Microbial functional diversity across biogeochemical provinces in the central Pacific Ocean.

Enzymes catalyze key reactions within Earth's life-sustaining biogeochemical cycles. Here, we use metaproteomics to examine the enzymatic capabilities of the microbial community (0.2 to 3 µm) along a 5,000-km-long, 1-km-deep transect in the central Pacific Ocean. Eighty-five percent of total protein abundance was of bacterial origin, with Archaea contributing 1.6%. Over 2,000 functional KEGG Ontology (KO) groups were identified, yet only 25 KO groups contributed over half of the protein abundance, simultaneously indicating abundant key functions and a long tail of diverse functions. Vertical attenuation of individual proteins displayed stratification of nutrient transport, carbon utilization, and environmental stress. The microbial community also varied along horizontal scales, shaped by environmental features specific to the oligotrophic North Pacific Subtropical Gyre, the oxygen-depleted Eastern Tropical North Pacific, and nutrient-rich equatorial upwelling. Some of the most abundant proteins were associated with nitrification and C1 metabolisms, with observed interactions between these pathways. The oxidoreductases nitrite oxidoreductase (NxrAB), nitrite reductase (NirK), ammonia monooxygenase (AmoABC), manganese oxidase (MnxG), formate dehydrogenase (FdoGH and FDH), and carbon monoxide dehydrogenase (CoxLM) displayed distributions indicative of biogeochemical status such as oxidative or nutritional stress, with the potential to be more sensitive than chemical sensors. Enzymes that mediate transformations of atmospheric gases like CO, CO2, NO, methanethiol, and methylamines were most abundant in the upwelling region. We identified hot spots of biochemical transformation in the central Pacific Ocean, highlighted previously understudied metabolic pathways in the environment, and provided rich empirical data for biogeochemical models critical for forecasting ecosystem response to climate change.

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.

Development of an Ocean Protein Portal for Interactive Discovery and Education.

Proteins are critical in catalyzing chemical reactions, forming key cellular structures, and in regulating cellular processes. Investigation of marine microbial proteins by metaproteomics methods enables the discovery of numerous aspects of microbial biogeochemical processes. However, these datasets present big data challenges as they often involve many samples collected across broad geospatial and temporal scales, resulting in thousands of protein identifications, abundances, and corresponding annotation information. The Ocean Protein Portal (OPP) was created to enable data sharing and discovery among multiple scientific domains and serve both research and education functions. The portal focuses on three use case questions: "Where is my protein of interest?", "Who makes it?", and "How much is there?" and provides profile and section visualizations, real-time taxonomic analysis, and links to metadata, sequence analysis, and other external resources to enable connections to be made between biogeochemical and proteomics datasets.

METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies-Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses.

We present METATRYP version 2 software that identifies shared peptides across the predicted proteomes of organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine METATRYP database with predicted proteomes from environmental sequencing confirms a low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed to facilitate ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa analyzed, sharing <1% peptides with taxa outside of the betacoronavirus group, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.

Progress and Challenges in Ocean Metaproteomics and Proposed Best Practices for Data Sharing.

Ocean metaproteomics is an emerging field enabling discoveries about marine microbial communities and their impact on global biogeochemical processes. Recent ocean metaproteomic studies have provided insight into microbial nutrient transport, colimitation of carbon fixation, the metabolism of microbial biofilms, and dynamics of carbon flux in marine ecosystems. Future methodological developments could provide new capabilities such as characterizing long-term ecosystem changes, biogeochemical reaction rates, and in situ stoichiometries. Yet challenges remain for ocean metaproteomics due to the great biological diversity that produces highly complex mass spectra, as well as the difficulty in obtaining and working with environmental samples. This review summarizes the progress and challenges facing ocean metaproteomic scientists and proposes best practices for data sharing of ocean metaproteomic data sets, including the data types and metadata needed to enable intercomparisons of protein distributions and annotations that could foster global ocean metaproteomic capabilities.

Effects of phosphorus starvation versus limitation on the marine cyanobacterium Prochlorococcus†MED4 I: uptake physiology.

Recent measurements of natural populations of the marine cyanobacterium Prochlorococcus indicate this numerically dominant phototroph assimilates phosphorus (P) at significant rates in P-limited oceanic regions. To better understand uptake capabilities of Prochlorococcus under different P stress conditions, uptake kinetic experiments were performed on Prochlorococcus MED4 grown in P-limited chemostats and batch cultures. Our results indicate that MED4 has a small cell-specific Vmax but a high specific affinity (αP ) for P, making it competitive with other marine cyanobacteria at low P concentrations. Additionally, MED4 regulates its uptake kinetics in response to P stress by significantly increasing Vmax and αP for both inorganic and organic P (PO4 and ATP). The Michaelis-Menten constant, KM , for PO4 remained constant under different P stress conditions, whereas the KM for ATP was higher when cells were stressed for PO4 , pointing to additional processes involved in uptake of ATP. MED4 cleaves the PO4 moieties from ATP, likely with a 5'-nucleotidase-like enzyme rather than alkaline phosphatase. MED4 exhibited distinct physiological differences between cells under steady-state P limitation versus those transitioning from P-replete to P-starved conditions. Thus, MED4 employs a variety of strategies to deal with changing P sources in the oceans and displays complexity in P stress acclimation and regulatory mechanisms.

