The global distribution and climate resilience of marine heterotrophic prokaryotes.

Heterotrophic Bacteria and Archaea (prokaryotes) are a major component of marine food webs and global biogeochemical cycles. Yet, there is limited understanding about how prokaryotes vary across global environmental gradients, and how their global abundance and metabolic activity (production and respiration) may be affected by climate change. Using global datasets of prokaryotic abundance, cell carbon and metabolic activity we reveal that mean prokaryotic biomass varies by just under 3-fold across the global surface ocean, while total prokaryotic metabolic activity increases by more than one order of magnitude from polar to tropical coastal and upwelling regions. Under climate change, global prokaryotic biomass in surface waters is projected to decline ~1.5% per °C of warming, while prokaryotic respiration will increase ~3.5% ( ~ 0.85 Pg C yr-1). The rate of prokaryotic biomass decline is one-third that of zooplankton and fish, while the rate of increase in prokaryotic respiration is double. This suggests that future, warmer oceans could be increasingly dominated by prokaryotes, diverting a growing proportion of primary production into microbial food webs and away from higher trophic levels as well as reducing the capacity of the deep ocean to sequester carbon, all else being equal.

Metaproteomic analysis decodes trophic interactions of microorganisms in the dark ocean.

Proteins in the open ocean represent a significant source of organic matter, and their profiles reflect the metabolic activities of marine microorganisms. Here, by analyzing metaproteomic samples collected from the Pacific, Atlantic and Southern Ocean, we reveal size-fractionated patterns of the structure and function of the marine microbiota protein pool in the water column, particularly in the dark ocean (>200 m). Zooplankton proteins contributed three times more than algal proteins to the deep-sea community metaproteome. Gammaproteobacteria exhibited high metabolic activity in the deep-sea, contributing up to 30% of bacterial proteins. Close virus-host interactions of this taxon might explain the dominance of gammaproteobacterial proteins in the dissolved fraction. A high urease expression in nitrifiers suggested links between their dark carbon fixation and zooplankton urea production. In summary, our results uncover the taxonomic contribution of the microbiota to the oceanic protein pool, revealing protein fluxes from particles to the dissolved organic matter pool.

Functional vertical connectivity of microbial communities in the ocean.

Sinking particles are a critical conduit for the transport of surface microbes to the ocean's interior. Vertical connectivity of phylogenetic composition has been shown; however, the functional vertical connectivity of microbial communities has not yet been explored in detail. We investigated protein and taxa profiles of both free-living and particle-attached microbial communities from the surface to 3000 m depth using a combined metaproteomic and 16S rRNA amplicon sequencing approach. A clear compositional and functional vertical connectivity of microbial communities was observed throughout the water column with Oceanospirillales, Alteromonadales, and Rhodobacterales as key taxa. The surface-derived particle-associated microbes increased the expression of proteins involved in basic metabolism, organic matter processing, and environmental stress response in deep waters. This study highlights the functional vertical connectivity between surface and deep-sea microbial communities via sinking particles and reveals that a considerable proportion of the deep-sea microbes might originate from surface waters and have a major impact on the biogeochemical cycles in the deep sea.

Substrate uptake patterns shape niche separation in marine prokaryotic microbiome.

Marine heterotrophic prokaryotes primarily take up ambient substrates using transporters. The patterns of transporters targeting particular substrates shape the ecological role of heterotrophic prokaryotes in marine organic matter cycles. Here, we report a size-fractionated pattern in the expression of prokaryotic transporters throughout the oceanic water column due to taxonomic variations, revealed by a multi-"omics" approach targeting ATP-binding cassette (ABC) transporters and TonB-dependent transporters (TBDTs). Substrate specificity analyses showed that marine SAR11, Rhodobacterales, and Oceanospirillales use ABC transporters to take up organic nitrogenous compounds in the free-living fraction, while Alteromonadales, Bacteroidetes, and Sphingomonadales use TBDTs for carbon-rich organic matter and metal chelates on particles. The expression of transporter proteins also supports distinct lifestyles of deep-sea prokaryotes. Our results suggest that transporter divergency in organic matter assimilation reflects a pronounced niche separation in the prokaryote-mediated organic matter cycles.

Decoupling between the genetic potential and the metabolic regulation and expression in microbial organic matter cleavage across microbiomes.

