Johnson
Winifred M.
Johnson
Winifred M.
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ArticleExtraction efficiency and quantification of dissolved metabolites in targeted marine metabolomics(John Wiley & Sons, 2017-03-20) Johnson, Winifred M. ; Kido Soule, Melissa C. ; Kujawinski, Elizabeth B.The field of metabolomics seeks to characterize the suite of small molecules that comprise the end-products of cellular regulation. Metabolomics has been used in biomedical applications as well as environmental studies that explore ecological and biogeochemical questions. We have developed a targeted metabolomics method using electrospray ionization–liquid chromatography tandem mass spectrometry to analyze metabolites dissolved in seawater. Preparation of samples from the marine environment presents challenges because dilute metabolites must be concentrated and desalted. We present the extraction efficiencies of 89 metabolites in our targeted method using solid phase extraction (SPE). In addition, we calculate the limits of detection and quantification for the metabolites in the method and compare the instrument response factors in five different matrices ranging from deionized water to spent medium from cultured marine microbes. High background organic matter content reduces the instrument response factor for only a small group of metabolites, yet enhances the extraction efficiency for other metabolites on the SPE cartridge used here, a modified styrene-divinylbenzene polymer called PPL. Aromatic or larger uncharged compounds, in particular, are reproducibly well retained on the PPL polymer. This method is suitable for the detection of dissolved metabolites in marine samples, with limits of detection ranging from < 1 pM to ∼ 2 nM dependent on the dual impacts of seawater matrix on extraction efficiency and on instrument response factors.
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ArticleAuxotrophic interactions: A stabilizing attribute of aquatic microbial communities?(Oxford University Press, 2020-06-10) Johnson, Winifred M. ; Alexander, Harriet ; Bier, Raven L. ; Miller, Dan R. ; Muscarella, Mario E. ; Pitz, Kathleen J. ; Smith, HeidiAuxotrophy, or an organism's requirement for an exogenous source of an organic molecule, is widespread throughout species and ecosystems. Auxotrophy can result in obligate interactions between organisms, influencing ecosystem structure and community composition. We explore how auxotrophy-induced interactions between aquatic microorganisms affect microbial community structure and stability. While some studies have documented auxotrophy in aquatic microorganisms, these studies are not widespread, and we therefore do not know the full extent of auxotrophic interactions in aquatic environments. Current theoretical and experimental work suggests that auxotrophy links microbial community members through a complex web of metabolic dependencies. We discuss the proposed ways in which auxotrophy may enhance or undermine the stability of aquatic microbial communities, highlighting areas where our limited understanding of these interactions prevents us from being able to predict the ecological implications of auxotrophy. Finally, we examine an example of auxotrophy in harmful algal blooms to place this often theoretical discussion in a field context where auxotrophy may have implications for the development and robustness of algal bloom communities. We seek to draw attention to the relationship between auxotrophy and community stability in an effort to encourage further field and theoretical work that explores the underlying principles of microbial interactions.
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ArticleThe osmolyte ties that bind: genomic insights into synthesis and breakdown of organic osmolytes in marine microbes(Frontiers Media, 2021-07-14) McParland, Erin L. ; Alexander, Harriet ; Johnson, Winifred M.The production and consumption of organic matter by marine organisms plays a central role in the marine carbon cycle. Labile organic compounds (metabolites) are the major currency of energetic demands and organismal interaction, but these compounds remain elusive because of their rapid turnover and concomitant minuscule concentrations in the dissolved organic matter pool. Organic osmolytes are a group of small metabolites synthesized at high intracellular concentrations (mM) to regulate cellular osmolarity and have the potential to be released as abundant dissolved substrates. Osmolytes may represent an essential currency of exchange among heterotrophic prokaryotes and primary and secondary producers in marine food webs. For example, the well-known metabolite dimethylsulfoniopropionate (DMSP) is used as an osmolyte by some phytoplankton and can be subsequently metabolized by 60% of the marine bacterial community, supplying up to 13% of the bacterial carbon demand and 100% of the bacterial sulfur demand. While marine osmolytes have been studied for decades, our understanding of their cycling and significance within microbial communities is still far from comprehensive. Here, we surveyed the genes responsible for synthesis, breakdown, and transport of 14 key osmolytes. We systematically searched for these genes across marine bacterial genomes (n = 897) and protistan transcriptomes (n = 652) using homologous protein profiles to investigate the potential for osmolyte metabolisms. Using the pattern of gene presence and absence, we infer the metabolic potential of surveyed microbes to interact with each osmolyte. Specifically, we identify: (1) complete pathways for osmolyte synthesis in both prokaryotic and eukaryotic marine microbes, (2) microbes capable of transporting osmolytes but lacking complete synthesis and/or breakdown pathways, and (3) osmolytes whose synthesis and/or breakdown appears to be specialized and is limited to a subset of organisms. The analysis clearly demonstrates that the marine microbial loop has the genetic potential to actively recycle osmolytes and that this abundant group of small metabolites may function as a significant source of nutrients through exchange among diverse microbial groups that significantly contribute to the cycling of labile carbon.
