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.

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.

Nitrification plays a key role in the marine nitrogen (N) cycle, including in oceanic oxygen minimum zones (OMZs), which are hot spots for denitrification and anaerobic ammonia oxidation (anammox). Recent evidence suggests that nitrification links the source (remineralized organic matter) and sink (denitrification and anammox) of fixed N directly in the steep oxycline in the OMZs. We performed shipboard incubations with 15N tracers to characterize the depth distribution of nitrification in the Eastern Tropical North Pacific (ETNP). Additional experiments were conducted to investigate photoinhibition. Allylthiourea (ATU) was used to distinguish the contribution of archaeal and bacterial ammonia oxidation. The abundance of archaeal and β‐proteobacterial ammonia monooxygenase gene subunit A (amoA) was determined by quantitative polymerase chain reaction. The rates of ammonia and nitrite oxidation showed distinct subsurface maxima, with the latter slightly deeper than the former. The ammonia oxidation maximum coincided with the primary nitrite concentration maximum, archaeal amoA gene maximum, and the subsurface nitrous oxide maximum. Negligible rates of ammonia oxidation were found at anoxic depths, where high rates of nitrite oxidation were measured. Archaeal amoA gene abundance was generally 1 to 2 orders of magnitude higher than bacterial amoA gene abundance, and inhibition of ammonia‐oxidizing bacteria with 10 μM ATU did not affect ammonia oxidation rates, indicating the dominance of archaea in ammonia oxidation. These results depict highly dynamic activities of ammonia and nitrite oxidation in the oxycline of the ETNP OMZ.

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.

Nitrification, the oxidation of ammonium ( NH4+) to nitrite ( NO2−) and to nitrate ( NO3−), is a component of the nitrogen (N) cycle internal to the fixed N pool. In oxygen minimum zones (OMZs), which are hotspots for oceanic fixed N loss, nitrification plays a key role because it directly supplies substrates for denitrification and anaerobic ammonia oxidation (anammox), and may compete for substrates with these same processes. However, the control of oxygen and substrate concentrations on nitrification are not well understood. We performed onboard incubations with 15N‐labeled substrates to measure rates of NH4+ and NO2− oxidation in the eastern tropical South Pacific (ETSP). The spatial and depth distributions of NH4+ and NO2− oxidation rates were primarily controlled by NH4+ and NO2− availability, oxygen concentration, and light. In the euphotic zone, nitrification was partially photoinhibited. In the anoxic layer, NH4+ oxidation was negligible or below detection, but high rates of NO2− oxidation were observed. NH4+ oxidation displayed extremely high affinity for both NH4+ and oxygen. The positive linear correlations between NH4+ oxidation rates and in situ NH4+ concentrations and ammonia monooxygenase subunit A (amoA) gene abundances in the upper oxycline indicate that the natural assemblage of ammonia oxidizers responds to in situ NH4+ concentrations or supply by adjusting their population size, which determines the NH4+ oxidation potential. The depth distribution of archaeal and bacterial amoA gene abundances and N2O concentration, along with independently reported simultaneous direct N2O production rate measurements, suggests that AOA were predominantly responsible for NH4+ oxidation, which was a major source of N2O production at oxygen concentrations > 5 µM.

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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.

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.