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Identifying mechanisms by which bacterial species evolve and maintain genomic diversity is particularly challenging for the uncultured lineages that dominate the surface ocean. A longitudinal analysis of bacterial genes, genomes, and transcripts during a coastal phytoplankton bloom revealed two co-occurring, highly related Rhodobacteraceae species from the deeply branching and uncultured NAC11-7 lineage. These have identical 16S rRNA gene amplicon sequences, yet their genome contents assembled from metagenomes and single cells indicate species-level divergence. Moreover, shifts in relative dominance of the species during dynamic bloom conditions over 7 weeks confirmed the syntopic species’ divergent responses to the same microenvironment at the same time. Genes unique to each species and genes shared but divergent in per-cell inventories of mRNAs accounted for 5% of the species’ pangenome content. These analyses uncover physiological and ecological features that differentiate the species, including capacities for organic carbon utilization, attributes of the cell surface, metal requirements, and vitamin biosynthesis. Such insights into the coexistence of highly related and ecologically similar bacterial species in their shared natural habitat are rare.

Bacteria that assemble in phycospheres surrounding living phytoplankton cells metabolize a substantial proportion of ocean primary productivity. Yet the type and extent of interactions occurring among species that colonize these micron-scale “hot spot” environments are challenging to study. We identified genes that mediate bacterial interactions in phycosphere communities by culturing a transposon mutant library of copiotrophic bacterium Ruegeria pomeroyi DSS-3 with the diatom Thalassiosira pseudonana CCMP1335 as the sole source of organic matter in the presence or absence of other heterotrophic bacterial species. The function of genes having significant effects on R. pomeroyi fitness indicated explicit cell–cell interactions initiated in the multibacterial phycospheres. We found that R. pomeroyi simultaneously competed for shared substrates while increasing reliance on substrates that did not support the other species’ growth. Fitness outcomes also indicated that the bacterium competed for nitrogen in the forms of ammonium and amino acids; obtained purines, pyrimidines, and cofactors via crossfeeding; both initiated and defended antagonistic interactions; and sensed an environment with altered oxygen and superoxide levels. The large genomes characteristic of copiotrophic marine bacteria are hypothesized to enable responses to dynamic ecological challenges occurring at the scale of microns. Here, we discover >200 nonessential genes implicated in the management of fitness costs and benefits of membership in a globally significant bacterial community.

Dissolved primary production released into seawater by marine phytoplankton is a major source of carbon fueling heterotrophic bacterial production in the ocean. The composition of the organic compounds released by healthy phytoplankton is poorly known and difficult to assess with existing chemical methods. Here, expression of transporter and catabolic genes by three model marine bacteria (Ruegeria pomeroyi DSS-3, Stenotrophomonas sp. SKA14, and Polaribacter dokdonensis MED152) was used as a biological sensor of metabolites released from the picoeukaryote Micromonas commoda RCC299. Bacterial expression responses indicated that the three species together recognized 38 picoeukaryote metabolites. This was consistent with the Micromonas expression of genes for starch metabolism and synthesis of peptidoglycan-like intermediates. A comparison of the hypothesized Micromonas exometabolite pool with that of the diatom Thalassiosira pseudonana CCMP1335, analyzed previously with the same biological sensor method, indicated that both phytoplankton released organic acids, nucleosides, and amino acids, but differed in polysaccharide and organic nitrogen release. Future ocean conditions are expected to favor picoeukaryotic phytoplankton over larger-celled microphytoplankton. Results from this study suggest that such a shift could alter the substrate pool available to heterotrophic bacterioplankton.

Metabolite exchange within marine microbial communities transfers carbon and other major elements through global cycles and forms the basis of microbial interactions. Yet lack of gene annotations and concern about the quality of existing ones remain major impediments to revealing the metabolite-microbial network. We employed an arrayed mutant library of the marine bacteriumRuegeria pomeroyiDSS-3 to experimentally annotate substrates of organic compound transporter systems, using mutant growth and compound drawdown analyses to link transporters to their substrates. Mutant experiments verified substrates for thirteenR. pomeroyitransporters. Four were previously hypothesized based on gene expression data (taurine, glucose/xylose, isethionate, and cadaverine/putrescine/spermidine); five were previously hypothesized based on homology to experimentally annotated transporters in other bacteria (citrate, glycerol,N-acetylglucosamine, fumarate/malate/succinate, and dimethylsulfoniopropionate); and four had no previous annotations (thymidine, carnitine, cysteate, and 3-hydroxybutyrate transporter). These bring the total number of experimentally-verified organic carbon influx transporters to 17 of 126 in theR. pomeroyigenome. In a longitudinal study of a coastal phytoplankton bloom, expression patterns of the experimentally annotated transporters linked them to different stages of the bloom, and also led to the hypothesis that citrate and 3-hydroxybutyrate were among the most highly available bacterial substrates. Improved functional knowledge of these gatekeepers of organic carbon uptake is facilitating better characterization of the surface ocean metabolite network.

