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Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling

ABSTRACT: Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance a versatile approach to elaborate cellular metabolism in obligate anaerobes using the pathogen Clostridioides difficile, an amino acid and carbohydrate-fermenting Clostridia. High-Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13C substrates, informed dynamic flux balance analysis (dFBA) of the pathogen’s genome-scale metabolism. Analyses identified dynamic recruitment of oxidative and supporting reductive pathways, with integration of high-flux amino acid and glycolytic metabolism at alanine’s biosynthesis to support efficient energy generation, nitrogen handling, and biomass generation. Model predictions informed an approach leveraging the sensitivity of 13C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U-13C]glucose and [15N]leucine, confirming the formation of [13C,15N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems.

ORGANISM(S): C. Difficile Clostridioides Difficile

TISSUE(S): Bacterial Cells

SUBMITTER: Aidan Pavao

PROVIDER: ST002433 | MetabolomicsWorkbench | Thu Jan 05 00:00:00 GMT 2023

REPOSITORIES: MetabolomicsWorkbench

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Project description:Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80? KlCAR1 reference strain) and IMS0367 (Klcar1? Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.

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Elucidating dynamic anaerobe metabolism with HRMAS 13C NMR and genome-scale modeling

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