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

Seryoung Kim, Kohei Ihara, Satoshi Katsube, Hatsuhiro Hori, Tasuke Ando, Emiko Isogai, Hiroshi Yoneyama

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

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 [3H]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 [3H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [3H]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.

Original languageEnglish
Pages (from-to)632-643
Number of pages12
JournalMicrobiologyOpen
Volume4
Issue number4
DOIs
Publication statusPublished - 2015 Aug 1

Keywords

  • Escherichia coli
  • Exporter
  • L-alanine

ASJC Scopus subject areas

  • Microbiology

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