1887

Abstract

has an -alanine export system that protects the cells from toxic accumulation of intracellular -alanine in the presence of-alanyl--alanine (-Ala--Ala). When a DadA-deficient strain was incubated with 6.0 mM -Ala--Ala, we detected -alanine and -alanine using high-performance 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-amino acid oxidase resulted in the disappearance of a signal corresponding to -alanine. Additionally, the culture supernatant enabled a -alanine auxotroph to grow without -alanine supplementation, confirming that the signal detected by HPLC was authentic -alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, or , the extracellular level of -alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow fromalanine to -alanine enhanced -alanine secretion. When high-density DadA-deficient cells preloaded with -Ala--Ala were treated with 20 µM carbonyl cyanide -chlorophenyl hydrazone (CCCP), secretion of both -alanine and-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 -alanine and -alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the -alanine exporter AlaE accumulated [H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [H]D-alanine was strongly inhibited by CCCP. These results indicate that has a transport system(s) that exports -alanine and that this function is most likely modulated by proton electrochemical potential.

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2016-07-01
2020-04-05
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