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Dataset: Reef biogeochemistry, metabolomics, and metagenomics, This comparative 'omics dataset was collected over 15 days in June 2019 along Florida's Coral Reef. We assessed 85 reefs for the prevalence of stony coral tissue loss disease (SCTLD), nutrients (total organic carbon (TOC), total organic nitrogen (TON), inorganic nutrients), and abundances of microbial functional groups (Prochlorococcus, Synechococcus, picoeukaryotes, and heterotrophic microbes (unpigmented bacteria and archaea)), from reef depth waters. At 45 of the reefs, high-resolution photomosaics were used to examine the composition of benthic organisms. At 13 geographically dispersed reefs, we collected seawater (1.7 liters in biological triplicates) for both targeted and untargeted metabolomics analyses. Seawater (2 liters in duplicate) was collected at 26 sites, including the 13 examined for metabolomics, for taxonomic (bacteria and archaea 16S ribosomal RNA gene) and functional (shotgun metagenome) microbiome analyses, and chlorophyll. Given the stony coral tissue loss disease outbreak, we also targeted healthy and diseased coral tissue and near-coral seawater for taxonomic microbiome (16S rRNA gene) analysis (11 sites). Significance: Microorganisms and the dissolved metabolites they process are central to the functioning of ocean ecosystems. These 'invisible' ocean components are poorly understood in biodiverse and productive coral reef ecosystems, where they contribute to nutrient cycling and signaling cues between reef organisms. Microbes and dissolved metabolites offer a new means to examine reef features and have applications for conservation, monitoring, and restoration efforts in these changing ecosystems. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/890979, NSF Division of Ocean Sciences (NSF OCE) OCE-1736288

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Cavaco, M. A., Bhatia, M. P., Hawley, A. K., Torres-Beltran, M., Johnson, W. M., Longnecker, K., Konwar, K., Kujawinski, E. B., & Hallam, S. J. Pathway-centric analysis of microbial metabolic potential and expression along nutrient and energy gradients in the western Atlantic Ocean. Frontiers in Marine Science, 9, (2022): 867310, https://doi.org/10.3389/fmars.2022.867310., Microbial communities play integral roles in driving nutrient and energy transformations in the ocean, collectively contributing to fundamental biogeochemical cycles. Although it is well known that these communities are stratified within the water column, there remains limited knowledge of how metabolic pathways are distributed and expressed. Here, we investigate pathway distribution and expression patterns from surface (5 m) to deep dark ocean (4000 m) at three stations along a 2765 km transect in the western South Atlantic Ocean. This study is based on new data, consisting of 43 samples for 16S rRNA gene sequencing, 20 samples for metagenomics and 19 samples for metatranscriptomics. Consistent with previous observations, we observed vertical zonation of microbial community structure largely partitioned between light and dark ocean waters. The metabolic pathways inferred from genomic sequence information and gene expression stratified with depth. For example, expression of photosynthetic pathways increased in sunlit waters. Conversely, expression of pathways related to carbon conversion processes, particularly those involving recalcitrant and organic carbon degradation pathways (i.e., oxidation of formaldehyde) increased in dark ocean waters. We also observed correlations between indicator taxa for specific depths with the selective expression of metabolic pathways. For example, SAR202, prevalent in deep waters, was strongly correlated with expression of the methanol oxidation pathway. From a biogeographic perspective, microbial communities along the transect encoded similar metabolic potential with some latitudinal stratification in gene expression. For example, at a station influenced by input from the Amazon River, expression of pathways related to oxidative stress was increased. Finally, when pairing distinct correlations between specific particulate metabolites (e.g., DMSP, AMP and MTA) and both the taxonomic microbial community and metatranscriptomic pathways, This work was funded by the NSF Division of Ocean Sciences (Grant no. OCE-1154320 to EK and KL) and a small (“Microbial controls on marine organic carbon cycling”) and large (“Marine microbial communities from the Southern Atlantic Ocean transect to study dissolved organic matter and carbon cycling”) community sequencing grants from the Joint Genome Institute (US Department of Energy, Walnut Creek, CA) to SH and MB. MB was supported by an NSERC post-doctoral fellowship and a CIFAR Global Scholars fellowship. MC was supported by a Campus Alberta Innovates Program (CAIP) chair to MB.

