Silva, P. A. | bioRxiv Science

bioRxiv2022

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

Anaerobic microbial metabolism drives critical functions within global ecosystems, host-microbiota interactions, and industrial applications, yet remains ill-defined. Here we advance versatile approaches to elaborate dynamic metabolism in living cells of 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 uniformly labeled 13C substrates informed dynamic flux balance analysis (dFBA) of the pathogens genome-scale metabolism. Predictions identified metabolic integration of glycolytic and amino acid fermentation pathways at alanines biosynthesis, to support efficient energy generation, maintenance of redox balance, nitrogen handling, and biomass generation. Model predictions advanced an approach using 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. We illustrate experimental and computational approaches to elaborate complex anaerobic metabolism for diverse applications.

microbiology↗

bioRxiv2021

Real-time HRMAS 13C NMR of obligately anaerobic cells identifies new metabolic targets in the pathogen Clostridioides difficile

Anaerobic microbial metabolism drives critical aspects of host-microbiome interactions and supports many economically important industrial applications. Yet, the metabolic pathways of anaerobic bacteria and their associated constraints for maintaining energy and redox balance are often poorly described. We employ High-Resolution Magic Angle Spinning Carbon-13 (13C) Nuclear Magnetic Resonance spectroscopy with dynamic flux balance analysis to resolve real-time metabolism in living cells of the obligately anaerobic pathogen Clostridioides difficile. Using 13C-labeled carbon sources, we elaborate the time-dependent progression of reductive and oxidative anaerobic fermentation pathways. Analyses identified new integration points for redox and nitrogen coupling between carbohydrate and amino acid metabolism, particularly in the production of 13C-alanine from 13C-glucose to provide an ammonia sink from co-occurring amino acid fermentation. Analyses conducted in the presence or absence of selenium, a required co-factor for the proline Stickland reductase, demonstrate further capacity to modulate cellular metabolism and resulting metabolites. Findings informed a genome-scale metabolic model of C. difficile, identifying alanine and associated electron carrier pools as critical metabolic integration points in energy flow and biomass expansion. We illustrate use of HRMAS NMR as a new analytical platform to resolve complex interactions in anaerobic metabolism and inform new metabolic targets to counter C. difficile infections.

microbiology↗