Metagenomics, metatranscriptomics, and metaproteomics are used to explore the microbial capability of enzyme secretion, but the links between protein-encoding genes and corresponding transcripts/proteins across ecosystems are underexplored. By conducting a multi-omics comparison focusing on key enzymes (carbohydrate-active enzymes [CAZymes] and peptidases) cleaving the main biomolecules across distinct microbiomes living in the ocean, soil, and human gut, we show that the community structure, functional diversity, and secretion mechanisms of microbial secretory CAZymes and peptidases vary drastically between microbiomes at metagenomic, metatranscriptomic, and metaproteomic levels. Such variations lead to decoupled relationships between CAZymes and peptidases from genetic potentials to protein expressions due to the different responses of key players toward organic matter sources and concentrations. Our results highlight the need for systematic analysis of the factors shaping patterns of microbial cleavage on organic matter to better link omics data to ecosystem processes. IMPORTANCE: Omics tools are used to explore adaptive mechanism of microbes in diverse systems, but the advantages and limitations of different omics tools remain skeptical. Here, we reported distinct profiles in microbial secretory enzyme composition revealed by different omics methods. In general, the predicted function from metagenomic analysis decoupled from the expression of corresponding transcripts/proteins. Linking omics results to taxonomic origin, functional capability, substrate specificity, secretion preference, and enzymatic activity measurement suggested the substrate's source, concentration and stoichiometry impose strong filtering on the expression of extracellular enzymes, which may overwrite the genetic potentials. Our results present an integrated perspective on the need for multi-dimensional characterization of microbial adaptation in a changing environment.

The microbial carbon pump and climate change.

The ocean has been a regulator of climate change throughout the history of Earth. One key mechanism is the mediation of the carbon reservoir by refractory dissolved organic carbon (RDOC), which can either be stored in the water column for centuries or released back into the atmosphere as CO2 depending on the conditions. The RDOC is produced through a myriad of microbial metabolic and ecological processes known as the microbial carbon pump (MCP). Here, we review recent research advances in processes related to the MCP, including the distribution patterns and molecular composition of RDOC, links between the complexity of RDOC compounds and microbial diversity, MCP-driven carbon cycles across time and space, and responses of the MCP to a changing climate. We identify knowledge gaps and future research directions in the role of the MCP, particularly as a key component in integrated approaches combining the mechanisms of the biological and abiotic carbon pumps for ocean negative carbon emissions.

Recovering high-quality bacterial genomes from cross-contaminated cultures: a case study of marine Vibrio campbellii.

BACKGROUND: Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains. RESULTS: We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen. CONCLUSIONS: Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.

Influence of Salinity on the Extracellular Enzymatic Activities of Marine Pelagic Fungi.

Even though fungi are ubiquitous in the biosphere, the ecological knowledge of marine fungi remains rather rudimentary. Also, little is known about their tolerance to salinity and how it influences their activities. Extracellular enzymatic activities (EEAs) are widely used to determine heterotrophic microbes' enzymatic capabilities and substrate preferences. Five marine fungal species belonging to the most abundant pelagic phyla (Ascomycota and Basidiomycota) were grown under non-saline and saline conditions (0 g/L and 35 g/L, respectively). Due to their sensitivity and specificity, fluorogenic substrate analogues were used to determine hydrolytic activity on carbohydrates (ß-glucosidase, ß-xylosidase, and N-acetyl-ß-D-glucosaminidase); peptides (leucine aminopeptidase and trypsin); lipids (lipase); organic phosphorus (alkaline phosphatase), and sulfur compounds (sulfatase). Afterwards, kinetic parameters such as maximum velocity (Vmax) and half-saturation constant (Km) were calculated. All fungal species investigated cleaved these substrates, but some species were more efficient than others. Moreover, most enzymatic activities were reduced in the saline medium, with some exceptions like sulfatase. In non-saline conditions, the average Vmax ranged between 208.5 to 0.02 µmol/g biomass/h, and in saline conditions, 88.4 to 0.02 µmol/g biomass/h. The average Km ranged between 1553.2 and 0.02 µM with no clear influence of salinity. Taken together, our results highlight a potential tolerance of marine fungi to freshwater conditions and indicate that changes in salinity (due to freshwater input or evaporation) might impact their enzymatic activities spectrum and, therefore, their contribution to the oceanic elemental cycles.

Bacterial degradation of ctenophore Mnemiopsis leidyi organic matter.

