Functional annotation and importance of marine bacterial transporters of plankton exometabolites

William F. Schroer, Hannah E. Kepner, Mario Uchimiya, Catalina Mejia, Lidimarie Trujillo Rodriguez, Christopher R. Reisch & Mary Ann Moran

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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 currencies of carbon flux. We employed an arrayed mutant library of the marine bacterium Ruegeria pomeroyi DSS-3 to experimentally annotate substrates of organic compound transporter systems, using mutant growth and compound drawdown analyses to link transporters to their cognate substrates. Mutant experiments verified substrates for thirteen R. pomeroyi transporters. 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). These bring the total number of experimentally-verified organic carbon influx transporters to 18 of 126 in the R. pomeroyi genome. 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 annotation of the gatekeepers of organic carbon uptake is critical for deciphering carbon flux and fate in microbial ecosystems.

Ecological divergence of syntopic marine bacterial species is shaped by gene content and expression

Brent Nowinski, Xiaoyuan Feng, Christina M. Preston, James M. Birch, Haiwei Luo, William B. Whitman & Mary Ann Moran

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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.

A Mutant Fitness Assay Identifies Bacterial Interactions in a Model Ocean Hot-Spot

Jeremy E. Schreier, Christa B. Smith, Thomas R. Ioerger, Mary Ann Moran

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Ecological interactions that occur between phycosphere-associated bacteria at the micron scale have the potential to influence a major fraction of annual carbon flux at the global scale. Despite the importance of microbial carbon flow, studying the ecology of these microenvironments remains challenging. We identified genes that mediate bacterial interactions in phycosphere communities by culturing a transposon mutant library of Ruegeroai 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 that significantly impacted R. pomeroyi fitness indicated explicit cell-cell interactions occurring in the multi-bacterial 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 elucidated bacteria competition for nitrogen, cross-feeding of amino acids, purines, pyrimidines, and cofactors, antagonistic interactions between bacterial species, and evidence for altered oxygen and superoxide levels driven by microbial activity. These results provided insights into the potential mechanisms of interspecies interactions in a model marine hot-spot of biogeochemical importance.

Bacterial transcriptional response to labile exometabolites from photosynthetic picoeukaryote Micromonas commoda

Frank X. Ferrer-González, Maria Hamilton, Christa B. Smith, Jeremy E. Schreier, Malin Olofsson & Mary Ann Moran

ISME Communications

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Much of the growth of heterotrophic marine bacteria is dependent on the dissolved organic carbon released by phytoplankton, yet the composition of algal exudates and how this may differ among taxa is still poorly understood. Here, we have utilized transcriptomic data from co-cultures of model marine bacteria grown with the picoeukaryote alga, Micromonas commoda, to characterize the exometabolite pool of this phytoplankton. We predicted that the bacteria recognized 38 different metabolites released by M. commoda, based on the expression of catabolic and transporter genes. Several of these metabolites, including some sulfur- and nitrogen- containing compounds, were unique to this alga, when compared to the exometabolites released by the diatom Thalassiosira pseudonana, as assessed previously using the same method. These results suggest that as phytoplankton communities shift towards dominance by smaller picophytoplankton over larger diatoms in a warming ocean, the pool of substrates available to heterotrophic bacteria may also be altered, with implications for global biogeochemical cycles.

Microbial Metabolites in the Marine Carbon Cycle

M. A. Moran, E. B. Kujawinski, W. F. Schroer, S. A. Amin, N. R. Bates, E. M. Bertrand, R. Braakman, C. T. Brown, M. W. Covert, S. C. Doney, S. T. Dyhrman, A. S. Edison, A. M. Eren, N. M. Levine, L. Li, A. C. Ross, M. A. Saito, A. E. Santoro, D. Segré, A. Shade, M. B. Sullivan, A. Vardi

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One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that is 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 (DOC), their flux and fate underpins the central role of the ocean in sustaining life on Earth. 

The Ocean’s Labile DOC Supply Chain

M. A. Moran, F. X. Ferrer-González, H. Fu, B. Nowinski, M. Olofsson, M. A. Powers, J. E. Schreier, W. F. Schroer, C. B. Smith, and M. Uchimiya

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Microbes of the surface ocean release, consume, and exchange labile metabolites at time scales of minutes to days. The details of this important step in the global carbon cycle remain poorly resolved, largely due to the methodological challenges of studying a diverse pool of metabolites that are produced and consumed nearly simultaneously. In this perspective, a new compilation of published data builds on previous studies to obtain an updated estimate of the fraction of marine net primary production that passes through the labile dissolved organic carbon (DOC) pool. In agreement with previous studies, our data mining and modeling approaches hypothesize that about half of ocean net primary production is processed through the labile DOC pool. The fractional contributions from three major sources are estimated at 0.4 for living phytoplankton, 0.4 for dead and dying phytoplankton, and 0.2 for heterotrophic microbes and mesoplankton.

Growth-Stage Related Shifts in Diatom Endometabolome Composition Set the Stage for Bacterial Heterotrophy

M. Olofsson, F. X. Ferrer-González, M. Uchimiya, J. E. Schreier, N. R. Holderman, C. B. Smith, A. S. Edison, M. A. Moran

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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.

Niche Dimensions of a Marine Bacterium are Identified Using Invasion Studies in Coastal Seawater

B. Nowinski, M. A. Moran

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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.

Diel Investments in Metabolite Production and Consumption in a Model Microbial System

M. Uchimiya, W. Schroer, M. Olofsson, A. S. Edison, M. A. Moran

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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. 

Resource Partitioning of Phytoplankton Metabolites that Support Bacterial Heterotrophy

Ferrer-González, F. X.,  B. Widner, N. R. Holderman, J. Glushka, A. S. Edison, E. B. Kujawinski, and  M. A. Moran

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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 (MS) and nuclear magnetic resonance (NMR) 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.