Cory C. Padilla

NC10 bacteria in marine oxygen minimum zones

Bacteria of the NC10 phylum link anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophy pathway. A niche for NC10 in the pelagic ocean has not been confirmed. We show that NC10 bacteria are present and transcriptionally active in oceanic oxygen minimum zones (OMZs) off northern Mexico and Costa Rica. NC10 16S rRNA genes were detected at all sites, peaking in abundance in the anoxic zone with elevated nitrite and methane concentrations. Phylogenetic analysis of particulate methane monooxygenase genes further confirmed the presence of NC10. rRNA and mRNA transcripts assignable to NC10 peaked within the OMZ and included genes of the putative nitrite-dependent intra-aerobic pathway, with high representation of transcripts containing the unique motif structure of the nitric oxide (NO) reductase of NC10 bacteria, hypothesized to participate in O2-producing NO dismutation. These findings confirm pelagic OMZs as a niche for NC10, suggesting a role for this group in OMZ nitrogen, methane and oxygen cycling.

SAR11 bacteria linked to ocean anoxia and nitrogen loss

Bacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. SAR11 bacteria are also abundant in oxygen minimum zones (OMZs), where oxygen falls below detection and anaerobic microbes have vital roles in converting bioavailable nitrogen to N2 gas. Anaerobic metabolism has not yet been observed in SAR11, and it remains unknown how these bacteria contribute to OMZ biogeochemical cycling. Here, genomic analysis of single cells from the world’s largest OMZ revealed previously uncharacterized SAR11 lineages with adaptations for life without oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalysing the nitrite-producing first step of denitrification and constituted ~40% of OMZ nar transcripts, with transcription peaking in the anoxic zone of maximum nitrate reduction activity. These results link SAR11 to pathways of ocean nitrogen loss, redefining the ecological niche of Earth’s most abundant organismal group.

Standard filtration practices may significantly distort planktonic microbial diversity estimates

Fractionation of biomass by filtration is a standard method for sampling planktonic microbes. It is unclear how the taxonomic composition of filtered biomass changes depending on sample volume. Using seawater from a marine oxygen minimum zone, we quantified the 16S rRNA gene composition of biomass on a prefilter (1.6 μm pore-size) and a downstream 0.2 μm filter over sample volumes from 0.05 to 5 L. Significant community shifts occurred in both filter fractions, and were most dramatic in the prefilter community. Sequences matching Vibrionales decreased from ~40 to 60% of prefilter datasets at low volumes (0.05–0.5 L) to less than 5% at higher volumes, while groups such at the Chromatiales and Thiohalorhabdales followed opposite trends, increasing from minor representation to become the dominant taxa at higher volumes. Groups often associated with marine particles, including members of the Deltaproteobacteria, Planctomycetes, and Bacteroidetes, were among those showing the greatest increase with volume (4 to 27-fold). Taxon richness (97% similarity clusters) also varied significantly with volume, and in opposing directions depending on filter fraction, highlighting potential biases in community complexity estimates. These data raise concerns for studies using filter fractionation for quantitative comparisons of aquatic microbial diversity, for example between free-living and particle-associated communities.

Cryptic oxygen cycling in anoxic marine zones

Significance Anoxic marine zones (AMZs) create expansive habitats for microbes whose anaerobic metabolisms help drive global nutrient cycles, for example, by removing nitrogen from the oceans by producing N2 gas. AMZ cycles may also be shaped by oxygen intrusion from outside the AMZ, creating opportunities for aerobic microbial metabolisms. Here we show that aerobic processes in AMZs are linked to oxygen production within the anoxic zone. Oxygen is produced during daytime in a layer of photosynthetic cyanobacteria near the top of the AMZ and then rapidly consumed by aerobic processes without accumulating. Oxygen turnover and carbon fixation rates are comparable to those of microbial N2 production, suggesting an important role for internal oxygen cycling in AMZ transformations of matter and energy. Oxygen availability drives changes in microbial diversity and biogeochemical cycling between the aerobic surface layer and the anaerobic core in nitrite-rich anoxic marine zones (AMZs), which constitute huge oxygen-depleted regions in the tropical oceans. The current paradigm is that primary production and nitrification within the oxic surface layer fuel anaerobic processes in the anoxic core of AMZs, where 30–50% of global marine nitrogen loss takes place. Here we demonstrate that oxygenic photosynthesis in the secondary chlorophyll maximum (SCM) releases significant amounts of O2 to the otherwise anoxic environment. The SCM, commonly found within AMZs, was dominated by the picocyanobacteria Prochlorococcus spp. Free O2 levels in this layer were, however, undetectable by conventional techniques, reflecting a tight coupling between O2 production and consumption by aerobic processes under apparent anoxic conditions. Transcriptomic analysis of the microbial community in the seemingly anoxic SCM revealed the enhanced expression of genes for aerobic processes, such as nitrite oxidation. The rates of gross O2 production and carbon fixation in the SCM were found to be similar to those reported for nitrite oxidation, as well as for anaerobic dissimilatory nitrate reduction and sulfate reduction, suggesting a significant effect of local oxygenic photosynthesis on Pacific AMZ biogeochemical cycling.

