Acetic Acid Acts as a Volatile Signal To Stimulate Bacterial Biofilm Formation

UNLABELLED Volatiles are small air-transmittable chemicals with diverse biological activities. In this study, we showed that volatiles produced by the bacterium Bacillus subtilis had a profound effect on biofilm formation of neighboring B. subtilis cells that grew in proximity but were physically separated. We further demonstrated that one such volatile, acetic acid, is particularly potent in stimulating biofilm formation. Multiple lines of genetic evidence based on B. subtilis mutants that are defective in either acetic acid production or transportation suggest that B. subtilis uses acetic acid as a metabolic signal to coordinate the timing of biofilm formation. Lastly, we investigated how B. subtilis cells sense and respond to acetic acid in regulating biofilm formation. We showed the possible involvement of three sets of genes (ywbHG, ysbAB, and yxaKC), all encoding putative holin-antiholin-like proteins, in cells responding to acetic acid and stimulating biofilm formation. All three sets of genes were induced by acetate. A mutant with a triple mutation of those genes showed a severe delay in biofilm formation, whereas a strain overexpressing ywbHG showed early and robust biofilm formation. Results of our studies suggest that B. subtilis and possibly other bacteria use acetic acid as a metabolic signal to regulate biofilm formation as well as a quorum-sensing-like airborne signal to coordinate the timing of biofilm formation by physically separated cells in the community. IMPORTANCE Volatiles are small, air-transmittable molecules produced by all kingdoms of organisms including bacteria. Volatiles possess diverse biological activities and play important roles in bacteria-bacteria and bacteria-host interactions. Although volatiles can be used as a novel and important way of cell-cell communication due to their air-transmittable nature, little is known about how the volatile-mediated signaling mechanism works. In this study, we demonstrate that the bacterium Bacillus subtilis uses one such volatile, acetic acid, as a quorum-sensing-like signal to coordinate the timing of the formation of structurally complex cell communities, also known as biofilms. We further characterized the molecular mechanisms of how B. subtilis responds to acetic acid in stimulating biofilm formation. Our study also suggests that acetic acid may be used as a volatile signal for cross-species communication.