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

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 nuclear magnetic resonance (NMR) spectroscopy of C. difficile, grown with fermentable 13 C 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 13 C NMR spectroscopy to simultaneously track cellular carbon and nitrogen flow from [U- 13 C]glucose and [ 15 N]leucine, confirming the formation of [ 13 C, 15 N]alanine. Findings identify metabolic strategies used by C. difficile to support its rapid colonization and expansion in gut ecosystems.