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

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 MLA 301Δ alaE was 4‐ and >4000‐fold lower, respectively, than in the alaE ‐positive parent cells MLA 301, 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 MLA 301Δ alaE and MLA 301 ΔalaE / pA laE cells producing plasmid‐borne AlaE, accumulated approximately 200% and 50%, respectively, of the [ 3 H] l ‐alanine detected in MLA 301 cells, suggesting that AlaE exports l ‐alanine. When 200 mmol/L l ‐alanine‐loaded inverted membrane vesicles prepared from MLA 301 ΔalaE / pA laE were placed in a solution containing 200 mmol/L or 0.34  μ mol/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.