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Item Aerobic bacterial methane synthesis(Proceedings of the National Academy of Sciences, 2021-06) Wang, Qian; Alowaifeer, Abdullah; Kerner, Patricia; Balasubramanian, Narayanaganesh; Patterson, Angela; Christian, William; Tarver, Angela; Dore, John E.; Hatzenpichler, Roland; Bothner, Brian; McDermott, Timothy R.Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the “methane paradox” because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.Item Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress(American Society for Microbiology, 2021-02) Zhuang, Weiping; Balasubramanian, Narayanaganesh; Wang, Lu; Wang, Qian; McDermott, Timothy R.; Copie, Valerie; Wang, Gejiao; Bothner, BrianArsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited.Item Low-Temperature Biosurfactants from Polar Microbes(MDPI AG, 2020-08) Trudgeon, Benjamin; Dieser, Markus; Balasubramanian, Narayanaganesh; Messmer, Mitch; Foreman, Christine M.Surfactants, both synthetic and natural, are used in a wide range of industrial applications, including the degradation of petroleum hydrocarbons. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants, yet microorganisms fromcold environments have been largely overlooked for their biotechnological potential as biosurfactant producers. In this study, four cold-adapted bacterial isolates from Antarctica are investigated for their ability to produce biosurfactants. Here we report on the physical properties and chemical structure of biosurfactants from the genera Janthinobacterium, Psychrobacter, and Serratia. These organisms were able to grow on diesel, motor oil, and crude oil at 4 C. Putative identification showed the presence of sophorolipids and rhamnolipids. Emulsion index test (E24) activity ranged from 36.4–66.7%. Oil displacement tests were comparable to 0.1–1.0% sodium dodecyl sulfate (SDS) solutions. Data presented herein are the first report of organisms of the genus Janthinobacterium to produce biosurfactants and their metabolic capabilities to degrade diverse petroleum hydrocarbons. The organisms’ ability to produce biosurfactants and grow on different hydrocarbons as their sole carbon and energy source at low temperatures (4 C) makes them suitable candidates for the exploration of hydrocarbon bioremediation in low-temperature environments.Item Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis(Frontiers Media SA, 2020-02) Steward, Katherine F.; Eilers, Brian; Tripet, Brian; Fuchs, Amanda; Dorle, Michael; Rawle, Rachel; Soriano, Berliza; Balasubramanian, Narayanaganesh; Copie, Valerie; Bothner, Brian; Hatzenpichler, RolandBioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.Item Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis(2020-02) Steward, Katherine F.; Eilers, Brian J.; Tripet, Brian P.; Fuchs, Amanda; Dorle, Michael; Rawle, Rachel A.; Soriano, Berliza; Balasubramanian, Narayanaganesh; Copie, Valerie; Bothner, Brian; Hatzenpichler, RolandBioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.Item Quorum sensing inhibition as a promising method to control biofilm growth in metalworking fluids(2019-04) Ozcan, Safiye S.; Dieser, Markus; Parker, Albert E.; Balasubramanian, Narayanaganesh; Foreman, Christine M.Microbial contamination in metalworking systems is a critical problem. This study determined the microbial communities in metalworking fluids (MWFs) from two machining shops and investigated the effect of quorum sensing inhibition (QSI) on biofilm growth. In both operations, biofilm-associated and planktonic microbial communities were dominated by Pseudomonadales (60.2–99.7%). Rapid recolonization was observed even after dumping spent MWFs and meticulous cleaning. Using Pseudomonas aeruginosa PAO1 as a model biofilm organism, patulin (40 µM) and furanone C-30 (75 µM) were identified as effective QSI agents. Both agents had a substantially higher efficacy compared to α-amylase (extracellular polymeric substance degrading enzyme) and reduced biofilm formation by 63% and 76%, respectively, in MWF when compared to untreated controls. Reduced production of putatively identified homoserine lactones and quinoline in MWF treated with QS inhibitors support the effect of QSI on biofilm formation. The results highlight the effectiveness of QSI as a potential strategy to eradicate biofilms in MWFs.