Flexible B12 ecophysiology of Phaeocystis antarctica due to a fusion B12-independent methionine synthase with widespread homologues.

Coastal Antarctic marine ecosystems are significant in carbon cycling because of their intense seasonal phytoplankton blooms. Southern Ocean algae are primarily limited by light and iron (Fe) and can be co-limited by cobalamin (vitamin B12). Micronutrient limitation controls productivity and shapes the composition of blooms which are typically dominated by either diatoms or the haptophyte Phaeocystis antarctica. However, the vitamin requirements and ecophysiology of the keystone species P. antarctica remain poorly characterized. Using cultures, physiological analysis, and comparative omics, we examined the response of P. antarctica to a matrix of Fe-B12 conditions. We show that P. antarctica is not auxotrophic for B12, as previously suggested, and identify mechanisms underlying its B12 response in cultures of predominantly solitary and colonial cells. A combination of proteomics and proteogenomics reveals a B12-independent methionine synthase fusion protein (MetE-fusion) that is expressed under vitamin limitation and interreplaced with the B12-dependent isoform under replete conditions. Database searches return homologues of the MetE-fusion protein in multiple Phaeocystis species and in a wide range of marine microbes, including other photosynthetic eukaryotes with polymorphic life cycles as well as bacterioplankton. Furthermore, we find MetE-fusion homologues expressed in metaproteomic and metatranscriptomic field samples in polar and more geographically widespread regions. As climate change impacts micronutrient availability in the coastal Southern Ocean, our finding that P. antarctica has a flexible B12 metabolism has implications for its relative fitness compared to B12-auxotrophic diatoms and for the detection of B12-stress in a more diverse set of marine microbes.

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.

Why Environmental Biomarkers Work: Transcriptome-Proteome Correlations and Modeling of Multistressor Experiments in the Marine Bacterium Trichodesmium.

Ocean microbial communities are important contributors to the global biogeochemical reactions that sustain life on Earth. The factors controlling these communities are being increasingly explored using metatranscriptomic and metaproteomic environmental biomarkers. Using published proteomes and transcriptomes from the abundant colony-forming cyanobacterium Trichodesmium (strain IMS101) grown under varying Fe and/or P limitation in low and high CO2, we observed robust correlations of stress-induced proteins and RNAs (i.e., involved in transport and homeostasis) that yield useful information on the nutrient status under low and/or high CO2. Conversely, transcriptional and translational correlations of many other central metabolism pathways exhibit broad discordance. A cellular RNA and protein production/degradation model demonstrates how biomolecules with small initial inventories, such as environmentally responsive proteins, achieve large increases in fold-change units as opposed to those with a higher basal expression and inventory such as metabolic systems. Microbial cells, due to their immersion in the environment, tend to show large adaptive responses in both RNA and protein that result in transcript-protein correlations. These observations and model results demonstrate multi-omic coherence for environmental biomarkers and provide the underlying mechanism for those observations, supporting the promise for global application in detecting responses to environmental stimuli in a changing ocean.

NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica.

Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.

Online Nanoflow Two-Dimension Comprehensive Active Modulation Reversed Phase-Reversed Phase Liquid Chromatography High-Resolution Mass Spectrometry for Metaproteomics of Environmental and Microbiome Samples.

Metaproteomics is a powerful analytical approach that can assess the functional capabilities deployed by microbial communities in both environmental and biomedical microbiome settings. Yet, the mass spectra resulting from these mixed biological communities are challenging to obtain due to the high number of low intensity peak features. The use of multiple dimensions of chromatographic separation prior to mass spectrometry analyses has been applied to proteomics previously but can require increased sampling handling and instrument time. Here, we demonstrate an automated online comprehensive active modulation two-dimensional liquid chromatography method for metaproteome sample analysis. A high pH PLRP-S column was used in the first dimension followed by low pH separation in the second dimension using dual modulating C18 traps and a C18 column. This method increased the number of unique peptides found in ocean metaproteome samples by more than 50% when compared to a one-dimension separation while using the same amount of sample and instrument time.

