Microbial diversity and abundance vary along salinity, oxygen, and particle size gradients in the Chesapeake Bay.

Marine snow and other particles are abundant in estuaries, where they drive biogeochemical transformations and elemental transport. Particles range in size, thereby providing a corresponding gradient of habitats for marine microorganisms. We used standard normalized amplicon sequencing, verified with microscopy, to characterize taxon-specific microbial abundances, (cells per litre of water and per milligrams of particles), across six particle size classes, ranging from 0.2 to 500 µm, along the main stem of the Chesapeake Bay estuary. Microbial communities varied in salinity, oxygen concentrations, and particle size. Many taxonomic groups were most densely packed on large particles (in cells/mg particles), yet were primarily associated with the smallest particle size class, because small particles made up a substantially larger portion of total particle mass. However, organisms potentially involved in methanotrophy, nitrite oxidation, and sulphate reduction were found primarily on intermediately sized (5-180 µm) particles, where species richness was also highest. All abundant ostensibly free-living organisms, including SAR11 and Synecococcus, appeared on particles, albeit at lower abundance than in the free-living fraction, suggesting that aggregation processes may incorporate them into particles. Our approach opens the door to a more quantitative understanding of the microscale and macroscale biogeography of marine microorganisms.

Increased cyanophage infection at the bottom of the euphotic zone, especially in the fall.

Picocyanobacteria contribute greatly to offshore primary production with cells extending through the deep euphotic zone. Literature indicates high viral infection of cyanobacteria in ocean transition zones. We postulate that the bottom of the euphotic zone is a transition zone, where communities transition from phototrophic to aphotic processes. We use single-copy core genes to examine cyanophage to cyanobacteria ratios in cellular metagenomes in the subtropical North Atlantic and Pacific. Cyanophage to cyanobacteria terL/rpoB ratios generally increase to >10 in the deep euphotic zone. As light levels decrease in the fall, Prochlorococcus in the deep euphotic zone experience reduced light levels. We find clear differences between spring (Geotraces GA02) and fall (GA03) in the North Atlantic, with terL/rpoB ratios increasing to >40 in the fall. When examining 23 months of the North Pacific Hawaii Ocean Timeseries, the depth of elevated cyanophage to cyanobacteria ratios in cellular metagenomes negatively correlated with surface photosynthetic radiation (PAR), particularly with the change in PAR, which reflected the season. In fall, all picocyanobacteria ecotypes were found at depths enriched with viruses, while in summer, only low light ecotypes were affected. Thus, we find high cyanophage infection both in the deep euphotic zone and during seasonal transitions.

Associations between picocyanobacterial ecotypes and cyanophage host genes across ocean basins and depth.

