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 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)

Cryospheric science : the power of glacial microbes

Author Posting. © 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 Nature Geoscience 10 (2017): 329-330, doi:10.1038/ngeo2933.Organic carbon fluxes from glaciers are a key control on biogeochemical cycles in polar regions. Two analyses of carbon cycling in glaciers show the importance of glacier-surface microbial communities in setting these inputs

Chemical composition and potential environmental impacts of water-soluble polar crude oil components inferred from ESI FT-ICR MS

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 10 (2015): e0136376, doi:10.1371/journal.pone.0136376.Polar petroleum components enter marine environments through oil spills and natural seepages each year. Lately, they are receiving increased attention due to their potential toxicity to marine organisms and persistence in the environment. We conducted a laboratory experiment and employed state-of-the-art Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to characterize the polar petroleum components within two operationally-defined seawater fractions: the water-soluble fraction (WSF), which includes only water-soluble molecules, and the water-accommodated fraction (WAF), which includes WSF and microscopic oil droplets. Our results show that compounds with higher heteroatom (N, S, O) to carbon ratios (NSO:C) than the parent oil were selectively partitioned into seawater in both fractions, reflecting the influence of polarity on aqueous solubility. WAF and WSF were compositionally distinct, with unique distributions of compounds across a range of hydrophobicity. These compositional differences will likely result in disparate impacts on environmental health and organismal toxicity, and thus highlight the need to distinguish between these often-interchangeable terminologies in toxicology studies. We use an empirical model to estimate hydrophobicity character for individual molecules within these complex mixtures and provide an estimate of the potential environmental impacts of different crude oil components.This study is funded by the Gulf of Mexico Research Initiative (GOMRI) Project # 161684 to Dr. Elizabeth B. Kujawinski

AutoTuner: high fidelity and robust parameter selection for metabolomics data processing

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in McLean, C., & Kujawinski, E. B. AutoTuner: high fidelity and robust parameter selection for metabolomics data processing. Analytical Chemistry, 92(8), (2020): 5724-5732, doi:10.1021/acs.analchem.9b04804.Untargeted metabolomics experiments provide a snapshot of cellular metabolism but remain challenging to interpret due to the computational complexity involved in data processing and analysis. Prior to any interpretation, raw data must be processed to remove noise and to align mass-spectral peaks across samples. This step requires selection of dataset-specific parameters, as erroneous parameters can result in noise inflation. While several algorithms exist to automate parameter selection, each depends on gradient descent optimization functions. In contrast, our new parameter optimization algorithm, AutoTuner, obtains parameter estimates from raw data in a single step as opposed to many iterations. Here, we tested the accuracy and the run-time of AutoTuner in comparison to isotopologue parameter optimization (IPO), the most commonly used parameter selection tool, and compared the resulting parameters’ influence on the properties of feature tables after processing. We performed a Monte Carlo experiment to test the robustness of AutoTuner parameter selection and found that AutoTuner generated similar parameter estimates from random subsets of samples. We conclude that AutoTuner is a desirable alternative to existing tools, because it is scalable, highly robust, and very fast (∼100–1000× speed improvement from other algorithms going from days to minutes). AutoTuner is freely available as an R package through BioConductor.We thank Titus Brown and Ben Temperton for advice on the algorithm validation, Arthur Eschenlauer for constructive feedback on the software design, Krista Longnecker for continuous support and discussions, Gabriel Leventhal for mathematics advice, the users of AutoTuner for debugging help through Github, and David Angeles-Albores and two anonymous reviewers for critical feedback on the manuscript. Funding support included the National GEM Consortium and NSF graduate research program fellowships (C.M.) and grants from the MIT Microbiome Center (Award 6936800, E.B.K.) and the Simons Foundation (Award ID #509034, E.B.K.)

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 WHOI’s 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

The effect of protozoan grazers on the cycling of polychlorinated biphenyls (PCBs) in marine systems

Thesis (Ph. D.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2000.Includes bibliographical references (p. 207-219).Processes affecting organic carbon distribution and composition can control the speciation of organic contaminants such as polychlorinated biphenyls (PCBs) and ultimately determine their residence time in a particular environment. In marine systems, the microbial loop influences organic carbon dynamics by recycling a significant fraction of dissolved and particulate organic matter. The goal of this thesis was to understand how these recycling processes affect chlorobiphenyl (CB) cycling in marine systems by monitoring CB dynamics among organic carbon pools represented by dissolved organic matter, bacterial prey and phagotrophic protozoan grazers. Initially, I studied the extent to which a protozoan grazer (Uronema sp.-10[micro]m ciliate) equilibrated with aqueous PCBs within 2-3 hours. Initial calculations predicted rapid equilibration via passive diffusion. Experimentally, no difference in equilibration time was noted between grazing and non-grazing protozoa, indicating that diffusion was the primary uptake pathway for these organisms. The results were extended to determine the transition size of an organism where the rates of diffusive and ingested uptake are equivalent (100-500[micro]m). Disassociation rate constants were estimated for complexes of CB congeners and dissolved organic carbon (DOC). CB-DOC complexes enhanced the diffusive uptake rate constant for Tenax resin and, by inference, protozoan grazers. In the second phase of this work, concentrations of surfactants, organic carbon and cells were monitored over time in protozoan cultures. The effects of bacterial growth substrate and protozoan species were examined. Surfactants increased during protozoan exponential growth while total DOC concentrations decreased. Production of(cont.) surface-active material in ciliate cultures was significantly higher than in flagellate cultures, and all protozoan cultures were higher than the bacterial control. Common headspace vessels were then used to compare and contrast the affinity of protozoan and bacterial culture filtrates (<0.2[micro]m) for PCBs relative to a seawater control. Affinities were normalized to bulk DOC and surfactant concentrations to determine underlying relationships among these parameters. Values of equilibrium partition coefficients (K[oc]) ranged from 10⁴·⁶ in Vineyard Sound seawater to 10⁵·⁴ and 10⁵·⁵ in protist cultures, indicating that "grazer-enhanced" DOM was a better sorbent for PCBs than DOM in bacterial controls and Vineyard Sound seawater.by Elizabeth Belle Kujawinski.Ph.D

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 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