed for the Mediterranean and the whole eastern Atlantic, and evidence is provided for its occurrence in the western Atlantic. The aim of the present work is to extend our previous data matrix and to clarify whether O. vulgaris exists in the northwestern Pacific. Therefore, the sequence variation in ostensible O. vulgaris from 13 localities in the Mediterranean (France), the Atlantic Ocean [Lanzarote, Senegal, South Africa (Atlantic, Indian Ocean), Tristan da Cunha, north, middle and south Brazil], the Caribbean Sea (Venezuela) and the Pacific Ocean (Taiwan, Japan and Costa Rica) was examined using the mitochondrial genes coding for the 16S rRNA and cytochrome oxidase subunit III (COIII). Sequence divergence was relatively low between populations of O. vulgaris from the Mediterranean, the eastern and western Atlantic (except north Brazil), Venezuela, Taiwan and Japan compared with other species of the genus Octopus . Trees constructed by using maximum likelihood, neighbour joining and maximum parsimony algorithms (PAUP) show the above-mentioned populations from the Mediterranean, the western and eastern Atlantic, Venezuela and the northwestern Pacific (Japan and Taiwan) as a monophyletic cluster. Thus, even if the Octopus vulgaris -like octopus from north Brazil should turn out a cryptic species, the data of this work not only support our hypothesis of the distribution of O. vulgaris in the Mediterranean, the eastern and western Atlantic but also show that O. vulgaris is present in the northwestern Pacific, namely in the waters of Taiwan and Japan.     

Population structure and phylogenetic characterization of marine benthic Archaea in deep-sea sediments.

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

Diversity of prokaryotes and methanogenesis in deep subsurface sediments from the Nankai Trough, Ocean Drilling Program Leg 190

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.     

Latitudinal distribution of prokaryotic picoplankton populations in the Atlantic Ocean

Members of the prokaryotic picoplankton are the main drivers of the biogeochemical cycles over large areas of the world's oceans. In order to ascertain changes in picoplankton composition in the euphotic and twilight zones at an ocean basin scale we determined the distribution of 11 marine bacterial and archaeal phyla in three different water layers along a transect across the Atlantic Ocean from South Africa (32.9°S) to the UK (46.4°N) during boreal spring. Depth profiles down to 500 m at 65 stations were analysed by catalysed reporter deposition fluorescence in situ hybridization (CARD‐FISH) and automated epifluorescence microscopy. There was no obvious overall difference in microbial community composition between the surface water layer and the deep chlorophyll maximum (DCM) layer. There were, however, significant differences between the two photic water layers and the mesopelagic zone. SAR11 (35 ± 9%) and Prochlorococcus (12 ± 8%) together dominated the surface waters, whereas SAR11 and Crenarchaeota of the marine group I formed equal proportions of the picoplankton community below the DCM (both ～15%). However, due to their small cell sizes Crenarchaeota contributed distinctly less to total microbial biomass than SAR11 in this mesopelagic water layer. Bacteria from the uncultured Chloroflexi‐related clade SAR202 occurred preferentially below the DCM (4–6%). Distinct latitudinal distribution patterns were found both in the photic zone and in the mesopelagic waters: in the photic zone, SAR11 was more abundant in the Northern Atlantic Ocean (up to 45%) than in the Southern Atlantic gyre (～25%), the biomass of Prochlorococcus peaked in the tropical Atlantic Ocean, and Bacteroidetes and Gammaproteobacteria bloomed in the nutrient‐rich northern temperate waters and in the Benguela upwelling. In mesopelagic waters, higher proportions of SAR202 were present in both central gyre regions, whereas Crenarchaeota were clearly more abundant in the upwelling regions and in higher latitudes. Other phylogenetic groups such as the Planctomycetes, marine group II Euryarchaeota and the uncultured clades SAR406, SAR324 and SAR86 rarely exceeded more than 5% of relative abundance.     

Population Structure and Phylogenetic Characterization of Marine Benthic Archaea in Deep-Sea Sediments

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary

The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.     

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.     

Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale

Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems.     