Background: Cyanophages, viruses that infect cyanobacteria, are globally abundant in the ocean's euphotic zone and are a potentially important cause of mortality for marine picocyanobacteria. Viral host genes are thought to increase viral fitness by either increasing numbers of genes for synthesizing nucleotides for virus replication, or by mitigating direct stresses imposed by the environment. The encoding of host genes in viral genomes through horizontal gene transfer is a form of evolution that links viruses, hosts, and the environment. We previously examined depth profiles of the proportion of cyanophage containing various host genes in the Eastern Tropical North Pacific Oxygen Deficient Zone (ODZ) and at the subtropical North Atlantic (BATS). However, cyanophage host genes have not been previously examined in environmental depth profiles across the oceans. Methodology: We examined geographical and depth distributions of picocyanobacterial ecotypes, cyanophage, and their viral-host genes across ocean basins including the North Atlantic, Mediterranean Sea, North Pacific, South Pacific, and Eastern Tropical North and South Pacific ODZs using phylogenetic metagenomic read placement. We determined the proportion of myo and podo-cyanophage containing a range of host genes by comparing to cyanophage single copy core gene terminase (terL). With this large dataset (22 stations), network analysis identified statistical links between 12 of the 14 cyanophage host genes examined here with their picocyanobacteria host ecotypes. Results: Picyanobacterial ecotypes, and the composition and proportion of cyanophage host genes, shifted dramatically and predictably with depth. For most of the cyanophage host genes examined here, we found that the composition of host ecotypes predicted the proportion of viral host genes harbored by the cyanophage community. Terminase is too conserved to illuminate the myo-cyanophage community structure. Cyanophage cobS was present in almost all myo-cyanophage and did not vary in proportion with depth. We used the composition of cobS phylotypes to track changes in myo-cyanophage composition. Conclusions: Picocyanobacteria ecotypes shift with changes in light, temperature, and oxygen and many common cyanophage host genes shift concomitantly. However, cyanophage phosphate transporter gene pstS appeared to instead vary with ocean basin and was most abundant in low phosphate regions. Abundances of cyanophage host genes related to nutrient acquisition may diverge from host ecotype constraints as the same host can live in varying nutrient concentrations. Myo-cyanophage community in the anoxic ODZ had reduced diversity. By comparison to the oxic ocean, we can see which cyanophage host genes are especially abundant (nirA, nirC, and purS) or not abundant (myo psbA) in ODZs, highlighting both the stability of conditions in the ODZ and the importance of nitrite as an N source to ODZ endemic LLV Prochlorococcus.

An analysis of protists in Pacific oxygen deficient zones: implications for Prochlorococcus and N2 -producing bacteria.

Ocean oxygen deficient zones (ODZs) host 30%-50% of marine N2 production. Cyanobacteria photosynthesizing in the ODZ create a secondary chlorophyll maximum and provide organic matter to N2 -producing bacteria. This chlorophyll maximum is thought to occur due to reduced grazing in anoxic waters. We first examine ODZ protists with long amplicon reads. We then use non-primer-based methods to examine the composition and relative abundance of protists in metagenomes from the Eastern Tropical North and South Pacific ODZs and compare these data to the oxic Hawaii Ocean Time-series (HOT) in the North Pacific. We identify and quantify protists in proportion to the total microbial community. From metagenomic data, we see a large drop in abundance of fungi and protists such as choanoflagellates, radiolarians, cercozoa and ciliates in the ODZs but not in the oxic mesopelagic at HOT. Diplonemid euglenozoa were the only protists that increased in the ODZ. Dinoflagellates and foraminifera reads were also present in the ODZ though less abundant compared to oxic waters. Denitrification has been found in foraminifera but not yet in dinoflagellates. DNA techniques cannot separate dinoflagellate cells and cysts. Metagenomic analysis found taxonomic groups missed by amplicon sequencing and identified trends in abundance.

Cyanophage host-derived genes reflect contrasting selective pressures with depth in the oxic and anoxic water column of the Eastern Tropical North Pacific.

Cyanophages encode host-derived genes that may increase their fitness. We examined the relative abundance of 18 host-derived cyanophages genes in metagenomes and viromes along depth profiles from the Eastern Tropical North Pacific Oxygen Deficient Zone (ETNP ODZ) where Prochlorococcus dominates a secondary chlorophyll maximum within the ODZ. Cyanophages at the oxic primary chlorophyll maximum encoded genes related to light and phosphate stress (psbA, psbD and pstS in T4-like and psbA in T7-like), but the proportion of cyanophage with these genes decreased with depth. The proportion of cyanophage with purine biosynthesis genes increased with depth in T4-like, but not T7-like cyanophages. No additional host-derived genes were found in deep T7-like cyanophages, suggesting that T4-like and T7-like cyanophages have different host-derived gene acquisition strategies, possibly linked to their different genome packaging mechanisms. In contrast to the ETNP, in the oxic North Atlantic T4-like cyanophages encoded psbA and pstS throughout the euphotic zone. Differences in pstS between the ETNP and the North Atlantic stations were consistent with differences in phosphate concentrations in those regimes. We suggest that the low proportion of cyanophage with psbA within the ODZ reflects the stably stratified low-light conditions occupied by their hosts, a Prochlorococcus ecotype endemic to ODZs.