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.

Siderophore-based microbial adaptations to iron scarcity across the eastern Pacific Ocean.

Nearly all iron dissolved in the ocean is complexed by strong organic ligands of unknown composition. The effect of ligand composition on microbial iron acquisition is poorly understood, but amendment experiments using model ligands show they can facilitate or impede iron uptake depending on their identity. Here we show that siderophores, organic compounds synthesized by microbes to facilitate iron uptake, are a dynamic component of the marine ligand pool in the eastern tropical Pacific Ocean. Siderophore concentrations in iron-deficient waters averaged 9 pM, up to fivefold higher than in iron-rich coastal and nutrient-depleted oligotrophic waters, and were dominated by amphibactins, amphiphilic siderophores with cell membrane affinity. Phylogenetic analysis of amphibactin biosynthetic genes suggests that the ability to produce amphibactins has transferred horizontally across multiple Gammaproteobacteria, potentially driven by pressures to compete for iron. In coastal and oligotrophic regions of the eastern Pacific Ocean, amphibactins were replaced with lower concentrations (1-2 pM) of hydrophilic ferrioxamine siderophores. Our results suggest that organic ligand composition changes across the surface ocean in response to environmental pressures. Hydrophilic siderophores are predominantly found across regions of the ocean where iron is not expected to be the limiting nutrient for the microbial community at large. However, in regions with intense competition for iron, some microbes optimize iron acquisition by producing siderophores that minimize diffusive losses to the environment. These siderophores affect iron bioavailability and thus may be an important component of the marine iron cycle.

Transcriptomic and proteomic responses of the oceanic diatom Pseudo-nitzschia granii to iron limitation.

Diatoms are a highly successful group of photosynthetic protists that often thrive under adverse environmental conditions. Members of the genus Pseudo-nitzschia are ecologically important diatoms which are able to subsist during periods of chronic iron limitation and form dense blooms following iron fertilization events. The cellular strategies within diatoms that orchestrate these physiological responses to variable iron concentrations remain largely uncharacterized. Using a combined transcriptomic and proteomic approach, we explore the exceptional ability of a diatom isolated from the iron-limited Northeast Pacific Ocean to reorganize its intracellular processes as a function of iron. We compared the molecular responses of Pseudo-nitzschia granii observed under iron-replete and iron-limited growth conditions to those of other model diatoms. Iron-coordinated molecular responses demonstrated some agreement between gene expression and protein abundance, including iron-starvation-induced-proteins, a putative iron transport system and components of photosynthesis and the Calvin cycle. Pseudo-nitzschia granii distinctly differentially expresses genes encoding proteins involved in iron-independent photosynthetic electron transport, urea acquisition and vitamin synthesis. We show that P. granii is unique among studied diatoms in its physiology stemming from distinct cellular responses, which may underlie its ability to subsist in low iron regions and rapidly bloom to outcompete other diatom taxa following iron enrichment.

Divergent responses of Atlantic coastal and oceanic Synechococcus to iron limitation.

Marine Synechococcus are some of the most diverse and ubiquitous phytoplankton, and iron (Fe) is an essential micronutrient that limits productivity in many parts of the ocean. To investigate how coastal and oceanic Atlantic Synechococcus strains acclimate to Fe availability, we compared the growth, photophysiology, and quantitative proteomics of two Synechococcus strains from different Fe regimes. Synechococcus strain WH8102, from a region in the southern Sargasso Sea that receives substantial dust deposition, showed impaired growth and photophysiology as Fe declined, yet used few acclimation responses. Coastal WH8020, from the dynamic, seasonally variable New England shelf, displayed a multitiered, hierarchical cascade of acclimation responses with different Fe thresholds. The multitiered response included changes in Fe acquisition, storage, and photosynthetic proteins, substitution of flavodoxin for ferredoxin, and modified photophysiology, all while maintaining remarkably stable growth rates over a range of Fe concentrations. Modulation of two distinct ferric uptake regulator (Fur) proteins that coincided with the multitiered proteome response was found, implying the coastal strain has different regulatory threshold responses to low Fe availability. Low nitrogen (N) and phosphorus (P) availability in the open ocean may favor the loss of Fe response genes when Fe availability is consistent over time, whereas these genes are retained in dynamic environments where Fe availability fluctuates and N and P are more abundant.