Metagenomic Binning Recovers a Transcriptionally Active Gammaproteobacterium Linking Methanotrophy to Partial Denitrification in an Anoxic Oxygen Minimum Zone

Diverse planktonic microorganisms play a crucial role in mediating methane flux from the ocean to the atmosphere. The distribution and composition of the marine methanotroph community is determined partly by oxygen availability. The low oxygen conditions of oxygen minimum zones (OMZs) may select for methanotrophs that oxidize methane using inorganic nitrogen compounds (e.g., nitrate, nitrite) in place of oxygen. However, environmental evidence for methane-nitrogen linkages in OMZs remains sparse, as does our knowledge of the genomic content and metabolic capacity of organisms catalyzing OMZ methane oxidation. Here, binning of metagenome sequences from a coastal anoxic OMZ recovered the first near complete (95%) draft genome representing the methanotroph clade OPU3. Phylogenetic reconstruction of concatenated single copy marker genes confirmed the OPU3-like bacterium as a divergent member of the type Ia methanotrophs, with an estimated genome size half that of other sequenced taxa in this group. The proportional abundance of this bacterium peaked at 4% of the total microbial community at the top of the anoxic zone in areas of nitrite and nitrate availability but declining methane concentrations. Genes mediating dissimilatory nitrate and nitrite reduction were identified in the OPU3 genome, and transcribed in conjunction with key enzymes catalyzing methane oxidation to formaldehyde and the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation, suggesting partial denitrification linked to methane oxidation. Together, these data provide the first field-based evidence for methanotrophic partial denitrification by the OPU3 cluster under anoxic conditions, supporting a role for OMZs as key sites in pelagic methane turnover.

Putative archaeal viruses from the mesopelagic ocean

Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce “MArVD”, for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments.

Single cell genomic and transcriptomic evidence for the use of alternative nitrogen substrates by anammox bacteria

Anaerobic ammonium oxidation (anammox) contributes substantially to ocean nitrogen loss, particularly in anoxic marine zones (AMZs). Ammonium is scarce in AMZs, raising the hypothesis that organic nitrogen compounds may be ammonium sources for anammox. Biochemical measurements suggest that the organic compounds urea and cyanate can support anammox in AMZs. However, it is unclear if anammox bacteria degrade these compounds to ammonium themselves, or rely on other organisms for this process. Genes for urea degradation have not been found in anammox bacteria, and genomic evidence for cyanate use for anammox is limited to a cyanase gene recovered from the sediment bacterium Candidatus Scalindua profunda. Here, analysis of Ca. Scalindua single amplified genomes from the Eastern Tropical North Pacific AMZ revealed genes for urea degradation and transport, as well as for cyanate degradation. Urease and cyanase genes were transcribed, along with anammox genes, in the AMZ core where anammox rates peaked. Homologs of these genes were also detected in meta-omic datasets from major AMZs in the Eastern Tropical South Pacific and Arabian Sea. These results suggest that anammox bacteria from different ocean regions can directly access organic nitrogen substrates. Future studies should assess if and under what environmental conditions these substrates contribute to the ammonium budget for anammox.

Piecewise Structural Equation Model (SEM) Disentangles the Environmental Conditions Favoring Diatom Diazotroph Associations (DDAs) in the Western Tropical North Atlantic (WTNA)

Diatom diazotroph associations (DDAs) are important components in the world’s oceans, especially in the western tropical north Atlantic (WTNA), where blooms have a significant impact on carbon and nitrogen cycling. However, drivers of their abundances and distribution patterns remain unknown. Here, we examined abundance and distribution patterns for two DDA populations in relation to the Amazon River (AR) plume in the WTNA. Quantitative PCR assays, targeting two DDAs (het-1 and het-2) by their symbiont’s nifH gene, served as input in a piecewise structural equation model (SEM). Collections were made during high (spring 2010) and low (fall 2011) flow discharges of the AR. The distributions of dissolved nutrients, chlorophyll-a, and DDAs showed coherent patterns indicative of areas influenced by the AR. A symbiotic Hemiaulus hauckii-Richelia (het-2) bloom (>106 cells L-1) occurred during higher discharge of the AR and was coincident with mesohaline to oceanic (30–35) sea surface salinities (SSS), and regions devoid of dissolved inorganic nitrogen (DIN), low concentrations of both DIP (>0.1 μmol L-1) and Si (>1.0 μmol L-1). The Richelia (het-1) associated with Rhizosolenia was only present in 2010 and at lower densities (10-1.76 × 105 nifH copies L-1) than het-2 and limited to regions of oceanic SSS (>36). The het-2 symbiont detected in 2011 was associated with H. membranaceus (>103 nifH copies L-1) and were restricted to regions with mesohaline SSS (31.8–34.3), immeasurable DIN, moderate DIP (0.1–0.60 μmol L-1) and higher Si (4.19–22.1 μmol L-1). The piecewise SEM identified a profound direct negative effect of turbidity on the het-2 abundance in spring 2010, while DIP and water turbidity had a more positive influence in fall 2011, corroborating our observations of DDAs at subsurface maximas. We also found a striking difference in the influence of salinity on DDA symbionts suggesting a niche differentiation and preferences in oceanic and mesohaline salinities by het-1 and het-2, respectively. The use of the piecewise SEM to disentangle the complex and concomitant hydrography of the WTNA acting on two biogeochemically relevant populations was novel and underscores its use to predict conditions favoring abundance and distributions of microbial populations.

Metabolic Analysis of Metatranscriptomic Data from Planktonic Communities

This paper describes an enhanced method for analyzing microbial metatranscriptomic (community RNA-seq) data using Expectation - Maximization (EM)-based differentiation and quantification of predicted gene, enzyme, and metabolic pathway activity. Here, we demonstrate the method by analyzing the metatranscriptome of planktonic communities in surface waters from the Northern Louisiana Shelf (Gulf of Mexico) during contrasting light and dark conditions. The analysis reveals that the level of transcripts encoding proteins of oxidative phosphorylation varys little between day and night. In contrast, transcripts of pyrimidine metabolism are significantly more abundant at night, whereas those of carbon fixation by photosynthetic organisms increase 2-fold in abundance from night to day.