Blooms of gelatinous zooplankton, an important source of protein-rich biomass in coastal waters, often collapse rapidly, releasing large amounts of labile detrital organic matter (OM) into the surrounding water. Although these blooms have the potential to cause major perturbations in the marine ecosystem, their effects on the microbial community and hence on the biogeochemical cycles have yet to be elucidated. We conducted microcosm experiments simulating the scenario experienced by coastal bacterial communities after the decay of a ctenophore (Mnemiopsis leidyi) bloom in the northern Adriatic Sea. Within 24 h, a rapid response of bacterial communities to the M. leidyi OM was observed, characterized by elevated bacterial biomass production and respiration rates. However, compared to our previous microcosm study of jellyfish (Aurelia aurita s.l.), M. leidyi OM degradation was characterized by significantly lower bacterial growth efficiency, meaning that the carbon stored in the OM was mostly respired. Combined metagenomic and metaproteomic analysis indicated that the degradation activity was mainly performed by Pseudoalteromonas, producing a large amount of proteolytic extracellular enzymes and exhibiting high metabolic activity. Interestingly, the reconstructed metagenome-assembled genome (MAG) of Pseudoalteromonas phenolica was almost identical (average nucleotide identity >99%) to the MAG previously reconstructed in our A. aurita microcosm study, despite the fundamental genetic and biochemical differences of the two gelatinous zooplankton species. Taken together, our data suggest that blooms of different gelatinous zooplankton are likely triggering a consistent response from natural bacterial communities, with specific bacterial lineages driving the remineralization of the gelatinous OM.IMPORTANCEJellyfish blooms are increasingly becoming a recurring seasonal event in marine ecosystems, characterized by a rapid build-up of gelatinous biomass that collapses rapidly. Although these blooms have the potential to cause major perturbations, their impact on marine microbial communities is largely unknown. We conducted an incubation experiment simulating a bloom of the ctenophore Mnemiopsis leidyi in the Northern Adriatic, where we investigated the bacterial response to the gelatinous biomass. We found that the bacterial communities actively degraded the gelatinous organic matter, and overall showed a striking similarity to the dynamics previously observed after a simulated bloom of the jellyfish Aurelia aurita s.l. In both cases, we found that a single bacterial species, Pseudoalteromonas phenolica, was responsible for most of the degradation activity. This suggests that blooms of different jellyfish are likely to trigger a consistent response from natural bacterial communities, with specific bacterial species driving the remineralization of gelatinous biomass.

Release of cell-free enzymes by marine pelagic fungal strains.

Fungi are ubiquitous organisms that secrete different enzymes to cleave large molecules into smaller ones so that can then be assimilated. Recent studies suggest that fungi are also present in the oceanic water column harboring the enzymatic repertoire necessary to cleave carbohydrates and proteins. In marine prokaryotes, the cell-free fraction is an important contributor to the oceanic extracellular enzymatic activities (EEAs), but the release of cell-free enzymes by marine fungi remains unknown. Here, to study the cell-free enzymatic activities of marine fungi and the potential influence of salinity on them, five strains of marine fungi that belong to the most abundant pelagic phyla (Ascomycota and Basidiomycota), were grown under non-saline and saline conditions (0 g/L and 35 g/L, respectively). The biomass was separated from the medium by filtration (0.2 µm), and the filtrate was used to perform fluorogenic enzymatic assays with substrate analogues of carbohydrates, lipids, organic phosphorus, sulfur moieties, and proteins. Kinetic parameters such as maximum velocity (Vmax) and half-saturation constant (Km) were obtained. The species studied were able to release cell-free enzymes, and this represented up to 85.1% of the respective total EEA. However, this differed between species and enzymes, with some of the highest contributions being found in those with low total EEA, with some exceptions. This suggests that some of these contributions to the enzymatic pool might be minimal compared to those with higher total EEA. Generally, in the saline medium, the release of cell-free enzymes degrading carbohydrates was reduced compared to the non-saline medium, but those degrading lipids and sulfur moieties were increased. For the remaining substrates, there was not a clear influence of the salinity. Taken together, our results suggest that marine fungi are potential contributors to the oceanic dissolved (i.e., cell-free) enzymatic pool. Our results also suggest that, under salinity changes, a potential effect of global warming, the hydrolysis of organic matter by marine fungal cell-free enzymes might be affected and hence, their potential contribution to the oceanic biogeochemical cycles.

Interplay between autotrophic and heterotrophic prokaryotic metabolism in the bathypelagic realm revealed by metatranscriptomic analyses.

