title: Secretion of d-alanine by Escherichia coli.
abstract: 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 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 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 d-alanine 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 high-density 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.
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Katsube
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Tasuke
authors.initials: T
authors.lastname: Ando
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Isogai
authors.affiliation: Laboratory of Animal Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1, Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoneyama
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D000409:Alanine / Q000737:chemistry / Q000378:metabolism*; D000411:Alanine Racemase / Q000235:genetics; D001692:Biological Transport / Q000235:genetics / Q000502:physiology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003605:D-Amino-Acid Oxidase / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*
chemical_list: D004151:Dipeptides; C002956:alanylalanine; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003605:D-Amino-Acid Oxidase; D000251:Adenosine Triphosphatases; D000411:Alanine Racemase; D000409:Alanine