Metabolic Studies of Fusobacterium varium Using NMR Spectroscopy

Abstract

A complex relationship exists between intestinal microorganisms and the human host; further knowledge of the microbial contributions is important to understand human health and disease. The metabolism of F. varium, a genome-sequenced constituent of the gut microbiota was investigated using NMR spectroscopy. The diverse amino acid requirements of F. varium were determined; while some amino acids were biosynthesized and utilized readily, others were required in the growth medium. The individual amino acid requirements were used to develop a chemically defined minimal medium for the determination of end products in lyophilized culture fluids by 1H NMR spectroscopy. Six carboxylate fermentation products (acetate, butyrate and smaller amounts of succinate, propionate, formate and D-lactate) were detected in the exometabolome of F. varium. Glucose, glycerol and metabolic intermediates were identified as energy sources, and their addition to the defined growth medium led to variations in the composition of the exometabolome, attributed to the availability of substrates and the need to maintain redox balance through regeneration of reduced coenzymes. For example, the oxidative steps required for glucose catabolism resulted in higher concentrations of reduced products (butyrate and lactate), whereas propionate was formed exclusively from threonine. Substrate-product relationships were established using isotopically labeled substrates. The pathway of glutamate catabolism was dramatically influenced by the presence of coenzyme B12, one of the B vitamins. Replacing the amino acid component of the defined medium resulted in meso-2,3-butanediol formation, a metabolite not previously identified in fusobacteria that may contribute to the pathogenesis of bowel disease.