BACKGROUND: Heterotrophic microbes inhabiting the dark ocean largely depend on the settling of organic matter from the sunlit ocean. However, this sinking of organic materials is insufficient to cover their demand for energy and alternative sources such as chemoautotrophy have been proposed. Reduced sulfur compounds, such as thiosulfate, are a potential energy source for both auto- and heterotrophic marine prokaryotes. METHODS: Seawater samples were collected from Labrador Sea Water (LSW, ~ 2000 m depth) in the North Atlantic and incubated in the dark at in situ temperature unamended, amended with 1 µM thiosulfate, or with 1 µM thiosulfate plus 10 µM glucose and 10 µM acetate (thiosulfate plus dissolved organic matter, DOM). Inorganic carbon fixation was measured in the different treatments and samples for metatranscriptomic analyses were collected after 1 h and 72 h of incubation. RESULTS: Amendment of LSW with thiosulfate and thiosulfate plus DOM enhanced prokaryotic inorganic carbon fixation. The energy generated via chemoautotrophy and heterotrophy in the amended prokaryotic communities was used for the biosynthesis of glycogen and phospholipids as storage molecules. The addition of thiosulfate stimulated unclassified bacteria, sulfur-oxidizing Deltaproteobacteria (SAR324 cluster bacteria), Epsilonproteobacteria (Sulfurimonas sp.), and Gammaproteobacteria (SUP05 cluster bacteria), whereas, the amendment with thiosulfate plus DOM stimulated typically copiotrophic Gammaproteobacteria (closely related to Vibrio sp. and Pseudoalteromonas sp.). CONCLUSIONS: The gene expression pattern of thiosulfate utilizing microbes specifically of genes involved in energy production via sulfur oxidation and coupled to CO2 fixation pathways coincided with the change in the transcriptional profile of the heterotrophic prokaryotic community (genes involved in promoting energy storage), suggesting a fine-tuned metabolic interplay between chemoautotrophic and heterotrophic microbes in the dark ocean. Video Abstract.

Seasonal patterns in microbial carbon and iron transporter expression in the Southern Ocean.

BACKGROUND: Heterotrophic microbes in the Southern Ocean are challenged by the double constraint of low concentrations of organic carbon (C) and iron (Fe). These essential elements are tightly coupled in cellular processes; however, the prokaryotic requirements of C and Fe under varying environmental settings remain poorly studied. Here, we used a combination of metatranscriptomics and metaproteomics to identify prokaryotic membrane transporters for organic substrates and Fe in naturally iron-fertilized and high-nutrient, low-chlorophyll waters of the Southern Ocean during spring and late summer. RESULTS: Pronounced differences in membrane transporter profiles between seasons were observed at both sites, both at the transcript and protein level. When specific compound classes were considered, the two approaches revealed different patterns. At the transcript level, seasonal patterns were only observed for subsets of genes belonging to each transporter category. At the protein level, membrane transporters of organic compounds were relatively more abundant in spring as compared to summer, while the opposite pattern was observed for Fe transporters. These observations suggest an enhanced requirement for organic C in early spring and for Fe in late summer. Mapping transcripts and proteins to 50 metagenomic-assembled genomes revealed distinct taxon-specific seasonal differences pointing to potentially opportunistic clades, such as Pseudomonadales and Nitrincolaceae, and groups with a more restricted repertoire of expressed transporters, such as Alphaproteobacteria and Flavobacteriaceae. CONCLUSION: The combined investigations of C and Fe membrane transporters suggest seasonal changes in the microbial requirements of these elements under different productivity regimes. The taxon-specific acquisition strategies of different forms of C and Fe illustrate how diverse microbes could shape transcript and protein expression profiles at the community level at different seasons. Our results on the C- and Fe-related metabolic capabilities of microbial taxa provide new insights into their potential role in the cycling of C and Fe under varying nutrient regimes in the Southern Ocean. Video Abstract.

Jellyfish detritus supports niche partitioning and metabolic interactions among pelagic marine bacteria.

