<span style="font-variant: small-caps">l</span>-Alanine Exporter AlaE Functions as One of the <span style="font-variant: small-caps">d</span>-Alanine Exporters in <i>Escherichia coli</i>

Abstract

d-amino acids have recently been found to be present in the extracellular milieu at millimolar levels and are therefore assumed to play a physiological function. However, the pathway (or potential pathways) by which these d-amino acids are secreted remains unknown. Recently, Escherichia coli has been found to possess one or more energy-dependent d-alanine export systems. To gain insight into these systems, we developed a novel screening system in which cells expressing a putative d-alanine exporter could support the growth of d-alanine auxotrophs in the presence of l-alanyl–l-alanine. In the initial screening, five d-alanine exporter candidates, AlaE, YmcD, YciC, YraM, and YidH, were identified. Transport assays of radiolabeled d-alanine in cells expressing these candidates indicated that YciC and AlaE resulted in lower intracellular levels of d-alanine. Further detailed transport assays of AlaE in intact cells showed that it exports d-alanine in an expression-dependent manner. In addition, the growth constraints on cells in the presence of 90 mM d-alanine were mitigated by the overexpression of AlaE, implying that AlaE could export free d-alanine in addition to l-alanine under conditions in which intracellular d/l-alanine levels are raised. This study also shows, for the first time, that YciC could function as a d-alanine exporter in intact cells.

Keywords

• d-amino acids%22%2C%22default_operator%22%3A%22AND%22%7D%7D%7D"> <span style="font-variant: small-caps">d</span>-amino acids
• d-alanine%22%2C%22default_operator%22%3A%22AND%22%7D%7D%7D"> <span style="font-variant: small-caps">d</span>-alanine
• exporter
• AlaE