Robust annotation of metabolites is a challenging task in metabolomics. Among available applications, 13C NMR experiment INADEQUATE determines direct 13C-13C connectivity unambiguously, offering indispensable information on molecular structure. Despite its great utility, it is not always practical to collect INADEQUATE data on every sample in a large metabolomics study because of its relatively long experiment time. Here, we propose an alternative approach that maintains the quality of information but saves experiment time. In this approach, individual samples in a study are first screened by 13C homonuclear J-resolved experiment (JRES). Next, JRES data are processed by statistical total correlation spectroscopy (STOCSY) to extract peaks that behave similarly among samples. Finally, INADEQUATE is collected on one internal pooled sample to select STOCSY peaks that originate from the same compound. We tested this concept using the 13C-labeled endometabolome of a model marine diatom strain incu-bated under various settings, intending to cover a range of metabolites produced under different external conditions. This scheme was able to extract known diatom metabolites proline, 2,3-dihydroxypropane-1-sulfonate (DHPS), b-1,3-glucan, choline, and glutamate. This pipeline also detected unknown compounds with structural information, which is valuable in metabolomics where a priori knowledge of metabolites is not always available. The ability of this scheme was seen even in sugar regions, which are usually chal-lenging in 1H NMR due to severe peak overlap. JRES and INADEQUATE were highly complementary; INADEQUATE provided directly-bonded 13C networks, whereas JRES linked INADEQUATE networks within the same compound but broken by nitrogen or sulfur atoms, highlighting the advantage of this integrated approach.(c) 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

We describe considerations and strategies for developing a nuclear magnetic resonance (NMR) sample preparation method to extract low molecular weight metabolites from high-salt spent media in a model coculture system of phytoplankton and marine bacteria. Phytoplankton perform half the carbon fixation and oxygen generation on Earth. A substantial fraction of fixed carbon becomes part of a metabolite pool of small molecules known as dissolved organic matter (DOM), which are taken up by marine bacteria proximate to phytoplankton. There is an urgent need to elucidate these metabolic exchanges due to widespread anthropogenic transformations on the chemical, phenotypic, and species composition of seawater. These changes are increasing water temperature and the amount of CO

Phytoplankton-derived metabolites fuel a large fraction of heterotrophic bacterial production in the global ocean, yet methodological challenges have limited our understanding of the organic molecules transferred between these microbial groups. In an experimental bloom study consisting of three heterotrophic marine bacteria growing together with the diatom Thalassiosira pseudonana, we concurrently measured diatom endometabolites (i.e., potential exometabolite supply) by nuclear magnetic resonance (NMR) spectroscopy and bacterial gene expression (i.e., potential exometabolite uptake) by metatranscriptomic sequencing. Twenty-two diatom endometabolites were annotated, with nine increasing in internal concentration in the late stage of the bloom, eight decreasing, and five showing no variation through the bloom progression. Some metabolite changes could be linked to shifts in diatom gene expression, as well as to shifts in bacterial community composition and their expression of substrate uptake and catabolism genes. Yet an overall low match indicated that endometabolome concentration was not a good predictor of exometabolite availability, and that complex physiological and ecological interactions underlie metabolite exchange. Six diatom endometabolites accumulated to higher concentrations in the bacterial co-cultures compared to axenic cultures, suggesting a bacterial influence on rates of synthesis or release of glutamate, arginine, leucine, 2,3-dihydroxypropane-1-sulfonate, glucose, and glycerol-3-phosphate. Better understanding of phytoplankton metabolite production, release, and transfer to assembled bacterial communities is key to untangling this nearly invisible yet pivotal step in ocean carbon cycling.

Small genes (40,000 small-gene families in ~2.3 million phage genome contigs. We find that small genes in phage genomes are approximately 3-fold more prevalent than in host prokaryotic genomes. Our approach enriches for small genes that are translated in microbiomes, suggesting the small genes identified are coding. More than 9,000 families encode potentially secreted or transmembrane proteins, more than 5,000 families encode predicted anti-CRISPR proteins, and more than 500 families encode predicted antimicrobial proteins. By combining homology and genomic-neighborhood analyses, we reveal substantial novelty and diversity within phage biology, including small phage genes found in multiple host phyla, small genes encoding proteins that play essential roles in host infection, and small genes that share genomic neighborhoods and whose encoded proteins may share related functions.