BACKGROUND: Jellyfish blooms represent a significant but largely overlooked source of labile organic matter (jelly-OM) in the ocean, characterized by a high protein content. Decaying jellyfish are important carriers for carbon export to the ocean's interior. To accurately incorporate them into biogeochemical models, the interactions between microbes and jelly-OM have yet to be fully characterized. We conducted jelly-OM enrichment experiments in microcosms to simulate the scenario experienced by the coastal pelagic microbiome after the decay of a jellyfish bloom. We combined metagenomics, endo- and exo-metaproteomic approaches to obtain a mechanistic understanding on the metabolic network operated by the jelly-OM degrading bacterial consortium. RESULTS: Our analysis revealed that OM released during the decay of jellyfish blooms triggers a rapid shuffling of the taxonomic and functional profile of the pelagic bacterial community, resulting in a significant enrichment of protein/amino acid catabolism-related enzymes in the jelly-OM degrading community dominated by Pseudoalteromonadaceae, Alteromonadaceae and Vibrionaceae, compared to unamended control treatments. In accordance with the proteinaceous character of jelly-OM, Pseudoalteromonadaceae synthesized and excreted enzymes associated with proteolysis, while Alteromonadaceae contributed to extracellular hydrolysis of complex carbohydrates and organophosphorus compounds. In contrast, Vibrionaceae synthesized transporter proteins for peptides, amino acids and carbohydrates, exhibiting a cheater-type lifestyle, i.e. benefiting from public goods released by others. In the late stage of jelly-OM degradation, Rhodobacteraceae and Alteromonadaceae became dominant, growing on jelly-OM left-overs or bacterial debris, potentially contributing to the accumulation of dissolved organic nitrogen compounds and inorganic nutrients, following the decay of jellyfish blooms. CONCLUSIONS: Our findings indicate that specific chemical and metabolic fingerprints associated with decaying jellyfish blooms are substantially different to those previously associated with decaying phytoplankton blooms, potentially altering the functioning and biogeochemistry of marine systems. We show that decaying jellyfish blooms are associated with the enrichment in extracellular collagenolytic bacterial proteases, which could act as virulence factors in human and marine organisms' disease, with possible implications for marine ecosystem services. Our study also provides novel insights into niche partitioning and metabolic interactions among key jelly-OM degraders operating a complex metabolic network in a temporal cascade of biochemical reactions to degrade pulses of jellyfish-bloom-specific compounds in the water column. Video Abstract.

Characterization of membrane vesicles in Alteromonas macleodii indicates potential roles in their copiotrophic lifestyle.

Bacterial membrane vesicles (MVs) are abundant in the oceans, but their potential functional roles remain unclear. In this study we characterized MV production and protein content of six strains of Alteromonas macleodii, a cosmopolitan marine bacterium. Alteromonas macleodii strains varied in their MV production rates, with some releasing up to 30 MVs per cell per generation. Microscopy imaging revealed heterogenous MV morphologies, including some MVs aggregated within larger membrane structures. Proteomic characterization revealed that A. macleodii MVs are rich in membrane proteins related to iron and phosphate uptake, as well as proteins with potential functions in biofilm formation. Furthermore, MVs harbored ectoenzymes, such as aminopeptidases and alkaline phosphatases, which comprised up to 20% of the total extracellular enzymatic activity. Our results suggest that A. macleodii MVs may support its growth through generation of extracellular 'hotspots' that facilitate access to essential substrates. This study provides an important basis to decipher the ecological relevance of MVs in heterotrophic marine bacteria.

A ubiquitous gammaproteobacterial clade dominates expression of sulfur oxidation genes across the mesopelagic ocean.

The deep ocean (>200 m depth) is the largest habitat on Earth. Recent evidence suggests sulfur oxidation could be a major energy source for deep ocean microbes. However, the global relevance and the identity of the major players in sulfur oxidation in the oxygenated deep-water column remain elusive. Here we combined single-cell genomics, community metagenomics, metatranscriptomics and single-cell activity measurements on samples collected beneath the Ross Ice Shelf in Antarctica to characterize a ubiquitous mixotrophic bacterial group (UBA868) that dominates expression of RuBisCO genes and of key sulfur oxidation genes. Further analyses of the gene libraries from the 'Tara Oceans' and 'Malaspina' expeditions confirmed the ubiquitous distribution and global relevance of this enigmatic group in the expression of sulfur oxidation and dissolved inorganic carbon fixation genes across the global mesopelagic ocean. Our study also underscores the unrecognized importance of mixotrophic microbes in the biogeochemical cycles of the deep ocean.

Prokaryotic Life in the Deep Ocean's Water Column.

The oceanic waters below a depth of 200 m represent, in terms of volume, the largest habitat of the biosphere, harboring approximately 70% of the prokaryotic biomass in the oceanic water column. These waters are characterized by low temperature, increasing hydrostatic pressure, and decreasing organic matter supply with depth. Recent methodological advances in microbial oceanography have refined our view of the ecology of prokaryotes in the dark ocean. Here, we review the ecology of prokaryotes of the dark ocean, present data on the biomass distribution and heterotrophic and chemolithoautotrophic prokaryotic production in the major oceanic basins, and highlight the phylogenetic and functional diversity of this part of the ocean. We describe the connectivity of surface and deep-water prokaryotes and the molecular adaptations of piezophilic prokaryotes to high hydrostatic pressure. We also highlight knowledge gaps in the ecology of the dark ocean's prokaryotes and their role in the biogeochemical cycles in the largest habitat of the biosphere.

