Identification of an l ‐alanine export system in Escherichia coli and isolation and characterization of export‐deficient mutants

An Escherichia coli strain that exhibits a double auxotrophy for l ‐alanine and d ‐alanine was constructed. During growth in the presence of the dipeptide l ‐alanyl‐ l ‐alanine (Ala–Ala), this was fully consumed with concomitant extracellular accumulation of l ‐alanine in a twofold molar concentration compared with the dipeptide. This finding indicates that the strain not only can hardly degrade l ‐alanine but has an export system(s) for l ‐alanine. To obtain access to the system, we chemically mutagenized the l ‐alanine‐nonmetabolizing strain and isolated mutants with increased Ala–Ala sensitivity. Two such mutants accumulated l ‐alanine up to 150–190 mM in the cytoplasm with a reduced rate of l ‐alanine export relative to the parent strain in the presence of Ala–Ala. Furthermore, when chloramphenicol was added together with Ala–Ala, the parent strain accumulated l ‐alanine in the cytoplasm to a level similar to that observed in the mutants in the absence of chloramphenicol. In contrast, the intracellular l ‐alanine level in the mutants did not change irrespective of chloramphenicol treatment. From these results, we conclude that E. coli has an inducible l ‐alanine export carrier, together with a second, as yet unidentified, mechanism of alanine export.