Phytoplankton release massive amounts of dissolved organic matter (DOM) into the water column during recurring blooms in coastal waters and inland seas. The released DOM encompasses a complex mixture of both known and unknown compounds, and is a rich nutrient source for heterotrophic bacteria. The metabolic activity of bacteria during and after phytoplankton blooms can hence be expected to reflect the characteristics of the released DOM. We therefore investigated if bacterioplankton could be used as “living sensors” of phytoplankton DOM quantity and/or quality, by applying gene expression analyses to identify bacterial metabolisms induced by DOM. We used transcriptional analysis of two Baltic Sea bacterial isolates (Polaribactersp. BAL334 [Flavobacteriia] andBrevundimonassp. BAL450 [Alphaproteobacteria]) growing with DOM from axenic cultures of the dinoflagellateProrocentrum minimum. We observed pronounced differences between the two bacteria both in growth and the expressed metabolic pathways in cultures exposed to dinoflagellate DOM compared with controls. Differences in metabolic responses between the two isolates were caused both by differences in gene repertoire between them (e.g. in the SEED categories for membrane transport, motility and photoheterotrophy) and the regulation of expression (e.g. fatty acid metabolism), emphasizing the importance of separating the responses of different taxa in analyses of community sequence data. Similarities between the bacteria included substantially increased expression of genes for Ton and Tol transport systems in both isolates, which are commonly associated with uptake of complex organic molecules.Polaribactersp. BAL334 showed stronger metabolic responses to DOM harvested from exponential than stationary phase dinoflagellates (128 compared to 26 differentially expressed genes), whereasBrevundimonassp. BAL450 responded more to the DOM from stationary than exponential phase dinoflagellates (33 compared to 6 differentially expressed genes). These findings suggest that shifts in bacterial metabolisms during different phases of phytoplankton blooms can be detected in individual bacterial species and can provide insights into their involvement in DOM transformations.

trial protocol 2

Marine microorganisms play crucial roles in Earth's element cycles through the production and consumption of organic matter. One of the elements whose fate is governed by microbial activities is sulfur, an essential constituent of biomass and a crucial player in climate processes. With sulfur already being well studied in the ocean in its inorganic forms, organic sulfur compounds are emerging as important chemical links between marine phytoplankton and bacteria. The high concentration of inorganic sulfur in seawater, which can readily be reduced by phytoplankton, provides a freely available source of sulfur for biomolecule synthesis. Mechanisms such as exudation and cell lysis release these phytoplankton-derived sulfur metabolites into seawater, from which they are rapidly assimilated by marine bacteria and archaea. Energy-limited bacteria use scavenged sulfur metabolites as substrates or for the synthesis of vitamins, cofactors, signalling compounds and antibiotics. In this Review, we examine the current knowledge of sulfur metabolites released into and taken up from the marine dissolved organic matter pool by microorganisms, and the ecological links facilitated by their diversity in structures, oxidation states and chemistry.

Unlike biologically available nitrogen and phosphorus, which are often at limiting concentrations in surface seawater, sulfur in the form of sulfate is plentiful and not considered to constrain marine microbial activity. Nonetheless, in a model system in which a marine bacterium obtains all of its carbon from co-cultured phytoplankton, bacterial gene expression suggests that at least seven dissolved organic sulfur (DOS) metabolites support bacterial heterotrophy. These labile exometabolites of marine dinoflagellates and diatoms include taurine, N-acetyltaurine, isethionate, choline-O-sulfate, cysteate, 2,3-dihydroxypropane-1-sulfonate (DHPS), and dimethylsulfoniopropionate (DMSP). Leveraging from the compounds identified in this model system, we assessed the role of sulfur metabolites in the ocean carbon cycle by mining the Tara Oceans dataset for diagnostic genes. In the 1.4 million bacterial genome equivalents surveyed, estimates of the frequency of genomes harboring the capability for DOS metabolite utilization ranged broadly, from only 1 out of every 190 genomes (for the C2 sulfonate isethionate) to 1 out of every 5 (for the sulfonium compound DMSP). Bacteria able to participate in DOS transformations are dominated by Alphaproteobacteria in the surface ocean, but by SAR324, Acidimicrobiia, and Gammaproteobacteria at mesopelagic depths, where the capability for utilization occurs in higher frequency than in surface bacteria for more than half the sulfur metabolites. The discovery of an abundant and diverse suite of marine bacteria with the genetic capacity for DOS transformation argues for an important role for sulfur metabolites in the pelagic ocean carbon cycle.