Genetic analysis of housekeeping genes reveals a deep-sea ecotype of Alteromonas macleodii in the Mediterranean Sea

The genetic diversity of 19 strains belonging to Alteromonas macleodii isolated from different geographic areas (Pacific and Indian Ocean, and different parts of the Mediterranean Sea) and at different depths (from the surface down to 3500 m) has been studied. Fragments of the 16S rRNA gene, the internal transcribed spacer (ITS) between 16S and 23S rDNA genes, the gyrB and the rpoB genes, have been sequenced for each strain. Amplified fragment length polymorphisms were used to characterize similarity at the level of the whole genome. Most of the diversity reflected the existence of a cluster of strains isolated from deep Mediterranean waters and two isolates from the Black Sea. Particularly the isolates from the deep sites were consistently different from all the others indicating the existence of a specific ecotype adapted to these conditions. Amplification of gyrB gene and ITS directly from DNA retrieved from deep Mediterreanean waters and one Atlantic sample showed that presence of this deep-sea ecotype is widespread and is not a product of culture bias. On the other hand, strains isolated from surface tropical waters showed a remarkable level of resemblance to the first isolate of this species obtained from Hawaii in 1972. The results indicate the existence of both lineages of global distribution and ecotypes adapted to specific conditions such as deep or more diluted (the Black Sea) waters.     

Photosynthetic picoeukaryote community structure in the South East Pacific Ocean encompassing the most oligotrophic waters on Earth

Photosynthetic picoeukaryotes (PPEs), comprising organisms < 3 μm in size, are important primary producers in marine food webs and include representatives from all known algal lineages. Little is known, however, regarding the composition and distribution of PPE communities, particularly at large spatial scales, or in relation to the underlying biotic and abiotic factors that influence this structure. Here, we analysed PPE community structure along a transect in the South East Pacific Ocean (BIOSOPE cruise) that encompassed a large trophic gradient, including hyper‐oligotrophic waters in the South Pacific Gyre (SPG), considered to be some of the ‘clearest’ natural waters on Earth. Using dot blot hybridizations with 16S rRNA oligonucleotide probes, we established that the PPE community was dominated by members of the classes Prymnesiophyceae and Chrysophyceae throughout the transect. Moreover, clone library construction followed by phylogenetic analysis of sequenced clones revealed several novel 16S rRNA gene lineages, including new clades of prymnesiophytes (designated Prym 16S‐III) and prasinophytes (Pras 16S‐VIII). Pras 16S‐VIII was found at all five stations at which clone libraries were constructed, representing a range of trophic conditions, including the South Pacific Gyre, suggesting members of this clade have a broad distribution in this part of the South East Pacific at least. In contrast, Prym 16S‐III sequences were largely restricted to oligotrophic stations of the SPG. Subsequent multivariate statistical analyses showed that, within the measured factors, chemical and biological factors seem to influence PPE community structure more than physical parameters. However, more than 50% of the variation in distribution of PPE classes remained unexplained.     

Microbial diversity in Cenozoic sediments recovered from the Lomonosov Ridge in the Central Arctic Basin

The current understanding of microbes inhabiting deeply buried marine sediments is based largely on samples collected from continental shelves in tropical and temperate latitudes. The geographical range of marine subsurface coring was expanded during the Integrated Ocean Drilling Program Arctic Coring Expedition (IODP ACEX). This expedition to the ice-covered central Arctic Ocean successfully cored the entire 428 m sediment stack on the Lomonosov Ridge during August and September 2004. The recovered cores vary from siliciclastic sediment low in organic carbon (< 0.2%) to organic rich (∼3%) black sediments that rapidly accumulated in the early middle Eocene. Three geochemical environments were characterized based on chemical analyses of porewater: an upper ammonium oxidation zone, a carbonate dissolution zone and a deep (> 200 m below sea floor) sulfate reduction zone. The diversity of microbes within each zone was assessed using 16S rRNA phylogenetic markers. Bacterial 16S rRNA genes were successfully amplified from each of the biogeochemical zones, while archaea was only amplified from the deep sulfate reduction zone. The microbial communities at each zone are phylogenetically different and are most closely related to those from other deep subsurface environments.