Project Summary This project aims to elucidate the physiological origin of the growth inhibitory effect of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate on exemplary bacterial species in the gut microbiota. Our preliminary data indicate that the affectors of SCFA toxicity are a reduction in intracellular pH and an accumulation of the anion form of these acids in the cytoplasm. A battery of high-throughput methods (quantitative metabolomics, quantitative proteomics, tRNA aminoacylation arrays, ratiometric fluorescence) are proposed together with classical biochemical approaches to identify the locations of the growth bottlenecks, and link their growth inhibitory effects quantitatively to the two identified affectors. These methods will be combined with orthogonal perturbations, one creating “overdose of useless metabolites” but not affecting intracellular pH, the other reducing intracellular pH but not affecting metabolite abundances, to quantify the effect of each perturbation on bacterial growth. The results obtained will be used to develop quantitative models that predict the % growth-reduction for a given SCFA level in the environment. The above studies will be done for each of the three major SCFAs and for four exemplary gut bacterial species: the best characterized model organism Echerichia coli, a pathogenic strain of Salmonella Typhimurium, and Bacteroides thetaiotaomircon and Eubacteria rectale, abundant members from the respective phyla of Bacteroidetes and Firmicutes which comprise the vast majority of the gut microbiota. We will additionally characterize key mutants that exhibit reduced SCFA-sensitivity. Comparisons of results for these different species and mutants will provide us with a comprehensive picture for strategies gut bacteria use to cope with SCFA toxicity, as well as the compromises these strategies impose on the growth physiology of the organisms in unstressed conditions.