Using modern low-oxygen marine ecosystems to understand the nitrogen cycle of the Paleo- and Mesoproterozoic oceans.

During the productive Paleoproterozoic (2.4-1.8 Ga) and less productive Mesoproterozoic (1.8-1.0 Ga), the ocean was suboxic to anoxic and multicellular organisms had not yet evolved. Here, we link geologic information about the Proterozoic ocean to microbial processes in modern low-oxygen systems. High iron concentrations and rates of Fe cycling in the Proterozoic are the largest differences from modern oxygen-deficient zones. In anoxic waters, which composed most of the Paleoproterozoic and ~40% of the Mesoproterozoic ocean, nitrogen cycling dominated. Rates of N2 production by denitrification and anammox were likely linked to sinking organic matter fluxes and in situ primary productivity under anoxic conditions. Additionally autotrophic denitrifiers could have used reduced iron or methane. 50% of the Mesoproterozoic ocean may have been suboxic, promoting nitrification and metal oxidation in the suboxic water and N2 O and N2 production by partial and complete denitrification in anoxic zones in organic aggregates. Sulfidic conditions may have composed ~10% of the Mesoproterozoic ocean focused along continental margins. Due to low nitrate concentrations in offshore regions, anammox bacteria likely dominated N2 production immediately above sulfidic zones, but in coastal regions, higher nitrate concentrations probably promoted complete S-oxidizing autotrophic denitrification at the sulfide interface.

Cyanobacteria and cyanophage contributions to carbon and nitrogen cycling in an oligotrophic oxygen-deficient zone.

Up to half of marine N losses occur in oxygen-deficient zones (ODZs). Organic matter flux from productive surface waters is considered a primary control on N2 production. Here we investigate the offshore Eastern Tropical North Pacific (ETNP) where a secondary chlorophyll a maximum resides within the ODZ. Rates of primary production and carbon export from the mixed layer and productivity in the primary chlorophyll a maximum were consistent with oligotrophic waters. However, sediment trap carbon and nitrogen fluxes increased between 105 and 150 m, indicating organic matter production within the ODZ. Metagenomic and metaproteomic characterization indicated that the secondary chlorophyll a maximum was attributable to the cyanobacterium Prochlorococcus, and numerous photosynthesis and carbon fixation proteins were detected. The presence of chemoautotrophic ammonia-oxidizing archaea and the nitrite oxidizer Nitrospina and detection of nitrate oxidoreductase was consistent with cyanobacterial oxygen production within the ODZ. Cyanobacteria and cyanophage were also present on large (>30 µm) particles and in sediment trap material. Particle cyanophage-to-host ratio exceeded 50, suggesting that viruses help convert cyanobacteria into sinking organic matter. Nitrate reduction and anammox proteins were detected, congruent with previously reported N2 production. We suggest that autochthonous organic matter production within the ODZ contributes to N2 production in the offshore ETNP.

Complete arsenic-based respiratory cycle in the marine microbial communities of pelagic oxygen-deficient zones.

Microbial capacity to metabolize arsenic is ancient, arising in response to its pervasive presence in the environment, which was largely in the form of As(III) in the early anoxic ocean. Many biological arsenic transformations are aimed at mitigating toxicity; however, some microorganisms can respire compounds of this redox-sensitive element to reap energetic gains. In several modern anoxic marine systems concentrations of As(V) are higher relative to As(III) than what would be expected from the thermodynamic equilibrium, but the mechanism for this discrepancy has remained unknown. Here we present evidence of a complete respiratory arsenic cycle, consisting of dissimilatory As(V) reduction and chemoautotrophic As(III) oxidation, in the pelagic ocean. We identified the presence of genes encoding both subunits of the respiratory arsenite oxidase AioA and the dissimilatory arsenate reductase ArrA in the Eastern Tropical North Pacific (ETNP) oxygen-deficient zone (ODZ). The presence of the dissimilatory arsenate reductase gene arrA was enriched on large particles (>30 um), similar to the forward bacterial dsrA gene of sulfate-reducing bacteria, which is involved in the cryptic cycling of sulfur in ODZs. Arsenic respiratory genes were expressed in metatranscriptomic libraries from the ETNP and the Eastern Tropical South Pacific (ETSP) ODZ, indicating arsenotrophy is a metabolic pathway actively utilized in anoxic marine water columns. Together these results suggest arsenic-based metabolisms support organic matter production and impact nitrogen biogeochemical cycling in modern oceans. In early anoxic oceans, especially during periods of high marine arsenic concentrations, they may have played a much larger role.