Effects of phosphorus starvation versus limitation on the marine cyanobacterium Prochlorococcus†MED4 II: gene expression.

Phosphorus (P) availability drives niche differentiation in the most abundant phytoplankter in the oceans, the marine cyanobacterium Prochlorococcus. We compared the molecular response of Prochlorococcus strain MED4 to P starvation in batch culture to P-limited growth in chemostat culture. We also identified an outer membrane porin, PMM0709, which may allow transport of organic phosphorous compounds, rather than phosphate as previously suggested. The expression of three P uptake genes, pstS, the high-affinity phosphate-binding component of the phosphate transporter, phoA, an alkaline phosphatase, and porin PMM0709, were strongly upregulated (between 10- and 700-fold) under both P starvation and limitation. pstS exhibits high basal expression under P-replete conditions and is likely necessary for P uptake regardless of P availability. A P-stress regulatory gene, ptrA, was upregulated in response to both P starvation and limitation although a second regulatory gene, phoB, was not. Elevated expression levels (> 10-fold) of phoR, a P-sensing histidine kinase, were only observed under conditions of P limitation. We suggest Prochlorococcus in P-limited systems are physiologically distinct from cells subjected to abrupt P depletion. Detection of expression of both pstS and phoR in field populations will enable discernment of the present P status of Prochlorococcus in the oligotrophic oceans.

Dinoflagellates alter their carbon and nutrient metabolic strategies across environmental gradients in the central Pacific Ocean.

Marine microeukaryotes play a fundamental role in biogeochemical cycling through the transfer of energy to higher trophic levels and vertical carbon transport. Despite their global importance, microeukaryote physiology, nutrient metabolism and contributions to carbon cycling across offshore ecosystems are poorly characterized. Here, we observed the prevalence of dinoflagellates along a 4,600-km meridional transect extending across the central Pacific Ocean, where oligotrophic gyres meet equatorial upwelling waters rich in macronutrients yet low in dissolved iron. A combined multi-omics and geochemical analysis provided a window into dinoflagellate metabolism across the transect, indicating a continuous taxonomic dinoflagellate community that shifted its functional transcriptome and proteome as it extended from the euphotic to the mesopelagic zone. In euphotic waters, multi-omics data suggested that a combination of trophic modes were utilized, while mesopelagic metabolism was marked by cytoskeletal investments and nutrient recycling. Rearrangement in nutrient metabolism was evident in response to variable nitrogen and iron regimes across the gradient, with no associated change in community assemblage. Total dinoflagellate proteins scaled with particulate carbon export, with both elevated in equatorial waters, suggesting a link between dinoflagellate abundance and total carbon flux. Dinoflagellates employ numerous metabolic strategies that enable broad occupation of central Pacific ecosystems and play a dual role in carbon transformation through both photosynthetic fixation in the euphotic zone and remineralization in the mesopelagic zone.

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.

Genomic potential for arsenic efflux and methylation varies among global Prochlorococcus populations.

The globally significant picocyanobacterium Prochlorococcus is the main primary producer in oligotrophic subtropical gyres. When phosphate concentrations are very low in the marine environment, the mol:mol availability of phosphate relative to the chemically similar arsenate molecule is reduced, potentially resulting in increased cellular arsenic exposure. To mediate accidental arsenate uptake, some Prochlorococcus isolates contain genes encoding a full or partial efflux detoxification pathway, consisting of an arsenate reductase (arsC), an arsenite-specific efflux pump (acr3) and an arsenic-related repressive regulator (arsR). This efflux pathway was the only previously known arsenic detox pathway in Prochlorococcus. We have identified an additional putative arsenic mediation strategy in Prochlorococcus driven by the enzyme arsenite S-adenosylmethionine methyltransferase (ArsM) which can convert inorganic arsenic into more innocuous organic forms and appears to be a more widespread mode of detoxification. We used a phylogenetically informed approach to identify Prochlorococcus linked arsenic genes from both pathways in the Global Ocean Sampling survey. The putative arsenic methylation pathway is nearly ubiquitously present in global Prochlorococcus populations. In contrast, the complete efflux pathway is only maintained in populations which experience extremely low PO4:AsO4, such as regions in the tropical and subtropical Atlantic. Thus, environmental exposure to arsenic appears to select for maintenance of the efflux detoxification pathway in Prochlorococcus. The differential distribution of these two pathways has implications for global arsenic cycling, as their associated end products, arsenite or organoarsenicals, have differing biochemical activities and residence times.