Genomic and proteomic characterization of "Candidatus Nitrosopelagicus brevis": an ammonia-oxidizing archaeon from the open ocean.

Thaumarchaeota are among the most abundant microbial cells in the ocean, but difficulty in cultivating marine Thaumarchaeota has hindered investigation into the physiological and evolutionary basis of their success. We report here a closed genome assembled from a highly enriched culture of the ammonia-oxidizing pelagic thaumarchaeon CN25, originating from the open ocean. The CN25 genome exhibits strong evidence of genome streamlining, including a 1.23-Mbp genome, a high coding density, and a low number of paralogous genes. Proteomic analysis recovered nearly 70% of the predicted proteins encoded by the genome, demonstrating that a high fraction of the genome is translated. In contrast to other minimal marine microbes that acquire, rather than synthesize, cofactors, CN25 encodes and expresses near-complete biosynthetic pathways for multiple vitamins. Metagenomic fragment recruitment indicated the presence of DNA sequences >90% identical to the CN25 genome throughout the oligotrophic ocean. We propose the provisional name "Candidatus Nitrosopelagicus brevis" str. CN25 for this minimalist marine thaumarchaeon and suggest it as a potential model system for understanding archaeal adaptation to the open ocean.

Physiological and proteomic characterization of light adaptations in marine Synechococcus.

Marine Synechococcus thrive over a range of light regimes in the ocean. We examined the proteomic, genomic and physiological responses of seven Synechococcus isolates to moderate irradiances (5-80 µE m-2 s-1 ), and show that Synechococcus spans a continuum of light responses ranging from low light optimized (LLO) to high light optimized (HLO). These light responses are linked to phylogeny and pigmentation. Marine sub-cluster 5.1A isolates with higher phycouribilin: phycoerythrobilin ratios fell toward the LLO end of the continuum, while sub-cluster 5.1B, 5.2 and estuarine Synechococcus with less phycouribilin fell toward the HLO end of the continuum. Global proteomes were highly responsive to light, with > 50% of abundant proteins varying more than twofold between the lowest and highest irradiance. All strains downregulated phycobilisome proteins with increasing irradiance. Regulation of proteins involved in photosynthetic electron transport, carbon fixation, oxidative stress protection (superoxide dismutases) and iron and nitrogen metabolism varied among strains, as did the number of high light inducible protein (Hlip) and DNA photolyase genes in their genomes. All but one LLO strain possessed the photoprotective orange carotenoid protein (OCP). The unique combinations of light responses in each strain gives rise to distinct photophysiological phenotypes that may affect Synechococcus distributions in the ocean.

Needles in the blue sea: sub-species specificity in targeted protein biomarker analyses within the vast oceanic microbial metaproteome.