Discovery of several novel, widespread, and ecologically distinct marine Thaumarchaeota viruses that encode amoC nitrification genes.

Much of the diversity of prokaryotic viruses has yet to be described. In particular, there are no viral isolates that infect abundant, globally significant marine archaea including the phylum Thaumarchaeota. This phylum oxidizes ammonia, fixes inorganic carbon, and thus contributes to globally significant nitrogen and carbon cycles in the oceans. Metagenomics provides an alternative to culture-dependent means for identifying and characterizing viral diversity. Some viruses carry auxiliary metabolic genes (AMGs) that are acquired via horizontal gene transfer from their host(s), allowing inference of what host a virus infects. Here we present the discovery of 15 new genomically and ecologically distinct Thaumarchaeota virus populations, identified as contigs that encode viral capsid and thaumarchaeal ammonia monooxygenase genes (amoC). These viruses exhibit depth and latitude partitioning and are distributed globally in various marine habitats including pelagic waters, estuarine habitats, and hydrothermal plume water and sediments. We found evidence of viral amoC expression and that viral amoC AMGs sometimes comprise up to half of total amoC DNA copies in cellular fraction metagenomes, highlighting the potential impact of these viruses on N cycling in the oceans. Phylogenetics suggest they are potentially tailed viruses and share a common ancestor with related marine Euryarchaeota viruses. This work significantly expands our view of viruses of globally important marine Thaumarchaeota.

Utilization of urea and cyanate in waters overlying and within the eastern tropical north Pacific oxygen deficient zone.

In marine oxygen deficient zones (ODZs), which contribute up to half of marine N loss, microbes use nitrogen (N) for assimilatory and dissimilatory processes. Here, we examine N utilization above and within the ODZ of the Eastern Tropical North Pacific Ocean, focusing on distribution, uptake and genes for the utilization of two simple organic N compounds, urea and cyanate. Ammonium, urea and cyanate concentrations generally peaked in the oxycline while uptake rates were highest in the surface. Within the ODZ, concentrations were lower, but urea N and C and cyanate C were taken up. All identified autotrophs had an N assimilation pathway that did not require external ammonium: ODZ Prochlorococcus possessed genes to assimilate nitrate, nitrite and urea; nitrite oxidizers (Nitrospina) possessed genes to assimilate nitrite, urea and cyanate; anammox bacteria (Scalindua) possessed genes to utilize cyanate; and ammonia-oxidizing Thaumarchaeota possessed genes to utilize urea. Urease genes were present in 20% of microbes, including SAR11, suggesting the urea utilization capacity was widespread. In the ODZ core, cyanate genes were largely (∼95%) associated with Scalindua, suggesting that, within this ODZ, cyanate N is primarily used for N loss via anammox (cyanammox), and that anammox does not require ammonium for N loss.

Niche Partitioning of the N Cycling Microbial Community of an Offshore Oxygen Deficient Zone.

