60 research outputs found
Intracellular metabolites in marine microorganisms during an experiment evaluating microbial mortality
© 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 WHOIâs Ocean Life Institute (to EBK and KL). The mass spectrometry samples were analyzed at the WHOI FT-MS Usersâ Facility with instrumentation funded by the National Science Foundation (OCE-0619608 and OCE-1058448)
Dissolved organic matter in newly formed sea ice and surface seawater
© The Author(s), 2015. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Geochimica et Cosmochimica Acta 171 (2015): 39-49, doi:10.1016/j.gca.2015.08.014.Changes in sea ice in the Arctic will have ramifications on regional and global carbon
cycling. Research to date has primarily focused on the regional impacts to biological activity and
global impacts on atmospheric processes. The current project considers the molecular-level
composition of organic carbon within sea ice compared to the organic matter in seawater. The
project revealed that the composition of organic matter within sea ice was more variable than the
composition of organic matter within the surface ocean. Furthermore, sea ice samples presented
two distinct patterns in the composition of organic matter with a portion of the sea ice samples
containing protein-like organic matter. Yet, the samples were collected in the early winter period
when little biological activity is expected. Thus, one hypothesis is that physical processes acting
during the formation of sea ice selectively transferred organic matter from seawater into sea ice.
The present project expands our understanding of dissolved organic matter in sea ice and surface
seawater and thereby increases our knowledge of carbon cycling in polar regions.This research was funded by a grant from WHOIâs Arctic Research Initiative to KL.2016-09-0
Using stable isotope probing to characterize differences between free-living and sediment-associated microorganisms in the subsurface
Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Taylor & Francis for personal use, not for redistribution. The definitive version was published in Geomicrobiology Journal (2013): 362-370, doi:10.1080/01490451.2012.689090.Aquifers are subterranean reservoirs of freshwater with heterotrophic bacterial communities attached to the sediments and free-living in the groundwater. In the present study, mesocosms were used to assess factors controlling the diversity and activity of the subsurface bacterial community. The assimilation of 13C, derived from 13C-acetate, was monitored to determine whether the sediment-associated and free-living bacterial community would respond similarly to the presence of protozoan grazers. We observed a dynamic response in the sediment-associated bacterial community and none in the free-living community. The disparity in these observations highlights the importance of the sediment-associated bacterial community in the subsurface carbon cycle.This research was supported by NSF grant EAR-0525166 to EBK
Mining mass spectrometry data : using new computational tools to find novel organic compounds in complex environmental mixtures
© The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Organic Geochemistry 110 (2017): 92-99, doi:10.1016/j.orggeochem.2017.05.008.Untargeted metabolomics datasets provide ample opportunity for discovery of novel metabolites. The major challenge is focusing data analysis on a short list of metabolites. Here, we apply a combination of computational tools that serve to reduce complex mass spectrometry data in order allow us to focus on new environmentally-relevant metabolites. In the first portion of the project, we explored mass spectrometry data from intracellular metabolites extracted from a model marine diatom, Thalassiosira pseudonana. The fragmentation data from these samples were analyzed using molecular networking, an on-line tool that clusters metabolites based on shared structural similarities. The features within each metabolite cluster were then putatively annotated using MetFrag, an in silico fragmentation tool. Using this combination of computational tools, we observed multiple lyso-sulfolipids, organic compounds not previously known to exist within cultured marine diatoms. In the second stage of the project, we searched our environmental data for these lyso-sulfolipids. The lyso-sulfolipid with a C14:0 fatty acid was found in dissolved and particulate samples from the western Atlantic Ocean, and a culture of cyanobacteria grown in our laboratory. Thus, the putative lyso-sulfolipids are present in both laboratory experiments and environmental samples. This project highlights the value of combining computational tools to detect and putatively identify organic compounds not previously recognized as important within T. pseudonana or the marine environment. Future applications of these tools to emerging metabolomics data will further open the black box of natural organic matter, identifying molecules that can be used to understand and monitor the global carbon cycle.The culture experiments and subsequent data analysis were funded by the Gordon and Betty Moore Foundation through Grant GBMF3304 to EBK
Using network analysis to discern compositional patterns in ultrahigh resolution mass spectrometry data of dissolved organic matter
Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Rapid Communications in Mass Spectrometry 30 (2016): 2388-2394, doi:10.1002/rcm.7719.Marine dissolved organic matter (DOM) has long been recognized as a large and dynamic component of the global carbon cycle. Yet, DOM is chemical varied and complex and these attributes present challenges to the researchers interested in addressing questions about the role of DOM in global biogeochemical cycles.