Proteomics has great potential for studies of marine microbial biogeochemistry, yet high microbial diversity in many locales presents us with unique challenges. We addressed this challenge with a targeted metaproteomics workflow for NtcA and P-II, two nitrogen regulatory proteins, and demonstrated its application for cyanobacterial taxa within microbial samples from the Central Pacific Ocean. Using METATRYP, an open-source Python toolkit, we examined the number of shared (redundant) tryptic peptides in representative marine microbes, with the number of tryptic peptides shared between different species typically being 1% or less. The related cyanobacteria Prochlorococcus and Synechococcus shared an average of 4.8 ± 1.9% of their tryptic peptides, while shared intraspecies peptides were higher, 13 ± 15% shared peptides between 12 Prochlorococcus genomes. An NtcA peptide was found to target multiple cyanobacteria species, whereas a P-II peptide showed specificity to the high-light Prochlorococcus ecotype. Distributions of NtcA and P-II in the Central Pacific Ocean were similar except at the Equator likely due to differential nitrogen stress responses between Prochlorococcus and Synechococcus. The number of unique tryptic peptides coded for within three combined oceanic microbial metagenomes was estimated to be ∼4 × 10(7) , 1000-fold larger than an individual microbial proteome and 27-fold larger than the human proteome, yet still 20 orders of magnitude lower than the peptide diversity possible in all protein space, implying that peptide mapping algorithms should be able to withstand the added level of complexity in metaproteomic samples.

Proteome trait regulation of marine Synechococcus elemental stoichiometry under global change.

Recent studies have demonstrated regional differences in marine ecosystem C:N:P with implications for carbon and nutrient cycles. Due to strong co-variance, temperature and nutrient stress explain variability in C:N:P equally well. A reductionistic approach can link changes in individual environmental drivers with changes in biochemical traits and cell C:N:P. Thus, we quantified effects of temperature and nutrient stress on Synechococcus chemistry using laboratory chemostats, chemical analyses, and data-independent acquisition mass spectrometry proteomics. Nutrient supply accounted for most C:N:Pcell variability and induced tradeoffs between nutrient acquisition and ribosomal proteins. High temperature prompted heat-shock, whereas thermal effects via the "translation-compensation hypothesis" were only seen under P-stress. A Nonparametric Bayesian Local Clustering algorithm suggested that changes in lipopolysaccharides, peptidoglycans, and C-rich compatible solutes may also contribute to C:N:P regulation. Physiological responses match field-based trends in ecosystem stoichiometry and suggest a hierarchical environmental regulation of current and future ocean C:N:P.

Vitamin B12 conveys a protective advantage to phycosphere-associated bacteria at high temperatures.

Many marine microbes require vitamin B12 (cobalamin) but are unable to synthesize it, necessitating reliance on other B12-producing microbes. Thus, phytoplankton and bacterioplankton community dynamics can partially depend on the production and release of a limiting resource by members of the same community. We tested the impact of temperature and B12 availability on the growth of two bacterial taxa commonly associated with phytoplankton: Ruegeria pomeroyi, which produces B12 and fulfills the B12 requirements of some phytoplankton, and Alteromonas macleodii, which does not produce B12 but also does not strictly require it for growth. For B12-producing R. pomeroyi, we further tested how temperature influences B12 production and release. Access to B12 significantly increased growth rates of both species at the highest temperatures tested (38 °C for R. pomeroyi, 40 °C for A. macleodii) and A. macleodii biomass was significantly reduced when grown at high temperatures without B12, indicating that B12 is protective at high temperatures. Moreover, R. pomeroyi produced more B12 at warmer temperatures but did not release detectable amounts of B12 at any temperature tested. Results imply that increasing temperatures and more frequent marine heatwaves with climate change will influence microbial B12 dynamics and could interrupt symbiotic resource sharing.

Functional control and metabolic integration of stolen organelles in a photosynthetic ciliate.