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur metabolite produced by many phytoplankton species and degraded by bacteria via two distinct pathways with climate-relevant implications. We assessed the diversity and abundance of bacteria possessing these pathways in the context of phytoplankton community composition over a 3-week time period spanning September-October, 2014 in Monterey Bay, CA. The dmdA gene from the DMSP demethylation pathway dominated the DMSP gene pool and was harboured mostly by members of the alphaproteobacterial SAR11 clade and secondarily by the Roseobacter group, particularly during the second half of the study. Novel members of the DMSP-degrading community emerged from dmdA sequences recovered from metagenome assemblies and single-cell sequencing, including largely uncharacterized gammaproteobacteria and alphaproteobacteria taxa. In the DMSP cleavage pathway, the SAR11 gene dddK was the most abundant early in the study, but was supplanted by dddP over time. SAR11 members, especially those harbouring genes for both DMSP degradation pathways, had a strong positive relationship with the abundance of dinoflagellates, and DMSP-degrading gammaproteobacteria co-occurred with haptophytes. This in situ study of the drivers of DMSP fate in a coastal ecosystem demonstrates for the first time correlations between specific groups of bacterial DMSP degraders and phytoplankton taxa.

Thousands of man-made synthetic chemicals are released to oceans and compose the anthropogenic dissolved organic carbon (ADOC). Little is known about the effects of this chronic pollution on marine microbiome activities. In this study, we measured the pollution level at three sites in the Northeast Subarctic Pacific Ocean (NESAP) and investigated how mixtures of three model families of ADOC at different environmentally relevant concentrations affected naturally occurring marine bacterioplankton communities' structure and metabolic functioning. The offshore northernmost site (North) had the lowest concentrations of hydrocarbons, as well as organophosphate ester plasticizers, contrasting with the two other continental shelf sites, the southern coastal site (South) being the most contaminated. At North, ADOC stimulated bacterial growth and promoted an increase in the contribution of some Gammaproteobacteria groups (e.g. Alteromonadales) to the 16 rRNA pool. These groups are described as fast responders after oil spills. In contrast, minor changes in South microbiome activities were observed. Gene expression profiles at Central showed the coexistence of ADOC degradation and stress-response strategies to cope with ADOC toxicities. These results show that marine microbial communities at three distinct domains in NESAP are influenced by background concentrations of ADOC, expanding previous assessments for polar and temperate waters., This publication is dedicated to the memory of our missed friend, mentor and colleague, Prof. Ronald P. Kiene. The authors thank the Capitan and crew of the R/V Oceanus. This work was funded by the Spanish MEIC through projects ISOMICS (CTM2015-65691-R) and SENTINEL (CTM2015-70535-P). The authors sincerely thank J.M. Gasol for support with flow cytometry counts and I. Forn for microscopy counts. The research group of Global Change and Genomic Biogeochemistry is supported by the Catalan Government (2017SGR800). IDAEA-CSIC is a Centre of Excellence Severo Ochoa (Spanish Ministry of Science and Innovation, Project CEX2018-000794-S)., With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI).

Marine Group II Euryarchaeota (Candidatus Poseidoniales), abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM), such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island (GA, USA) to metagenome-assembled genomes to determine the diversity of transcriptionally active Ca. Poseidoniales. Summer metatranscriptomes had the highest abundance of Ca. Poseidoniales transcripts, mostly from the O1 and O3 genera within Ca. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly from Ca. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the ß-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific to Ca. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment of Ca. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundant Ca. Poseidoniales in nearshore and inshore waters. Together, our data suggest that Ca. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.

Dissolved metabolites serve as nutrition, energy, and chemical signals for microbial systems. However, the full scope and magnitude of these processes in marine systems are unknown, largely due to insufficient methods, including poor extraction of small, polar compounds using common solid-phase extraction resins. Here, we utilized pre-extraction derivatization and ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to detect and quantify targeted dissolved metabolites in seawater and saline culture media. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-ISs) produced from 13C6-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples and evaluated matrix-derived biases. We have optimized this quantitative method for 73 common metabolites, of which 50 cannot be quantified without derivatization due to low extraction efficiencies. Of the 73 metabolites, 66 were identified in either culture media or seawater and 45 of those were quantified. This derivatization method is sensitive (detection limits = pM to nM), rapid (∼5 min per sample), and high throughput.

