Butyrate as a growth factor of Clostridium acetobutylicum

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. Proteomic data suggest that butyrate may lead to lysine butyrylation, a lesser-known post-translational modification, which might affect enzyme catalysis and thus cellular metabolism. Although high levels of butyrate induce toxic stress responses, it is not known if butyrate at non-toxic levels influences cellular processes such as growth, health, metabolism, and sporulation. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome in order to eliminate the butyrate synthetic pathway and thus butyrate formation. For rapid genome engineering, a xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. The addition of non-toxic levels of butyrate revealed that butyrate has a profound effect on the growth, health, and sporulation of C. acetobutylicum. By further deleting spo0A, the gene of the master regulator of sporulation, and followed by butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of the precursor of butyrate biosynthesis crotonate to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and other Clostridium organisms, and may thus be as a modulator of microbial population dynamics. HighlightsO_LIDeployed chromosomally integrated spCas9 for markerless one-step Clostridium acetobutylicum genome engineering. C_LIO_LIDeleted 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from Clostridium acetobutylicum to elucidate the roles of butyrate in cellular processes. C_LIO_LIDemonstrated butyrate as a growth factor stimulating cellular processes in Clostridium acetobutylicum and potentially other Clostridium species. C_LIO_LISuggested butyrate as a potential modulator of microbial population based on different responses of microbes against butyrate. C_LI