Microbial communities in marine oxygen deficient zones (ODZs) are responsible for up to half of marine N loss through conversion of nutrients to N2O and N2. This N loss is accomplished by a consortium of diverse microbes, many of which remain uncultured. Here, we characterize genes for all steps in the anoxic N cycle in metagenomes from the water column and >30 µm particles from the Eastern Tropical North Pacific (ETNP) ODZ. We use an approach that allows for both phylogenetic identification and semi-quantitative assessment of gene abundances from individual organisms, and place these results in context of chemical measurements and rate data from the same location. Denitrification genes were enriched in >30 µm particles, even in the oxycline, while anammox bacteria were not abundant on particles. Many steps in denitrification were encoded by multiple phylotypes with different distributions. Notably three N2O reductases (nosZ), each with no cultured relative, inhabited distinct niches; one was free-living, one dominant on particles and one had a C terminal extension found in autotrophic S-oxidizing bacteria. At some depths >30% of the community possessed nitrite reductase nirK. A nirK OTU linked to SAR11 explained much of this abundance. The only bacterial gene found for NO reduction to N2O in the ODZ was a form of qnorB related to the previously postulated "nitric oxide dismutase," hypothesized to produce N2 directly while oxidizing methane. However, similar qnorB-like genes are also found in the published genomes of many bacteria that do not oxidize methane, and here the qnorB-like genes did not correlate with the presence of methane oxidation genes. Correlations with N2O concentrations indicate that these qnorB-like genes likely facilitate NO reduction to N2O in the ODZ. In the oxycline, qnorB-like genes were not detected in the water column, and estimated N2O production rates from ammonia oxidation were insufficient to support the observed oxycline N2O maximum. However, both qnorB-like and nosZ genes were present within particles in the oxycline, suggesting a particulate source of N2O and N2. Together, our analyses provide a holistic view of the diverse players in the low oxygen nitrogen cycle.

Effect of the environment on horizontal gene transfer between bacteria and archaea.

BACKGROUND: Horizontal gene transfer, the transfer and incorporation of genetic material between different species of organisms, has an important but poorly quantified role in the adaptation of microbes to their environment. Previous work has shown that genome size and the number of horizontally transferred genes are strongly correlated. Here we consider how genome size confuses the quantification of horizontal gene transfer because the number of genes an organism accumulates over time depends on its evolutionary history and ecological context (e.g., the nutrient regime for which it is adapted). RESULTS: We investigated horizontal gene transfer between archaea and bacteria by first counting reciprocal BLAST hits among 448 bacterial and 57 archaeal genomes to find shared genes. Then we used the DarkHorse algorithm, a probability-based, lineage-weighted method (Podell & Gaasterland, 2007), to identify potential horizontally transferred genes among these shared genes. By removing the effect of genome size in the bacteria, we have identified bacteria with unusually large numbers of shared genes with archaea for their genome size. Interestingly, archaea and bacteria that live in anaerobic and/or high temperature conditions are more likely to share unusually large numbers of genes. However, high salt was not found to significantly affect the numbers of shared genes. Numbers of shared (genome size-corrected, reciprocal BLAST hits) and transferred genes (identified by DarkHorse) were strongly correlated. Thus archaea and bacteria that live in anaerobic and/or high temperature conditions are more likely to share horizontally transferred genes. These horizontally transferred genes are over-represented by genes involved in energy conversion as well as the transport and metabolism of inorganic ions and amino acids. CONCLUSIONS: Anaerobic and thermophilic bacteria share unusually large numbers of genes with archaea. This is mainly due to horizontal gene transfer of genes from the archaea to the bacteria. In general, these transfers are from archaea that live in similar oxygen and temperature conditions as the bacteria that receive the genes. Potential hotspots of horizontal gene transfer between archaea and bacteria include hot springs, marine sediments, and oil wells. Cold spots for horizontal transfer included dilute, aerobic, mesophilic environments such as marine and freshwater surface waters.

Concurrent activity of anammox and denitrifying bacteria in the Black Sea.