Organic carbon transfer between surface ocean photosynthetic and heterotrophic microbes is a central but poorly understood process in the global carbon cycle. In a model community in which diatom extracellular release of organic molecules sustained growth of a co-cultured bacterium, we determined quantitative changes in the diatom endometabolome and the bacterial uptake transcriptome over two diel cycles. Of the nuclear magnetic resonance (NMR) peaks in the diatom endometabolites, 38% had diel patterns with noon or mid-afternoon maxima; the remaining either increased (36%) or decreased (26%) through time. Of the genes in the bacterial uptake transcriptome, 94% had a diel pattern with a noon maximum; the remaining decreased over time (6%). Eight diatom endometabolites identified with high confidence were matched to the bacterial genes mediating their utilization. Modeling of these coupled inventories with only diffusion-based phytoplankton extracellular release could not reproduce all the patterns. Addition of active release mechanisms for physiological balance and bacterial recognition significantly improved model performance. Estimates of phytoplankton extracellular release range from only a few percent to nearly half of annual net primary production. Improved understanding of the factors that influence metabolite release and consumption by surface ocean microbes will better constrain this globally significant carbon flux.

Organic carbon transfer between photoautotrophic and heterotrophic microbes in the surface ocean mediated through metabolites dissolved in seawater is a central but poorly understood process in the global carbon cycle. In a synthetic microbial community in which diatom extracellular release of organic molecules sustained growth of a co-cultured bacterium, metabolite transfer was assessed over two diel cycles based on per cell quantification of phytoplankton endometabolites and bacterial transcripts. Of 31 phytoplankton endometabolites identified and classified into temporal abundance patterns, eight could be matched to patterns of bacterial transcripts mediating their uptake and catabolism. A model simulating the coupled endometabolite-transcription relationships hypothesized that one category of outcomes required an increase in phytoplankton metabolite synthesis in response to the presence of the bacterium. An experimental test of this hypothesis confirmed higher endometabolome accumulation in the presence of bacteria for all five compounds assigned to this category – leucine, glycerol-3-phosphate, glucose, and the organic sulfur compounds dihydroxypropanesulfonate and dimethylsulfoniopropionate. Partitioning of photosynthate into rapidly-cycling dissolved organic molecules at the expense of phytoplankton biomass production has implications for carbon sequestration in the deep ocean. That heterotrophic bacteria can impact this partitioning suggests a previously unrecognized influence on the ocean’s carbon reservoirs.Significance StatementMicrobes living in the surface ocean are critical players in the global carbon cycle, carrying out a particularly key role in the flux of carbon between the ocean and atmosphere. The release of metabolites by marine phytoplankton and their uptake by heterotrophic bacteria is one of the major routes of microbial carbon turnover. Yet the identity of these metabolites, their concentration in seawater, and the factors that affect their synthesis and release are poorly known. Here we provide experimental evidence that marine heterotrophic bacteria can affect phytoplankton production and extracellular release of metabolites. This microbial interaction has relevance for the partitioning of photosynthate between dissolved and particulate carbon reservoirs in the ocean, an important factor in oceanic carbon sequestration.

Marine Group IIEuryarchaeota(CandidatusPoseidoniales), abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM) such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island (GA, USA) to metagenome-assembled genomes to determine the diversity of transcriptionally-activeCa. Poseidoniales. Summer metatranscriptomes had the highest abundance ofCa. Poseidoniales transcripts, mostly from the O1 and O3 genera withinCa. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly fromCa. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the β-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific toCa. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment ofCa. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundantCa. Poseidoniales in nearshore and inshore waters. Together, our data suggestCa. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.

Dissolved metabolites support microbial systems via nutritional and energetic utilization and chemical signaling. However, the full scope and magnitude of these processes in marine systems is unknown largely because the mass spectrometric identification and quantification of these compounds from saline matrices has been hampered by poor extraction of small, polar compounds using common solid phase extraction resins (e.g. PPL resin, Bond Elut, Agilent). Here we utilized pre-extraction derivatization to improve the extraction, quantification, and chromatographic resolution of targeted dissolved metabolites in seawater and saline culture medium detected with ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MSMS) with Orbitrap detection. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-IS) produced from 13C-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples, and we evaluated matrix-derived biases. This method quantifies 73 metabolites in seawater, of which 50 have an extraction efficiency

Benzoyl chloride derivatization of dissolved metabolites in seawater and culture medium improves sensitivity, number of quantifiable compounds, and sample throughput. Samples are quantified using a standard curve and stable isotope labeled internal internal standards prepared from an isotopically labeled reagent. Samples are filtered (0.2 um), derivatized in basic solution and extracted from seawater/culture medium using a solid phase extraction resin (PPL, Bond Elut, Agilent). Benzoylated metabolites are detected using UPLC-MSMS with Orbitrap detection. Peaks are analyzed with Skyline.

We report 11 bacterial draft genome sequences and 38 metagenome-assembled genomes (MAGs) from marine phytoplankton exometabolite enrichments. The genomes and MAGs represent marine bacteria adapted to the metabolite environment of phycospheres, organic matter-rich regions surrounding phytoplankton cells, and are useful for exploring functional and taxonomic attributes of phytoplankton-associated bacterial communities.