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ArticleEvidence for quorum sensing and differential metabolite production by a marine bacterium in response to DMSP(Nature Publishing Group, 2016-02-16) Johnson, Winifred M. ; Kido Soule, Melissa C. ; Kujawinski, Elizabeth B.Microbes, the foundation of the marine foodweb, do not function in isolation, but rather rely on molecular level interactions among species to thrive. Although certain types of interactions between autotrophic and heterotrophic microorganisms have been well documented, the role of specific organic molecules in regulating inter-species relationships and supporting growth are only beginning to be understood. Here, we examine one such interaction by characterizing the metabolic response of a heterotrophic marine bacterium, Ruegeria pomeroyi DSS-3, to growth on dimethylsulfoniopropionate (DMSP), an abundant organosulfur metabolite produced by phytoplankton. When cultivated on DMSP, R. pomeroyi synthesized a quorum-sensing molecule, N-(3-oxotetradecanoyl)-l-homoserine lactone, at significantly higher levels than during growth on propionate. Concomitant with the production of a quorum-sensing molecule, we observed differential production of intra- and extracellular metabolites including glutamine, vitamin B2 and biosynthetic intermediates of cyclic amino acids. Our metabolomics data indicate that R. pomeroyi changes regulation of its biochemical pathways in a manner that is adaptive for a cooperative lifestyle in the presence of DMSP, in anticipation of phytoplankton-derived nutrients and higher microbial density. This behavior is likely to occur on sinking marine particles, indicating that this response may impact the fate of organic matter.
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ArticlePathway-centric analysis of microbial metabolic potential and expression along nutrient and energy gradients in the western Atlantic Ocean(Frontiers Media, 2022-05-19) Cavaco, Maria A. ; Bhatia, Maya P. ; Hawley, Alyse K. ; Torres-Beltrán, Mónica ; Johnson, Winifred M. ; Longnecker, Krista ; Konwar, Kishori ; Kujawinski, Elizabeth B. ; Hallam, Steven J.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 across depth and space, we were able to observe how changes in the marine metabolite pool may be influenced by microbial function and vice versa. Taken together, these results indicate that marine microbial communities encode a core repertoire of widely distributed metabolic pathways that are differentially regulated along nutrient and energy gradients. Such pathway distribution patterns are consistent with robustness in microbial food webs and indicate a high degree of functional redundancy.
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ThesisLinking microbial metabolism and organic matter cycling through metabolite distributions in the ocean(Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 2017-02) Johnson, Winifred M. ; Kujawinski, Elizabeth ; Lomas, MichaelKey players in the marine carbon cycle are the ocean-dwelling microbes that fix, remineralize, and transform organic matter. Many of the small organic molecules in the marine carbon pool have not been well characterized and their roles in microbial physiology, ecological interactions, and carbon cycling remain largely unknown. In this dissertation metabolomics techniques were developed and used to profile and quantify a suite of metabolites in the field and in laboratory experiments. Experiments were run to study the way a specific metabolite can influence microbial metabolite output and potentially processing of organic matter. Specifically, the metabolic response of the heterotrophic marine bacterium, Ruegeria pomeroyi, to the algal metabolite dimethylsulfoniopropionate (DMSP) was analyzed using targeted and untargeted metabolomics. The manner in which DMSP causes R. pomeroyi to modify its biochemical pathways suggests anticipation by R. pomeroyi of phytoplankton-derived nutrients and higher microbial density. Targeted metabolomics was used to characterize the latitudinal and vertical distributions of particulate and dissolved metabolites in samples gathered along a transect in the Western Atlantic Ocean. The assembled dataset indicates that, while many metabolite distributions co-vary with biomass abundance, other metabolites show distributions that suggest abiotic, species specific, or metabolic controls on their variability. On sinking particles in the South Atlantic portion of the transect, metabolites possibly derived from degradation of organic matter increase and phytoplankton-derived metabolites decrease. This work highlights the role DMSP plays in the metabolic response of a bacterium to the environment and reveals unexpected ways metabolite abundances vary between ocean regions and are transformed on sinking particles. Further metabolomics studies of the global distributions and interactions of marine biomolecules promise to provide new insights into microbial processes and metabolite cycling.
