D-alanine synthesis and exogenous alanine affect the antimicrobial susceptibility of .

D-alanine is an important amino acid for peptidoglycan biosynthesis in . In addition, D-alanine is used for the modification of teichoic acids to weaken the net surface negative charge, leading to decreased susceptibility to cationic antimicrobial agents. D-alanine synthesis is dependent on only two enzymes. One is alanine racemase, encoded by the gene, which reversibly converts L-alanine and D-alanine. The other is D-amino acid transaminase, encoded by the gene, which synthesizes D-amino acids from α-keto acids and other D-amino acids. In addition, the uptake of L- and D-alanine is dependent on the alanine transporter CycA. To reveal the relationship between D-alanine supply and antimicrobial susceptibility, we evaluated antimicrobial susceptibility in and inactivation mutants. These mutants, especially the Δ and Δ mutants, presented increased susceptibility to β-lactams, D-cycloserine, bacitracin, lysostaphin, and cationic antimicrobial agents such as aminoglycosides, nisin A, and daptomycin. The net negative charge of the cell surface increased in the Δ and Δ mutants. The changes in susceptibility to antimicrobial agents and cell surface charge were restored in their gene-complemented mutants. Furthermore, in an alanine-depleted medium, the MIC for oxacillin decreased significantly, and the MIC for gentamicin also decreased slightly. Clinical MRSA strains also showed significantly increased susceptibility to oxacillin in the alanine-depleted medium. These results indicate that D-alanine deficiency leads to impaired peptidoglycan and increased net surface negative charge, resulting in increased antimicrobial susceptibility.