Secretion of D-alanine by escherichia coli

Escherichia coli has an L-alanine export system that protects the cells from toxic accumulation of intracellular L-alanine in the presence of L-alanyl-L-alanine (L-Ala-L-Ala). When a DadA-deficient strain was incubated with 6.0 mM L-Ala-L-Ala, we detected L-alanine and D-alanine using highperformance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with D-amino acid oxidase resulted in the disappearance of a signal corresponding to D-alanine. Additionally, the culture supernatant enabled a D-alanine auxotroph to grow without D-alanine supplementation, confirming that the signal detected by HPLC was authentic D-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of Dalanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from L-alanine to D-alanine enhanced D-alanine secretion. When highdensity DadA-deficient cells preloaded with L-Ala-L-Ala were treated with 20 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both L-alanine and D-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the L-alanine and D-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the L-alanine exporter AlaE accumulated [³H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [³H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports D-alanine and that this function is most likely modulated by proton electrochemical potential.