To address this issue, we determined the intracellular level of l-alanine in the parent strain MLA301 in the presence or absence of chloramphenicol, a translational inhibitor (Fig. 4a). As expected, intracellular l-alanine was retained at a higher level in the presence of chloramphenicol, corresponding to R428 concentration a two- to fivefold increased concentration during the incubation time of between 5 and 10 min, compared with the level in the absence of chloramphenicol (Fig. 4a). It

should be noted that ethanol, which had been used to prepare a chloramphenicol stock solution, did not influence the intracellular level of l-alanine in this strain. This result clearly indicates that the expression of an l-alanine efflux system is induced under the conditions used. In contrast, LAX12 showed a similar intracellular I-BET-762 chemical structure l-alanine level irrespective of the presence or absence of chloramphenicol (Fig. 4b). Similarly, intracellular l-alanine in LAX16 did not change in the presence of chloramphenicol compared

with the level observed in the absence of chloramphenicol (data not shown). These results indicated that LAX12 and LAX16 lacked an inducible l-alanine export system. Because bacterial cells need to balance their metabolism, anabolism and catabolism, for healthy growth, even natural metabolites can cause growth arrest if they accumulate intracellularly to an extremely high level due to an imbalance. Indeed, such cases have been found for several amino acids, where the inability to export these compounds due to dysfunction of the relevant export systems leads to growth inhibition (Vrljic et al., 1996; Levetiracetam Simic et al., 2001; Kennerknecht et al., 2002). On the basis of this phenomenon, we isolated mutants, LAX12 and LAX16, lacking the ability to export l-alanine and showing extensive intracellular accumulation of l-alanine

when they were incubated in the presence of an l-alanine-containing dipeptide (Fig. 3a). Although the extent of growth inhibition of LAX12 and LAX16 in minimal medium containing Ala–Ala was somewhat different, both mutants started to grow after a period of cultivation (Fig. 2). The delayed growth might have been due to the appearance of revertants that had the same sensitivity to Ala–Ala as the parent strain. However, this possibility is very unlikely, because clones obtained after prolonged cultivation showed almost the same sensitivity to Ala–Ala as the respective original mutants (data not shown). Therefore, the growth delay in the presence of Ala–Ala seemed to be an inherent property of each mutant, and was not due to reversion. In a previous study on the l-cysteine export system of E. coli, a multicopy plasmid harboring the multidrug exporter bcr gene rendered the cells capable of exporting l-cysteine, suggesting that Bcr was involved in the export of the amino acid (Yamada et al., 2006).