Stealing prey plastids for metabolic gain is a common phenomenon among protists within aquatic ecosystems.1 Ciliates of the Mesodinium rubrum species complex are unique in that they also steal a transcriptionally active but non-dividing prey nucleus, the kleptokaryon, from certain cryptophytes.2 The kleptokaryon enables full control and replication of kleptoplastids but has a half-life of about 10 days.2 Once the kleptokaryon is lost, the ciliate experiences a slow loss of photosynthetic metabolism and eventually death.2,3,4 This transient ability to function phototrophically allows M. rubrum to form productive blooms in coastal waters.5,6,7,8 Here, we show, using multi-omics approaches, that an Antarctic strain of the ciliate not only depends on stolen Geminigera cryophila organelles for photosynthesis but also for anabolic synthesis of fatty acids, amino acids, and other essential macromolecules. Transcription of diverse pathways was higher in the kleptokaryon than that in G. cryophila, and many increased in higher light. Proteins of major biosynthetic pathways were found in greater numbers in the kleptokaryon relative to M. rubrum, implying anabolic dependency on foreign metabolism. We show that despite losing transcriptional control of the kleptokaryon, M. rubrum regulates kleptoplastid pigments with changing light, implying an important role for post-transcriptional control. These findings demonstrate that the integration of foreign organelles and their gene and protein expression, energy metabolism, and anabolism occur in the absence of a stable endosymbiotic association. Our results shed light on potential events early in the process of complex plastid acquisition and broaden our understanding of symbiogenesis.

Zinc stimulation of coastal productivity in low carbon dioxide environments.

Zinc (Zn) is a key micronutrient used by phytoplankton for carbon (C) acquisition, yet there have been few observations of its influence on natural oceanic phytoplankton populations. In this study, we observed Zn limitation of growth in the natural phytoplankton community of Terra Nova Bay, Antarctica, due to low (~220 µatm) pCO2 conditions, in addition to primary iron (Fe) limitation. Shipboard incubation experiments amended with Zn and Fe resulted in significantly higher chlorophyll a content and dissolved inorganic carbon drawdown compared to Fe addition alone. Zn and Fe response proteins detected in incubation and environmental biomass provided independent verification of algal co-stress for these micronutrients. These observations of Zn limitation under low pCO2 conditions demonstrate Zn can influence coastal primary productivity. Yet, as surface ocean pCO2 rises with continued anthropogenic emissions, the occurrence of Zn/C co-limitation will become rarer, impacting the biogeochemical cycling of Zn and other trace metal micronutrients.

Dynamic diel proteome and daytime nitrogenase activity supports buoyancy in the cyanobacterium Trichodesmium.

Cyanobacteria of the genus Trichodesmium provide about 80 Tg of fixed nitrogen to the surface ocean per year and contribute to marine biogeochemistry, including the sequestration of carbon dioxide. Trichodesmium fixes nitrogen in the daylight, despite the incompatibility of the nitrogenase enzyme with oxygen produced during photosynthesis. While the mechanisms protecting nitrogenase remain unclear, all proposed strategies require considerable resource investment. Here we identify a crucial benefit of daytime nitrogen fixation in Trichodesmium spp. that may counteract these costs. We analysed diel proteomes of cultured and field populations of Trichodesmium in comparison with the marine diazotroph Crocosphaera watsonii WH8501, which fixes nitrogen at night. Trichodesmium's proteome is extraordinarily dynamic and demonstrates simultaneous photosynthesis and nitrogen fixation, resulting in balanced particulate organic carbon and particulate organic nitrogen production. Unlike Crocosphaera, which produces large quantities of glycogen as an energy store for nitrogenase, proteomic evidence is consistent with the idea that Trichodesmium reduces the need to produce glycogen by supplying energy directly to nitrogenase via soluble ferredoxin charged by the photosynthesis protein PsaC. This minimizes ballast associated with glycogen, reducing cell density and decreasing sinking velocity, thus supporting Trichodesmium's niche as a buoyant, high-light-adapted colony forming cyanobacterium. To occupy its niche of simultaneous nitrogen fixation and photosynthesis, Trichodesmium appears to be a conspicuous consumer of iron, and has therefore developed unique iron-acquisition strategies, including the use of iron-rich dust. Particle capture by buoyant Trichodesmium colonies may increase the residence time and degradation of mineral iron in the euphotic zone. These findings describe how cellular biochemistry defines and reinforces the ecological and biogeochemical function of these keystone marine diazotrophs.

Adaptive responses of marine diatoms to zinc scarcity and ecological implications.