A device for assessing microbial activity under ambient hydrostatic pressure: The in situ microbial incubator (ISMI).

Microbes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high-pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk 3H-leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep-sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep-sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep-sea biotic processes.

Decoupling of respiration rates and abundance in marine prokaryoplankton.

The ocean-atmosphere exchange of CO2 largely depends on the balance between marine microbial photosynthesis and respiration. Despite vast taxonomic and metabolic diversity among marine planktonic bacteria and archaea (prokaryoplankton)1-3, their respiration usually is measured in bulk and treated as a 'black box' in global biogeochemical models4; this limits the mechanistic understanding of the global carbon cycle. Here, using a technology for integrated phenotype analyses and genomic sequencing of individual microbial cells, we show that cell-specific respiration rates differ by more than 1,000× among prokaryoplankton genera. The majority of respiration was found to be performed by minority members of prokaryoplankton (including the Roseobacter cluster), whereas cells of the most prevalent lineages (including Pelagibacter and SAR86) had extremely low respiration rates. The decoupling of respiration rates from abundance among lineages, elevated counts of proteorhodopsin transcripts in Pelagibacter and SAR86 cells and elevated respiration of SAR86 at night indicate that proteorhodopsin-based phototrophy3,5-7 probably constitutes an important source of energy to prokaryoplankton and may increase growth efficiency. These findings suggest that the dependence of prokaryoplankton on respiration and remineralization of phytoplankton-derived organic carbon into CO2 for its energy demands and growth may be lower than commonly assumed and variable among lineages.

Limited carbon cycling due to high-pressure effects on the deep-sea microbiome.

Deep-sea microbial communities are exposed to high-pressure conditions, which has a variable impact on prokaryotes depending on whether they are piezophilic (that is, pressure-loving), piezotolerant or piezosensitive. While it has been suggested that elevated pressures lead to higher community-level metabolic rates, the response of these deep-sea microbial communities to the high-pressure conditions of the deep sea is poorly understood. Based on microbial activity measurements in the major oceanic basins using an in situ microbial incubator, we show that the bulk heterotrophic activity of prokaryotic communities becomes increasingly inhibited at higher hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in situ hydrostatic pressure was about one-third of that measured in the same community at atmospheric pressure conditions. In the bathypelagic zone-between 1,000 and 4,000 m depth-~85% of the prokaryotic community was piezotolerant and ~5% of the prokaryotic community was piezophilic. Despite piezosensitive-like prokaryotes comprising only ~10% (mainly members of Bacteroidetes, Alteromonas) of the deep-sea prokaryotic community, the more than 100-fold metabolic activity increase of these piezosensitive prokaryotes upon depressurization leads to high apparent bulk metabolic activity. Overall, the heterotrophic prokaryotic activity in the deep sea is likely to be substantially lower than hitherto assumed, with major impacts on the oceanic carbon cycling.

Temporal variation in the prokaryotic community of a nearshore marine environment.

Prokaryotic communities inhabiting surface waters of temperate areas exhibit patterns of seasonal succession. Generally, studies describing these temporal changes are not performed in the proximity to the coast. In the present study, temporal variation of these communities was determined in surface waters at two stations located in the close proximity to the eastern shore of the northern Adriatic Sea. Sequencing of the V4 region of the 16S rRNA gene identified the highest community richness in December with distinct shifts in community structure between periods from April to May, June to October, and November to March. Temperature was shown to be the main environmental force explaining community temporal variation. The NS5 marine group, uncultured Cryomorphaceae, SAR86 clade, and Synechococcus were present throughout the year. Members without know relatives within Rhodobacteraceae and the NS4 marine group were more pronounced in the period from April to May, the AEGEAN-169 marine group, SAR11 subclade III, and HIMB11 in the period from June to October, and SAR11 subclade Ia and Archaea in the period from November to March. Litoricola and OM60 (NOR5) clade were characteristic for both the community sampled from April to May and November to March. Taken together, prokaryotic communities inhabiting nearshore surface waters exhibit a general pattern in community structure similar to other surface associated assemblages of temperate areas. However, the identified specific community composition and temporal patterns differ from other coastal areas.