This project analyzed organic matter extracts from seawater with direct infusion with electrospray ionization into a Fourier transform ion cyclotron resonance mass spectrometer (ESI FT-ICR-MS). We used network analysis to quantify the number of chemical transformations between mass-to-charge values in each sample. The network of chemical transformations was calculated using the MetaNetter plug-in within Cytoscape. The chemical transformations serve as markers for the shared structural characteristics of compounds within complex dissolved organic matter.
Network analysis revealed that transformations involving selected sulfur-containing moieties and isomers of amino acids were more prevalent in the deep sea than in the surface ocean. Common chemical transformations were not significantly different between the deep sea and surface ocean. Network analysis complements existing computational tools used to analyze ultrahigh resolution mass spectrometry data.
This combination of ultrahigh resolution mass spectrometry with novel computational tools has identified new potential building blocks of organic compounds in the deep sea, including the unexpected importance of dissolved organic sulfur components. The method described here can be readily applied by researchers to analyze heterogeneous and complex dissolved organic matter.This work was supported by WHOIâs Deep Ocean Exploration Institute (to EBK) and NSF OCE-1154320 (to EBK and KL).2017-08-1
Composition of dissolved organic matter in groundwater
Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 75 (2011): 2752-2761, doi:10.1016/j.gca.2011.02.020.Groundwater constitutes a globally important source of freshwater for drinking water and other agricultural and industrial purposes, and is a prominent source of freshwater flowing into the coastal ocean. Therefore, understanding the chemical components of groundwater is relevant to both coastal and inland communities. We used electrospray ionization coupled with Fourier-transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) to examine dissolved organic compounds in groundwater prior to and after passage through a sediment-filled column containing microorganisms. The data revealed that an unexpectedly high proportion of organic compounds contained nitrogen and sulfur, possibly due to transport of surface waters from septic systems and rain events. We matched 292 chemical features, based on measured mass:charge (m/z) values, to compounds stored in the Kyoto Encyclopedia of Genes and Genomes (KEGG). A subset of these compounds (88) had only one structural isomer in KEGG, thus supporting tentative identification. Most identified elemental formulas were linked with metabolic pathways that produce polyketides or with secondary metabolites produced by plants. The presence of polyketides in groundwater is notable because of their anti-bacterial and anti-cancer properties. However, their relative abundance must be quantified with appropriate analyses to assess any implications for public health.Funding was provided by NSF grants EAR-0525166 and OCE-0751897 to EBK
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Microbial diversity of sulfide structures from hydrothermal vent sites at 9°N, Guaymas Basin and the Juan de Fuca Ridge
The microbial diversity associated with sulfide structures from three
different hydrothermal vent fields was examined using phylogenetic analysis
based on the small subunit ribosomal RNA gene (16S rDNA). Samples were
collected from sites at 9°N, Guaymas Basin and the Juan de Fuca Ridge and
analyzed with denaturing gradient gel electrophoresis (DGGE). In addition,
clone libraries were constructed for two samples from Guaymas Basin. The
Bacteria from Juan de Fuca Ridge all clustered within the e-Proteobacteria, while
the Archaea identified from all three sites were Euryarchaeota. Among the
Euryarchaeota were sequences clustering within the Methanococcales,
Methanosarcinales, Methanobacteriales, and Methanomicrobiales; the later two
groups have not previously been identified as part of the microbial
community at deep-sea hydrothermal vents. Two groups of sequences were
identified that cluster with sequences previously only identified from
hydrothermal systems in the western Pacific and the Mid-Atlantic Ridge; these
groups may be endemic to deep-sea hydrothermal vents
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Bacterioplankton in the Oregon upwelling system: distribution, cell-specific leucine incorporation, and diversity
