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. Issue 4 (13th June 2015)

• Record Type: Journal Article
• Authors: Kim, Seryoung; Ihara, Kohei; Katsube, Satoshi; Hori, Hatsuhiro; Ando, Tasuke; Isogai, Emiko; Yoneyama, Hiroshi
• Is Part Of: MicrobiologyOpen. Volume 4:Issue 4(2015:Aug.)
• Page Start: 632
• Page End: 643
• Publication Date: 2015-06-13
• Subjects: Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.269
• Languages: English
• ISSNs: 2045-8827

Abstract

<abstract abstract-type="main" id="mbo3269-abs-0001"> <title>Abstract</title> <p>We previously reported that the <italic>alaE</italic> gene of <italic>Escherichia coli</italic> encodes the <sc>l</sc>‐alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of <sc>l</sc>‐alanine and <sc>l</sc>‐alanyl‐<sc>l</sc>‐alanine in <italic>alaE</italic>‐deficient <sc>l</sc>‐alanine‐nonmetabolizing cells MLA301Δ<italic>alaE</italic> was 4‐ and >4000‐fold lower, respectively, than in the <italic>alaE</italic>‐positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic‐level accumulation of intracellular <sc>l</sc>‐alanine and its derivatives. Furthermore, the growth of the <italic>alaE</italic>‐deficient mutant derived from the <sc>l</sc>‐alanine‐metabolizing strain was strongly inhibited in the presence of a physiological level of <sc>l</sc>‐alanyl‐<sc>l</sc>‐alanine. Intact MLA301Δ<italic>alaE</italic> and MLA301<italic>ΔalaE</italic>/pAlaE cells producing plasmid‐borne AlaE, accumulated approximately 200% and 50%, respectively, of the [<sup>3</sup>H]<sc>l</sc>‐alanine detected in MLA301 cells, suggesting that AlaE exports <sc>l</sc>‐alanine. When 200 mmol/L <sc>l</sc>‐alanine‐loaded inverted membrane vesicles prepared from MLA301<italic>ΔalaE</italic>/pAlaE were placed in a solution containing 200 mmol/L or 0.34 <italic>μ</italic>mol/L <sc>l</sc>‐alanine, energy‐dependent [<sup>3</sup>H]<sc>l</sc>‐alanine accumulation occurred under either condition. This energy‐dependent uphill accumulation of [<sup>3</sup>H]<sc>l</sc>‐alanine was strongly inhibited in the presence of carbonyl cyanide <italic>m</italic>‐chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE‐mediated <sc>l</sc>‐alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the <sc>l</sc>‐alanine exporter are discussed.</p> </abstract>