Niche theory is a foundational ecological concept that explains the distribution of species in natural environments. Identifying the dimensions of any organism’s niche is challenging because numerous environmental factors can affect organism viability. We used serial invasion experiments to introduce Ruegeria pomeroyi DSS-3, a heterotrophic marine bacterium, into a coastal phytoplankton bloom on 14 dates. RNA-sequencing analysis of R. pomeroyi was conducted after 90 min to assess its niche dimensions in this dynamic ecosystem. We identified ~100 external conditions eliciting transcriptional responses, which included substrates, nutrients, metals and biotic interactions such as antagonism, resistance and cofactor synthesis. The peak bloom was characterized by favourable states for most of the substrate dimensions, but low inferred growth rates of R. pomeroyi at this stage indicated that its niche was narrowed by factors other than substrate availability, most probably negative biotic interactions with the bloom dinoflagellate. Our findings indicate chemical and biological features of the ocean environment that can constrain where heterotrophic bacteria survive. Chemical and biological features of the ocean environment that impact on survival of heterotrophic bacteria are identified by invasion experiments with Ruegeria pomeroyi.

Ruegeria pomeroyi DSS-3 is an alphaproteobacterium isolated in 1998 from coastal Georgia, USA, seawater by enriching for growth on the organic sulfur compound dimethylsulfoniopropionate (DMSP) (González and Moran 1997, Buchan et al. 2001). R. pomeroyi is a member of the Roseobacter group, which consists of marine bacteria within the family Rhodobacteraceae that are important members of bacterioplankton communities in coastal and open oceans. R. pomeroyi grows quickly in the laboratory and has a genetic system. Since its genome sequence became available in 2004 (Moran et al., 2004), R. pomeroyi has been serving as a model organism for studies of the biogeochemical, ecological, and physiological strategies of heterotrophic marine bacteria. This document catalogs the current manually curated gene annotations of the Ruegeria pomeroyi DSS-3 genome, updated as of April, 2020.

The communities of bacteria that assemble around marine microphytoplankton are predictably dominated by Rhodobacterales, Flavobacteriales, and families within the Gammaproteobacteria. Yet whether this consistent ecological pattern reflects the result of resource-based niche partitioning or resource competition requires better knowledge of the metabolites linking microbial autotrophs and heterotrophs in the surface ocean. We characterized molecules targeted for uptake by three heterotrophic bacteria individually co-cultured with a marine diatom using two strategies that vetted the exometabolite pool for biological relevance by means of bacterial activity assays: expression of diagnostic genes and net drawdown of exometabolites, the latter detected with mass spectrometry and nuclear magnetic resonance using novel sample preparation approaches. Of the more than 36 organic molecules with evidence of bacterial uptake, 53% contained nitrogen (including nucleosides and amino acids), 11% were organic sulfur compounds (including dihydroxypropanesulfonate and dimethysulfoniopropionate), and 28% were components of polysaccharides (including chrysolaminarin, chitin, and alginate). Overlap in phytoplankton-derived metabolite use by bacteria in the absence of competition was low, and only guanosine, proline, and N-acetyl-d-glucosamine were predicted to be used by all three. Exometabolite uptake pattern points to a key role for ecological resource partitioning in the assembly marine bacterial communities transforming recent photosynthate.

Aquatic environments contain large communities of microorganisms whose synergistic interactions mediate the cycling of major and trace nutrients, including vitamins. B-vitamins are essential coenzymes that many organisms cannot synthesize. Thus, their exchange among de novo synthesizers and auxotrophs is expected to play an important role in the microbial consortia and explain some of the temporal and spatial changes observed in diversity. In this study, we analyzed metatranscriptomes of a natural marine microbial community, diel sampled quarterly over one year to try to identify the potential major B-vitamin synthesizers and consumers. Transcriptomic data showed that the best-represented taxa dominated the expression of synthesis genes for some B-vitamins but lacked transcripts for others. For instance, Rhodobacterales dominated the expression of vitamin-B12 synthesis, but not of vitamin-B7 , whose synthesis transcripts were mainly represented by Flavobacteria. In contrast, bacterial groups that constituted less than 4% of the community (e.g., Verrucomicrobia) accounted for most of the vitamin-B1 synthesis transcripts. Furthermore, ambient vitamin-B1 concentrations were higher in samples collected during the day, and were positively correlated with chlorophyll-a concentrations. Our analysis supports the hypothesis that the mosaic of metabolic interdependencies through B-vitamin synthesis and exchange are key processes that contribute to shaping microbial communities in nature.

