Effect of Medium Modifications on Anaerobic Bacterial Metabolism and Exometabolome Composition

Contributions from the community of intestinal anaerobic bacteria to human physiology, health and disease are increasingly recognized, and microbial metabolites are often important for host-microbe interactions. Fusobacterium varium, a genome-sequenced constituent of gut microbiotica, requires amino acids for growth and is a well-known producer of carboxylic acids, particularly butyrate. Quantitative determinations of six carboxylic acid fermentation products were readily performed by ¹H NMR analysis of lyophilized culture supernatants after growth of F. varium on defined glucose-amino acids medium. Isotopic labelling experiments demonstrated that the fermentation products were derived from both glucose and amino acids. Various subsets of the six fermentation products accumulated when glucose was replaced by other energy sources, such as acetoacetate, 3-hydroxybutyrate and crotonate, the intermediates of the core acetate-butyrate pathway in F. varium. On glucose-amino acids defined medium, degradation of ¹³C-labelled glutamate by the hydroxyglutarate pathway was shown by the position of isotopic label in acetate and butyrate. Upon addition of cobalt ion or coenzyme B₁₂ to the defined medium, the methylaspartate pathway, which requires coenzyme B₁₂ for the first step, became the major route of glutamate catabolism, showing a pathway dependence on the availability of a micronutrient. Substitution of amino acids in the defined medium by bactopeptone led to the appearance of a previously unrecognized metabolite of fusobacteria. The neutral metabolite was extracted from culture fluids, purified by vacuum distillation, and identified as meso-2,3-butanediol by NMR and mass spectrometry. meso-2,3-Butanediol production was stimulated by increased glucose concentrations; on the other hand, no meso-2,3-butanediol formed when glucose was replaced by pyruvate. Overall, the use of a variety of organic substrates as terminal electron acceptors by F. varium leads to the observed variable production of fermentation products. These exometabolome variations depend to a certain extent on the availability of substrates and the need to regenerate reduced redox coenzymes. As an normal inhabitant of mucosal surfaces, F. varium has been associated with ulcerative colitis; the identification of meso-2,3-butanediol as a metabolite may provide a link between F. varium and inflammatory bowel disease.