After the discovery of ANaerobic AMMonium OXidation (anammox) in the environment, the role of heterotrophic denitrification as the main marine pathway for fixed N loss has been questioned. A 3 part, 15 month time series investigating nitrite reductase (nirS) mRNA transcripts at a single location in the Black Sea was conducted in order to better understand the activity of anammox and denitrifying bacteria. Here we show that both of these groups were active, as well as being concurrent in the lower suboxic zone over this time span. Their distributions, however, differed in that only expression of denitrification-type nirS was seen in the upper suboxic zone, where geochemistry was variable. Depth profiles covering the suboxic zone showed that the four groups of anammox-type sequences were expressed consistently in the lower suboxic zone, and were consistent with anammox 16 S rDNA gene profiles. By contrast, denitrifier-type nirS sequence groups were mixed; some groups exhibited consistent expression in the lower suboxic zone, while others appeared less consistent. Co-occurrence of both anammox and denitrifier expression was common and ongoing. Both types of transcripts were also found in samples with low concentrations of sulfide (>2 µM). Six major groups of denitrifier-type nirS transcripts were identified, and several groups of denitrifier-type nirS transcripts were closely related to sequences from the Baltic Sea. An increase in denitrifier-type nirS transcript diversity and depth range in October 2007 corresponded to a small increase in mixed layer net community productivity (NCP) as measured by O(2)/Ar gas ratios, as well as to an increase in N(2) concentrations in the suboxic zone. Taken together, the variations in expression patterns between anammox and denitrification provide one possible explanation as to how near instantaneous rate measurements, such as isotope spike experiments, may regularly detect anammox activity but underreport denitrification.

Stimulation of autotrophic denitrification by intrusions of the bosporus plume into the anoxic black sea.

Autotrophic denitrification was measured in the southwestern coastal Black Sea, where the Bosporus Plume injects oxidized chemical species (especially O(2) and [Formula: see text]) into the oxic, suboxic, and anoxic layers. Prominent oxygen intrusions caused an overlap of [Formula: see text] and sulfide at the same station where autotrophic denitrification activity was detected with incubation experiments. Several bacteria that have been proposed to oxidize sulfide in other low oxygen environments were found in the Black Sea including SUP05, Sulfurimonas, Arcobacter, and BS-GSO2. Comparison of TRFLP profiles from this mixing zone station and the Western Gyre (a station not affected by the Bosporus Plume) indicate the greatest relative abundance of Sulfurimonas and Arcobacter at the appropriate depths at the mixing zone station. The autotrophic gammaproteobacterium BS-GSO2 correlated with ammonium fluxes rather than with sulfide fluxes and the maximum in SUP05 peak height was shallower than the depths where autotrophic denitrification was detected. Notably, anammox activity was not detected at the mixing zone station, though low levels of DNA from the anammox bacteria CandidatusScalindua were present. These results provide evidence for a modified ecosystem with different N(2) production pathways in the southwest coastal region compared to that found in the rest of the Black Sea. Moreover, the same Sulfurimonas phylotype (BS139) was previously detected on >30 µm particles in the suboxic zone of the Western Gyre along with DNA of potential sulfate reducers, so it is possible that particle-attached autotrophic denitrification may be an overlooked N(2) production pathway in the central Black Sea as well.

Free-living and aggregate-associated Planctomycetes in the Black Sea.

We examined the distribution of uncultured Planctomycetes phylotypes along depth profiles spanning the redox gradient of the Black Sea suboxic zone to gain insight into their respective ecological niches. Planctomycetes phylogeny correlated with depth and chemical profiles, implying similar metabolisms within phylogenetic groups. A suboxic zone sample was split into > 30 and < 30 µm fractions to examine putative aggregate-attached and free-living Planctomycetes. All identified Planctomycetes were present in the > 30 µm fraction except for members of the Scalindua genus, which were apparently free-living. Sequences from Candidatus Scalindua, known to carry out the anammox process, formed two distinct clusters with nonoverlapping depth ranges. One cluster, only 97.1% similar to the named species, was present at high nitrite/nitrate and low ammonium concentrations in the upper suboxic zone. We propose this sequence type be named 'Candidatus Scalindua richardsii'. A second cluster, containing sequences more similar to 'Candidatus Scalindua sorokinii', was present at high ammonium and low nitrite conditions in the lower suboxic zone. Sequences obtained from the sulfidic zone (1000 m depth) yielded Planctomycetes from two uncharacterized Planctomycetacia clusters and three potentially new genera as well as sequences from the uncultured OP3 phylum.