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Weber, L., Armenteros, M., Soule, M. K., Longnecker, K., Kujawinski, E. B., & Apprill, A. Extracellular reef metabolites across the protected Jardines de la Reina, Cuba Reef System. Frontiers in Marine Science, 7, (2020): 582161, https://doi.org/10.3389/fmars.2020.582161., Coral reef ecosystems are incredibly diverse marine biomes that rely on nutrient cycling by microorganisms to sustain high productivity in generally oligotrophic regions of the ocean. Understanding the composition of extracellular reef metabolites in seawater, the small organic molecules that serve as the currency for microorganisms, may provide insight into benthic-pelagic coupling as well as the complexity of nutrient cycling in coral reef ecosystems. Jardines de la Reina (JR), Cuba is an ideal environment to examine extracellular metabolites across protected and high-quality reefs. Here, we used liquid chromatography mass spectrometry (LC-MS) to quantify specific known metabolites of interest (targeted metabolomics approach) and to survey trends in metabolite feature composition (untargeted metabolomics approach) from surface and reef depth (6 – 14 m) seawater overlying nine forereef sites in JR. We found that untargeted metabolite feature composition was surprisingly similar between reef depth and surface seawater, corresponding with other biogeochemical and physicochemical measurements and suggesting that environmental conditions were largely homogenous across forereefs within JR. Additionally, we quantified 32 of 53 detected metabolites using the targeted approach, including amino acids, nucleosides, vitamins, and other metabolic intermediates. Two of the quantified metabolites, riboflavin and xanthosine, displayed interesting trends by depth. Riboflavin concentrations were higher in reef depth compared to surface seawater, suggesting that riboflavin may be produced by reef organisms at depth and degraded in the surface through photochemical oxidation. Xanthosine concentrations were significantly higher in surface reef seawater. 5′-methylthioadenosine (MTA) concentrations increased significantly within the central region of the archipelago, displaying biogeographic patterns that warrant further investigation. Here we lay the groundwork for future investigation, This work was supported by the Dalio Foundation (now “OceanX”) and the National Science Foundation (OCE-1736288) (award to Amy Apprill). The mass spectrometry samples were analyzed at the WHOI FT-MS Users’ Facility with instrumentation funded by the National Science Foundation (grant OCE-1058448 to EK and MK) and the Simons Foundation (Award ID #509042, EK). Lastly, a portion of the publication fees was supported by the Massachusetts Institute of Technology (MIT) Open Access Article Publication Subvention fund from MIT Libraries.

Coral reef ecosystems are incredibly diverse marine biomes that rely on nutrient cycling by microorganisms to sustain high productivity in generally oligotrophic regions of the ocean. Understanding the composition of extracellular reef metabolites in seawater, the small organic molecules that serve as the currency for microorganisms, may provide insight into benthic-pelagic coupling as well as the complexity of nutrient cycling in coral reef ecosystems. Jardines de la Reina (JR), Cuba is an ideal environment to examine extracellular metabolites across protected and high-quality reefs. Here, we used liquid chromatography mass spectrometry (LC-MS) to quantify specific known metabolites of interest (targeted metabolomics approach) and to survey trends in metabolite feature composition (untargeted metabolomics approach) from surface and reef depth (6 – 14 m) seawater overlying nine forereef sites in JR. We found that untargeted metabolite feature composition was surprisingly similar between reef depth and surface seawater, corresponding with other biogeochemical and physicochemical measurements and suggesting that environmental conditions were largely homogenous across forereefs within JR. Additionally, we quantified 32 of 53 detected metabolites using the targeted approach, including amino acids, nucleosides, vitamins, and other metabolic intermediates. Two of the quantified metabolites, riboflavin and xanthosine, displayed interesting trends by depth. Riboflavin concentrations were higher in reef depth compared to surface seawater, suggesting that riboflavin may be produced by reef organisms at depth and degraded in the surface through photochemical oxidation. Xanthosine concentrations were significantly higher in surface reef seawater. 5′-methylthioadenosine (MTA) concentrations increased significantly within the central region of the archipelago, displaying biogeographic patterns that warrant further investigation. Here we lay the groundwork for future investigation, National Science Foundation (Grant OCE-1736288)

