Background

Bacteria live in polymicrobial communities where a great diversity of species coexist and compete for the available resources. One of the many tactics that bacteria have devised to persist in a specific niche is the production of toxic extracellular metabolites as a strategy to interfere with growth and/or viability of other microbes. D-amino acids have been known for a long time to have a powerful effect in cell shape and viability in bacterial cultures (Bopp, 1965; Fox et al., 1944; Kobayashi et al., 1948; Yaw and Kakavas, 1952; Lark and Lark, 1959; Grula, 1960; Tuttle and Gest, 1960). However, it has not been until recently that D-amino acids have gained physiological meaning when it was reported that many taxonomically unrelated bacteria could release millimolar concentrations of non-canonical D-amino acids (NCDAAs) to the extracellular medium (Lam et al., 2009). Vibrio cholerae, the causative agent of the diarrheal disease cholerae, presents a periplasmic broad spectrum racemase called BsrV reported to produce a great variety of D-amino acids, mainly D-Met and D-Leu (Lam et al., 2009; Cava et al., 2011). Further studies demonstrated that the main mode of action of these D-amino acids was through their incorporation into the peptidoglycan polymer, an essential bacterial structure that plays a role in morphology determination and cell integrity (Caparros et al., 1992; Lam et al., 2009; Cava et al., 2011). Peptidoglycan is a macromolecule composed of glycan chains crosslinked by short peptides. Interestingly, NCDAAs can be incorporated into the peptidoglycan into the 4^th or the 5^th residue of the peptide stem of the muropeptide subunits and this editing has a key role in synchronizing cell wall metabolism with growth arrest (Lam et al., 2009; Cava et al., 2011).

A recent study demonstrated that the cell wall is not the only target of non-canonical D-amino acids (Alvarez et al., 2018). V. cholerae and many other bacteria produce a great variety of D-amino acids which have distinct functions (Lam et al., 2009; Alvarez et al., 2018). D-arginine stands out as a fitness modulator of bacterial subpopulations, since it shows a significantly higher growth inhibitory activity against a wide diversity of bacterial species compared with other D-amino acids. In contrast to D-methionine, which has a major modulatory role in cell wall biosynthesis, D-arginine growth inhibition is suppressed by mutations in the chaperone systems and the phosphate uptake machinery in several model organisms, strongly supporting different roles for NCDAAs in bacterial physiology (Alvarez et al., 2018).

Co-cultivation is an excellent method to assess the inhibitory effect of D-arginine in mixed bacterial populations. However, when the competing bacteria present very different growth rates (e.g., V. cholerae and Caulobacter crescentus used in this study), relative cell counting can be challenging. Besides, it might be difficult to assess the role of small metabolites in species competition when other mechanisms, such as cell-to-cell dependent interactions (e.g., T6SS), can occur simultaneously. Here we present a method to assess the effect of small metabolites on bacterial populations. The design is based in the compartmentalization of the competing subpopulations in two independent rooms separated by a filter that permits diffusion of small metabolites such as amino acids. Furthermore, this method can be used to demonstrate the metabolic cooperation between producer and non-producer bacteria (e.g., V. cholerae wild-type and ΔbsrV mutant) that share extracellular D-amino acids to outcompete other species in the environment. Finally, we also describe the methodology to determine the total D-amino acid concentration in the media.