Marine bacterioplankton play an important role in global elemental cycles because they return carbon dioxide and nutrients to the biosphere as they reduce organic matter. Furthermore, marine bacterioplankton are not uniformly active, and subpopulations of the in situ community may be more or less active at any given time. Defining whether or not a cell is 'active' is not without difficulty, and the result varies depending on the assay used, since different assays examine different physiological processes within a cell. Linking the level of activity of a cell with its phylogenetic identity is an additional important step in examination of the role of marine prokaryotes in global elemental cycles. In this project, flow cytometry was used in two ways to examine relative cell-specific metabolic activity in bacterioplankton cells: as relative cell-specific nucleic acid content via staining with SYBR Green I, and as
ability to reduce sufficient 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to be identified as having an active electron transport system. Based on flow cytometric sorting of cells labeled with ÂłH-leucine, the high nucleic acid (HNA) cells had higher cell-specific leucine incorporation rates than the low nucleic acid (LNA) cells. The HNA cells were also responsible for proportionately more of the leucine incorporation by the total heterotrophic population. While the CTC-positive cells had higher average cell-specific leucine incorporation rates than the HNA cells, their low abundances meant that they were responsible for less than 15% of the total leucine incorporation. The diversity ofBacteria observed within the HNA and LNA assemblages was examined using phylogenetic analysis based on the V3-V4-V5 variable regions of 16S rRNA genes. Most of the phylogenetic groups ofBacteria identified in this study were
present in both the HNA and LNA assemblage and had, at times, an active electron transport system (i.e., were able to reduce CTC). Finally, non-metric multidimensional scaling was presented as a new method to analyze DNA sequence data in conjunction with measured environmental parameters
Organic sulfur: a spatially variable and understudied component of marine organic matter
© 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 molecularâlevel 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 sulfurâcontaining compounds were structurally diverse, and showed higher numbers of sulfurâcontaining 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 âDeepDOMâ 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 FTâMS Users' Facility that is funded by the National Science Foundation (grant OCEâ0619608) and the Gordon and Betty Moore Foundation (GMBF1214). This project was funded by NSF grants OCEâ1154320 (to EBK and KL), the W.M. Marquet Award (to KL), and OCEâ1435708 (to GAC). The authors declare no conflicts of interest
Dissolved organic matter produced by Thalassiosira pseudonana
Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Marine Chemistry 168 (2015): 114-123, doi:10.1016/j.marchem.2014.11.003.Phytoplankton are significant producers of dissolved organic matter (DOM) in marine ecosystems but the identity and dynamics of this DOM remain poorly constrained. Knowledge on the identity and dynamics of DOM are crucial for understanding the molecular-level reactions at the base of the global carbon cycle. Here we apply emerging analytical and computational tools from metabolomics to investigate the composition of DOM produced by the centric diatom Thalassiosira pseudonana. We assessed both intracellular metabolites within T. pseudonana (the endo-metabolome) and extracellular metabolites released by T. pseudonana (the exo-metabolome). The intracellular metabolites had a more variable composition than the extracellular metabolites. We putatively identified novel compounds not previously associated with T. pseudonana as well as compounds that have previously been identified within T. pseudonanaâs metabolic capacity (e.g. dimethylsulfoniopropionate and degradation products of chitin). The resulting information will provide the basis for future experiments to assess the impact of T. pseudonana on the composition of dissolved organic matter in marine environments.Instrumentation in the WHOI FT-MS facility was funded by the National Science Foundation MRI program (OCE-0619608) and by the Gordon and Betty T. Moore Foundation (Grant #1214). This work was supported by NSF grant OCE-0928424 to EBK
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