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Longnecker, K., & Kujawinski, E. B. Intracellular metabolites in marine microorganisms during an experiment evaluating microbial mortality. Metabolites, 10(3), (2020): 105, doi: 10.3390/metabo10030105., Metabolomics is a tool with immense potential for providing insight into the impact of biological processes on the environment. Here, we used metabolomics methods to characterize intracellular metabolites within marine microorganisms during a manipulation experiment that was designed to test the impact of two sources of microbial mortality, protozoan grazing and viral lysis. Intracellular metabolites were analyzed with targeted and untargeted mass spectrometry methods. The treatment with reduced viral mortality showed the largest changes in metabolite concentrations, although there were organic compounds that shifted when the impact of protozoan grazers was reduced. Intracellular concentrations of guanine, phenylalanine, glutamic acid, and ectoine presented significant responses to changes in the source of mortality. Unexpectedly, variability in metabolite concentrations were not accompanied by increases in microbial abundance which indicates that marine microorganisms altered their internal organic carbon stores without changes in biomass or microbial growth. We used Weighted Correlation Network Analysis (WGCNA) to identify correlations between the targeted and untargeted mass spectrometry data. This analysis revealed multiple unknown organic compounds were correlated with compatible solutes, also called osmolytes or chemical chaperones, which emphasizes the dominant role of compatible solutes in marine microorganisms., This research was funded by the US National Science Foundation (OCE-1154320 to EBK and KL, OCE-1634016 to EBK) and WHOI’s Ocean Life Institute (to EBK and KL). The mass spectrometry samples were analyzed at the WHOI FT-MS Users’ Facility with instrumentation funded by the National Science Foundation (OCE-0619608 and OCE-1058448).

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Longnecker, K., Oswald, L., Soule, M. C. K., Cutter, G. A., & Kujawinski, E. B. Organic sulfur: a spatially variable and understudied component of marine organic matter. Limnology and Oceanography Letters, (2020), doi:10.1002/lol2.10149., Sulfur (S) is a major heteroatom in organic matter. This project evaluated spatial variability in the concentration and molecular‐level composition of organic sulfur along gradients of depth and latitude. We measured the concentration of total organic sulfur (TOS) directly from whole seawater. Our data reveal high variability in organic sulfur, relative to established variability in total organic carbon or nitrogen. The deep ocean contained significant amounts of organic sulfur, and the concentration of TOS in North Atlantic Deep Water (NADW) decreased with increasing age while total organic carbon remained stable. Analysis of dissolved organic matter extracts by ultrahigh resolution mass spectrometry revealed that 6% of elemental formulas contained sulfur. The sulfur‐containing compounds were structurally diverse, and showed higher numbers of sulfur‐containing elemental formulas as NADW moved southward. These measurements of organic sulfur in seawater provide the foundation needed to define the factors controlling organic sulfur in the global ocean., We thank Catherine Carmichael, Winifred Johnson, and Gretchen Swarr for assistance with sample collection and processing, and Joe Jennings for the analysis of inorganic nutrients. The help of the captain and crew of the R/V Knorr and the other cruise participants during the “DeepDOM” cruise is appreciated. Two anonymous reviewers and Patricia Soranno provided thorough comments that greatly improved the manuscript. The ultrahigh resolution mass spectrometry samples were analyzed at the WHOI FT‐MS Users' Facility that is funded by the National Science Foundation (grant OCE‐0619608) and the Gordon and Betty Moore Foundation (GMBF1214). This project was funded by NSF grants OCE‐1154320 (to EBK and KL), the W.M. Marquet Award (to KL), and OCE‐1435708 (to GAC). The authors declare no conflicts of interest.

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Saw, J. H. W., Nunoura, T., Hirai, M., Takaki, Y., Parsons, R., Michelsen, M., Longnecker, K., Kujawinski, E. B., Stepanauskas, R., Landry, Z., Carlson, C. A., & Giovannoni, S. J. Pangenomics analysis reveals diversification of enzyme families and niche specialization in globally abundant SAR202 bacteria. Mbio, 11(1), (2020): e02975-19, doi:10.1128/mBio.02975-19., It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns. IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Informat, We thank the captain, crew, ROV and CTD operation teams, and science party of the JAMSTEC RV Kairei cruises KR11-11, KR12-19, and KR14-01. We thank the staff of the Bigelow Laboratory for Ocean Sciences’ Single Cell Genomics Center for the generation of single-cell genomic data. We thank Mark Dasenko from the Center for Genome Research and Biocomputing at Oregon State University for sequencing six of the Illumina SAG libraries. We thank the captain, crew and CTD operations team of the RV Atlantic Explorer (cruise AE1712). T.N. was supported in part by a Grant-in-Aid for Scientific Research (B) (30070015) from the Japan Society for the Promotion of Science (JSPS). The mass spectrometry samples were analyzed at the WHOI FT-MS Users’ Facility; funding for data collection and analysis came from the National Science Foundation (NSF Grant OCE-1154320 to E.B.K. and K.L.). This work was funded by Simons Foundation International as part of the BIOS-SCOPE initiative (S.J.G., C.A.C., and E.B.K.), and by NSF grants OCE-1335810 and DEB-1441717 to R.S. This work was funded by National Science Foundation grant OCE-1436865.