Functional ocean biogeography Marine ecosystems are well represented in metagenomic and transcriptomic data. These data are not routinely used to test ecosystem models that explore ocean biogeography or biogeochemistry. Coles et al. built a model in which genes for a range of functions were assigned to different suites of simulated microbes (see the Perspective by Rynearson). Communities emerged from the model with realistic biogeographical and biogeochemical profiles when compared to microbial data collected from the Amazon River plume. However, functional composition trumped the details of taxonomy, and different, coevolving community compositions emerged that provided similar biogeochemical outcomes. Science , this issue p. 1149 ; see also p. 1129

The trophic linkage between marine bacteria and phytoplankton in the surface ocean is a key step in the global carbon cycle, with almost half of marine primary production transformed by heterotrophic bacterioplankton within hours to weeks of fixation. Early studies conceptualized this link as the passive addition and removal of organic compounds from a shared seawater reservoir. Here, we analysed transcript and intracellular metabolite patterns in a two-member model system and found that the presence of a heterotrophic bacterium induced a potential recognition cascade in a marine phytoplankton species that parallels better-understood vascular plant response systems. Bacterium Ruegeria pomeroyi DSS-3 triggered differential expression of >80 genes in diatom Thalassiosira pseudonana CCMP1335 that are homologs to those used by plants to recognize external stimuli, including proteins putatively involved in leucine-rich repeat recognition activity, second messenger production and protein kinase cascades. Co-cultured diatoms also downregulated lipid biosynthesis genes and upregulated chitin metabolism genes. From differential expression of bacterial transporter systems, we hypothesize that nine diatom metabolites supported the majority of bacterial growth, among them sulfonates, sugar derivatives and organic nitrogen compounds. Similar recognition responses and metabolic linkages as observed in this model system may influence carbon transformations by ocean plankton.

In their role as primary producers, marine phytoplankton modulate heterotrophic bacterial activities through differences in the types and amounts of organic matter they release. This study investigates the transcriptional response of bacterium Ruegeria pomeroyi, a member of the Roseobacter clade known to affiliate with diverse phytoplankton groups in the ocean, during a shift in phytoplankton taxonomy. The bacterium was initially introduced into a culture of the dinoflagellate Alexandrium tamarense, and then experienced a change in phytoplankton community composition as the diatom Thalassiosira pseudonana gradually outcompeted the dinoflagellate. Samples were taken throughout the 30-day experiment to track shifts in bacterial gene expression informative of metabolic and ecological interactions. Transcriptome data indicate fundamental differences in the exometabolites released by the two phytoplankton. During growth with the dinoflagellate, gene expression patterns indicated that the main sources of carbon and energy for R. pomeroyi were dimethysulfoniopropionate (DMSP), taurine, methylated amines, and polyamines. During growth with the diatom, dihydroxypropanesulfonate (DHPS), xylose, ectoine, and glycolate instead appeared to fuel the bulk of bacterial metabolism. Expression patterns of genes for quorum sensing, gene transfer agent, and motility suggest that bacterial processes related to cell communication and signaling differed depending on which phytoplankton species dominated the co-culture. A remodeling of the R. pomeroyi transcriptome implicating more than a quarter of the genome occurred through the change in phytoplankton regime.

Metatranscriptomics and metagenomics data sets benchmarked with internal standards were used to characterize the expression patterns for biogeochemically relevant bacterial and archaeal genes mediating carbon, nitrogen, phosphorus and sulfur uptake and metabolism through the salinity gradient of the Amazon River Plume. The genes were identified in 48 metatranscriptomic and metagenomic data sets summing to >500 million quality-controlled reads from six locations in the plume ecosystem. The ratio of transcripts per gene copy (a direct measure of expression made possible by internal standard additions) showed that the free-living bacteria and archaea exhibited only small changes in the expression levels of biogeochemically relevant genes through the salinity and nutrient zones of the plume. In contrast, the expression levels of genes in particle-associated cells varied over orders of magnitude among the stations, with the largest differences measured for genes mediating aspects of nitrogen cycling (nifH, amtB and amoA) and phosphorus acquisition (pstC, phoX and phoU). Taxa varied in their baseline gene expression levels and extent of regulation, and most of the spatial variation in the expression level could be attributed to changes in gene regulation after removing the effect of shifting taxonomic composition. We hypothesize that changes in microbial element cycling along the Amazon River Plume are largely driven by shifting activities of particle-associated cells, with most activities peaking in the mesohaline regions where N2 fixation rates are elevated.

One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that are generated from the activities of marine phytoplankton, bacteria, grazers and viruses. Here we discuss the sources of microbial metabolites in the surface ocean, their roles in ecology and biogeochemistry, and approaches that can be used to analyse them from chemistry, biology, modelling and data science. Although microbial-derived metabolites account for only a minor fraction of the total reservoir of marine dissolved organic carbon, their flux and fate underpins the central role of the ocean in sustaining life on Earth.

