Bacterial Competition Assay Based on Extracellular D-amino Acid Production

Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments. With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.