Dataset: Total Organic Sulfur, This dataset contains the concentration of total organic sulfur (TOS) in seawater from samples collected during the R/V Knorr cruise KN210-04 between 29 Mar 2013 and 06 May 2013 along the eastern coast of South America. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/745536, NSF Division of Ocean Sciences (NSF OCE) OCE-1154320

Dataset: CTD, Processed CTD data from all sensors mounted on the rosette from the KN210-04 cruise. Data include temperature, salinity, dissolved oxygen, fluorometry, turbidity, PAR, and SPAR. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/481164, NSF Division of Ocean Sciences (NSF OCE) OCE-1154320

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in [citation], doi:[doi]. Johnson, W. M., Longnecker, K., Soule, M. C. K., Arnold, W. A., Bhatia, M. P., Hallam, S. J., Van Mooy, B. A. S., & Kujawinski, E. B. Metabolite composition of sinking particles differs from surface suspended particles across a latitudinal transect in the South Atlantic. Limnology and Oceanography, (2019), doi:10.1002/lno.11255., Marine sinking particles transport carbon from the surface and bury it in deep‐sea sediments, where it can be sequestered on geologic time scales. The combination of the surface ocean food web that produces these particles and the particle‐associated microbial community that degrades them creates a complex set of variables that control organic matter cycling. We use targeted metabolomics to characterize a suite of small biomolecules, or metabolites, in sinking particles and compare their metabolite composition to that of the suspended particles in the euphotic zone from which they are likely derived. These samples were collected in the South Atlantic subtropical gyre, as well as in the equatorial Atlantic region and the Amazon River plume. The composition of targeted metabolites in the sinking particles was relatively similar throughout the transect, despite the distinct oceanic regions in which they were generated. Metabolites possibly derived from the degradation of nucleic acids and lipids, such as xanthine and glycine betaine, were an increased mole fraction of the targeted metabolites in the sinking particles relative to surface suspended particles, while algal‐derived metabolites like the osmolyte dimethylsulfoniopropionate were a smaller fraction of the observed metabolites on the sinking particles. These compositional changes are shaped both by the removal of metabolites associated with detritus delivered from the surface ocean and by production of metabolites by the sinking particle‐associated microbial communities. Furthermore, they provide a basis for examining the types and quantities of metabolites that may be delivered to the deep sea by sinking particles., The authors would like to thank the captain and crew of the R/V Knorr and R/V Atlantic Explorer, as well as Justin Ossolinski, Catherine Carmichael, and Sean Sylva for helping to make this data set possible. Special thanks to Colleen Durkin for sharing her data and providing feedback on the manuscript. Funding for this work came from the National Science Foundation (NSF Grant OCE‐1154320 to EBK and KL) and a WHOI Ocean Ventures Fund award to WMJ. The instruments in the WHOI FT‐MS Facility were purchased with support from the Gordon & Betty Moore Foundation and NSF. Support for WMJ was provided by a National Defense Science and Engineering Fellowship. Sequencing was performed under the auspices of the US Department of Energy (DOE) JGI Community Science Program (CSP) project (CSP 1685) supported by the Office of Science of US DOE Contract DE‐AC02‐ 05CH11231. Additional work related to sample collection and processing was supported by the G. Unger Vetlesen and Ambrose Monell Foundations, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institute for Advanced Study (CIFAR), and the Canada Foundation for Innovation through grants awarded to SJH. MPB was supported by a CIFAR Global Scholarship and NSERC postdoctoral fellowship.

Dataset: nutrients, Inorganic and organic nutrient data from Niskin bottles from the KN210-04 cruise. Data include concentration of: phosphate, nitrate+nitrite, silicate, nitrite, ammonium, non-purgeable organic carbon, and total nitrogen. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/473296, NSF Division of Ocean Sciences (NSF OCE) OCE-1154320