Microbes play a dominant role in the biogeochemistry of coastal waters, which receive organic matter from diverse sources. We present metagenomes and 45 metagenome-assembled genomes (MAGs) from Sapelo Island, Georgia, to further understand coastal microbial populations., Microbes play a dominant role in the biogeochemistry of coastal waters, which receive organic matter from diverse sources. We present metagenomes and 45 metagenome-assembled genomes (MAGs) from Sapelo Island, Georgia, to further understand coastal microbial populations. Notably, four MAGs are archaea, with two Thaumarchaeota and two marine group II Euryarchaeota.

In the Anthropocene, in which we now live, climate change is impacting most life on Earth. Microorganisms support the existence of all higher trophic life forms. To understand how humans and other life forms on Earth (including those we are yet to discover) can withstand anthropogenic climate change, it is vital to incorporate knowledge of the microbial ‘unseen majority’. We must learn not just how microorganisms affect climate change (including production and consumption of greenhouse gases) but also how they will be affected by climate change and other human activities. This Consensus Statement documents the central role and global importance of microorganisms in climate change biology. It also puts humanity on notice that the impact of climate change will depend heavily on responses of microorganisms, which are essential for achieving an environmentally sustainable future., Nature Reviews Microbiology, 17 (9), ISSN:1740-1526, ISSN:1740-1534

Metagenomic and metatranscriptomic time-series data covering a 52-day period in the fall of 2016 provide an inventory of bacterial and archaeal community genes, transcripts, and taxonomy during an intense dinoflagellate bloom in Monterey Bay, CA, USA. The dataset comprises 84 metagenomes (0.8 terabases), 82 metatranscriptomes (1.1 terabases), and 88 16S rRNA amplicon libraries from samples collected on 41 dates. The dataset also includes 88 18S rRNA amplicon libraries, characterizing the taxonomy of the eukaryotic community during the bloom. Accompanying the sequence data are chemical and biological measurements associated with each sample. These datasets will facilitate studies of the structure and function of marine bacterial communities during episodic phytoplankton blooms., Design Type(s)transcription profiling design • sequence assembly objective • biodiversity assessment objectiveMeasurement Type(s)transcription profiling assay • marine metagenome • microbial communityTechnology Type(s)RNA sequencing • DNA sequencing • amplicon sequencingFactor Type(s)assay protocol • temporal_instantSample Characteristic(s)marine metagenome • Monterey Bay • ocean biome Machine-accessible metadata file describing the reported data (ISA-Tab format)

Global environmental changes are challenging the structure and functioning of ecosystems. However, a mechanistic understanding of how global environmental changes will affect ecosystems is still lacking. The complex and interacting biological and physical processes spanning vast temporal and spatial scales that constitute an ecosystem make this a formidable problem. A unifying framework based on ecological theory, that considers fundamental and realized niches, combined with metabolic, evolutionary, and climate change studies, is needed to provide the mechanistic understanding required to evaluate and forecast the future of marine communities, ecosystems, and their services.

The fate of the sulfur moiety of dimethylsulfoniopropionate (DMSP) depends on the 'bacterial switch', a regulatory point between two metabolic pathways with different biogeochemical endpoints. Studies have focused on transcriptional patterns of known genes to determine physiological and environmental factors affecting this switch, but post-transcriptional regulation has been under-studied. Here we use a model bacterium containing both pathways to look for transcription of non-coding regulatory small RNAs (sRNAs) during DMSP metabolism. RNA-seq analysis of Ruegeria pomeroyi DSS-3 grown with DMSP, metabolic intermediates of DMSP degradation (MMPA or acetate), or methionine revealed 182 putative sRNAs, with 46 showing differential expression during growth on DMSP. A knockout mutant constructed for an upregulated sRNA had a phenotype that differed in its use of the two degradation pathways. Because transcription patterns of many differentially expressed sRNAs were not correlated with the transcription of their putative target gene, their effects on DMSP degradation would not be observable in the transcriptome. Overall, our results indicate that sRNAs are crucial but largely cryptic actors in regulating DMSP metabolism in this model marine bacterium and potentially other bacterial groups involved in the surface ocean sulfur cycle.

Microorganisms are the foundation of the marine carbon cycle. Photosynthetic cyanobacteria and eukaryotic phytoplankton in the surface ocean use solar energy to convert carbon dioxide into energy-rich organic compounds (“carbon fixation”) that are then released when the organisms are grazed or lysed. This in turn fuels a dynamic community of heterotrophic bacteria in both the surface and deep oceans. Over a year, oceanic phytoplankton species fix as much carbon as do land plants.