Scarce dissolved surface ocean concentrations of the essential algal micronutrient zinc suggest that Zn may influence the growth of phytoplankton such as diatoms, which are major contributors to marine primary productivity. However, the specific mechanisms by which diatoms acclimate to Zn deficiency are poorly understood. Using global proteomic analysis, we identified two proteins (ZCRP-A/B, Zn/Co Responsive Protein A/B) among four diatom species that became abundant under Zn/Co limitation. Characterization using reverse genetic techniques and homology data suggests putative Zn/Co chaperone and membrane-bound transport complex component roles for ZCRP-A (a COG0523 domain protein) and ZCRP-B, respectively. Metaproteomic detection of ZCRPs along a Pacific Ocean transect revealed increased abundances at the surface (<200 m) where dZn and dCo were scarcest, implying Zn nutritional stress in marine algae is more prevalent than previously recognized. These results demonstrate multiple adaptive responses to Zn scarcity in marine diatoms that are deployed in low Zn regions of the Pacific Ocean.

Technical updates to the bacterial method for nitrate isotopic analyses.

The bacterial conversion of aqueous nitrate (NO(3)(-)) to nitrous oxide (N(2)O) for isotopic analysis has found widespread use since its introduction (Sigman, D. M.; Casciotti, K. L.; Andreani, M.; Galanter, M.; Böhlke, J. K. Anal. Chem.2001, 73, 4145-4153; Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Böhlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). The bacterial strain Pseudomonas aureofaciens (ATTC no. 13985) was shown to convert NO(3)(-) to N(2)O while retaining both N and O isotopic signatures, and automation of the isotopic analysis of N(2)O greatly increased the throughput of the method (Casciotti, K. L.; Sigman, D. M.; Galanter Hastings, M.; Böhlke, J. K.; Hilkert, A. Anal. Chem.2002, 74, 4905-4912). Continued development of the denitrifier method has led to increased precision and throughput of NO(3)(-) isotopic analysis. Presented here are several recent procedural modifications and the demonstration of their effectiveness.

Characterization of the metalloproteome of Pseudoalteromonas (BB2-AT2): biogeochemical underpinnings for zinc, manganese, cobalt, and nickel cycling in a ubiquitous marine heterotroph.

Pseudoalteromonas (BB2-AT2) is a ubiquitous marine heterotroph, often associated with labile organic carbon sources in the ocean (e.g. phytoplankton blooms and sinking particles). Heterotrophs hydrolyze exported photosynthetic materials, components of the biological carbon pump, with the use of diverse metalloenzymes containing zinc (Zn), manganese (Mn), cobalt (Co), and nickel (Ni). Studies on the metal requirements and cytosolic utilization of metals for marine heterotrophs are scarce, despite their relevance to global carbon cycling. Here, we characterized the Zn, Mn, Co, and Ni metallome of BB2-AT2. We found that the Zn metallome is complex and cytosolic Zn is associated with numerous proteins for transcription (47.2% of the metallome, obtained from singular value decomposition of the metalloproteomic data), translation (33.5%), proteolysis (12.8%), and alkaline phosphatase activity (6.4%). Numerous proteolytic enzymes also appear to be putatively associated with Mn, and to a lesser extent, Co. Putative identification of the Ni-associated proteins, phosphoglucomutase and a protein in the cupin superfamily, provides new insights for Ni utilization in marine heterotrophs. BB2-AT2 relies on numerous transition metals for proteolytic and phosphatase activities, inferring an adaptative potential to metal limitation. Our field observations of increased alkaline phosphatase activity upon addition of Zn in field incubations suggest that such metal limitation operates in sinking particulate material collected from sediment traps. Taken together, this study improves our understanding of the Zn, Mn, Co, and Ni metallome of marine heterotrophic bacteria and provides novel and mechanistic frameworks for understanding the influence of nutrient limitation on biogeochemical cycling.