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ArticleMetabolite composition of sinking particles differs from surface suspended particles across a latitudinal transect in the South Atlantic(Wiley, 2019-07-31) Johnson, Winifred M. ; Longnecker, Krista ; Kido Soule, Melissa C. ; Arnold, William A. ; Bhatia, Maya P. ; Hallam, Steven J. ; Van Mooy, Benjamin A. S. ; Kujawinski, Elizabeth B.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.
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ArticleParticulate and dissolved metabolite distributions along a latitudinal transect of the western Atlantic Ocean(Association for the Sciences of Limnology and Oceanography, 2022-12-23) Johnson, Winifred M. ; Kido Soule, Melissa C. ; Longnecker, Krista ; Bhatia, Maya P. ; Hallam, Steven J. ; Lomas, Michael W. ; Kujawinski, Elizabeth B.Metabolites, or the small organic molecules that are synthesized by cells during metabolism, comprise a complex and dynamic pool of carbon in the ocean. They are an essential currency in interactions at the population and community levels of biological organization. Characterizing metabolite distributions inside microbial cells and dissolved in seawater is essential to understanding the controls on their production and fate, as well as their roles in shaping marine microbial food webs. Here, we apply a targeted metabolomics method to quantify particulate and dissolved distributions of a suite of biologically relevant metabolites including vitamins, amino acids, nucleic acids, osmolytes, and intermediates in biosynthetic pathways along a latitudinal transect in the western Atlantic Ocean. We find that, in the upper 200 m of the water column, most particulate or intracellular metabolites positively covary with the most abundant microbial taxa. In contrast, dissolved metabolites exhibited greater variability with differences in distribution between ocean regions. Although fewer particulate metabolites were detected below 200 m, the particulate metabolites identified in the deep ocean may be linked to adaptive physiological strategies of deep‐sea microbes. Based on the identified metabolite distributions, we propose relationships between certain metabolites and microbial populations, and find that dissolved metabolite distributions are not directly related to their particulate abundances.
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ArticleSeasonal and daily patterns in known dissolved metabolites in the northwestern Sargasso Sea(Association for the Sciences of Limnology and Oceanography, 2024-01-10) Longnecker, Krista ; Kido Soule, Melissa C. ; Swarr, Gretchen J. ; Parsons, Rachel J. ; Liu, Shuting ; Johnson, Winifred M. ; Widner, Brittany ; Curry, Ruth G. ; Carlson, Craig A. ; Kujawinski, Elizabeth B.Organic carbon in seawater plays a significant role in the global carbon cycle. The concentration and composition of dissolved organic carbon reflect the activity of the biological community and chemical reactions that occur in seawater. From 2016 to 2019, we repeatedly sampled the oligotrophic northwest Sargasso Sea in the vicinity of the Bermuda Atlantic Time-series Study site (BATS) to quantitatively follow known compounds within the pool of dissolved organic matter in the upper 1000 m of the water column. Most metabolites showed surface enrichment, and 83% of the metabolites had significantly lower concentrations with increasing depth. Dissolved metabolite concentrations most notably revealed temporal variability. Fourteen metabolites displayed seasonality that was repeated in each of the 4 yr sampled. Concentrations of vitamins, including pantothenic acid (vitamin B5) and riboflavin (vitamin B2), increased annually during winter periods when mixed layer depths were deepest. During diel sampling, light-sensitive riboflavin decreased significantly during daylight hours. The temporal variability in metabolites at BATS was less than the spatial variability in metabolites from a previous sample set collected over a broad latitudinal range in the western Atlantic Ocean. The metabolites examined in this study are all components of central carbon metabolism. By examining these metabolites at finer resolution and in a time-series, we begin to provide insights into the chemical compounds that may be exchanged by microorganisms in marine systems, data which are fundamental to understanding the chemical response of marine systems to future changes in climate.