Metabolic strategies of free-living and aggregate-associated bacterial communities inferred from biologic and chemical profiles in the Black Sea suboxic zone.

The Black Sea is a permanently anoxic basin with a well-defined redox gradient. We combine environmental 16S rRNA gene data from clone libraries, terminal restriction fragment length polymorphisms, and V6 hypervariable region pyrosequences to provide the most detailed bacterial survey to date. Furthermore, this data set is informed by comprehensive geochemical data; using this combination of information, we put forward testable hypotheses regarding possible metabolisms of uncultured bacteria from the Black Sea's suboxic zone (microaerophily, nitrate reduction, manganese cycling, and oxidation of methane, ammonium, and sulfide). Dominant bacteria in the upper suboxic zone included members of the SAR11, SAR324, and Microthrix groups and in the deep suboxic zone included members of BS-GSO-2, Marine Group A, and SUP05. A particulate fraction (30 µm filter) was used to distinguish between free-living and aggregate-attached communities in the suboxic zone. The particulate fraction contained greater diversity of V6 tag sequences than the bulk water samples. Lentisphaera, Epsilonproteobacteria, WS3, Planctomycetes, and Deltaproteobacteria were enriched in the particulate fraction, whereas SAR11 relatives dominated the free-living fraction. On the basis of the bacterial assemblages and simple modeling, we find that in suboxic waters, the interior of sinking aggregates potentially support manganese reduction, sulfate reduction, and sulfur oxidation.

Analysis of nitrite reductase (nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea suboxic zone.

Chemical profiles of the Black Sea suboxic zone show a distribution of nitrogen species which is traditionally associated with denitrification, i.e. a secondary nitrite maximum associated with nitrate depletion and a N(2) gas peak. To better understand the distribution and diversity of the denitrifier community in the Black Sea suboxic zone, we combined a cultivation approach with cloning and sequencing of PCR-amplified nitrite reductase (nirS and nirK) genes. The Black Sea suboxic zone appears to harbour a homogeneous community of denitrifiers. For nirK, over 94% of the sequences fell into only three distinct phylogenetic clusters, and for nirS, a single closely related sequence type accounted for 91% of the sequences retrieved. Both nirS and nirK genes showed a dramatic shift in community composition at the bottom of the suboxic zone, but overall, nirK-based community composition showed much greater variation across depths compared with the highly uniform distribution of nirS sequences throughout the suboxic zone. The dominant nirK and nirS sequences differed at the amino acid level by at least 17% and 8%, respectively, from their nearest database matches. Denitrifying isolates recovered from the suboxic zone shared 97% 16S rRNA gene sequence similarity with Marinobacter maritimus. Analysis of the recently discovered nirS gene from the anammox bacterium Candidatus'Kuenenia stuttgartiensis' revealed that mismatches with commonly used primers may have prevented the previous detection of this divergent sequence.

Whole-genome reciprocal BLAST analysis reveals that planctomycetes do not share an unusually large number of genes with Eukarya and Archaea.

The genome sequences of Rhodopirellula baltica, formerly Pirellula sp. strain 1, Blastopirellula marina, Gemmata obscuriglobus, and Kuenenia stuttgartiensis were used in a series of pairwise reciprocal best-hit analyses to evaluate the contested evolutionary position of Planctomycetes. Contrary to previous reports which suggested that R. baltica had a high percentage of genes with closest matches to Archaea and Eukarya, we show here that these Planctomycetes do not share an unusually large number of genes with the Archaea or Eukarya, compared with other Bacteria. Thus, best-hit analyses may assign phylogenetic affinities incorrectly if close relatives are absent from the sequence database.