title: Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.
abstract: We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [(3) H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.
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: Seryoung
authors.initials: S
authors.lastname: Kim
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: Kohei
authors.initials: K
authors.lastname: Ihara
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: Hatsuhiro
authors.initials: H
authors.lastname: Hori
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: D000409:Alanine / Q000378:metabolism* / Q000633:toxicity*; D026921:Amino Acid Transport Systems, Neutral / Q000172:deficiency / Q000378:metabolism*; D001692:Biological Transport; D004151:Dipeptides / Q000378:metabolism / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D008826:Microbial Sensitivity Tests; D010957:Plasmids; D018892:Proton-Motive Force
chemical_list: C560941:AlaE protein, E coli; D026921:Amino Acid Transport Systems, Neutral; D004151:Dipeptides; D029968:Escherichia coli Proteins; C002956:alanylalanine; D000409:Alanine
keywords: Escherichia coli; exporter; l-alanine