, 2001; Kennerknecht et al., 2002). Accordingly, this sensitivity was shown to be due to the excessive intracellular accumulation of the respective amino acids following

uptake and intracellular hydrolysis of FDA approved Drug Library purchase the peptide. This method can also be applied to E. coli, because incubation of the cells with a peptide results in the appearance of the constituent amino acids generated via a successive process of peptide uptake, intracellular hydrolysis and amino acid export (Payne & Bell, 1979). A wide range of wild-type and metabolically engineered strains of bacteria have been shown to produce alanine (Kinoshita et al., 1957; Katsumata & Hashimoto, 1996; Hashimoto & Katsumata, 1998; Hols et al., 1999). Escherichia coli, the wild-type strain of which does not intrinsically accumulate alanine in the medium (Kinoshita et al., 1957), has also been engineered to do so (Zhang et al., 2007).

It is thus easily considered that export systems for alanine would exist see more widely in the microbial world. However, no clarification of the l-alanine exporter in E. coli or in other bacteria has so far been reported. In this report, we describe the isolation of E. coli mutants with decreased l-alanine export activity and present lines of evidence that alanine export may occur by two mechanisms, one of which is due to an inducible export carrier. Escherichia coli strains used in this study were wild-type strain MG1655, d-alanine auxotroph MB2795 (alr∷FRT dadX∷FRT) (Strych et al., 2001) and l-alanine auxotroph HYE008 (avtA∷GM yfbQ∷KM Ala−), which had been

obtained by chemical mutagenesis of a double mutant deficient in avtA and yfbQ genes (unpublished data). The plasmids used were pYfdZ18cs-KM, a derivative tuclazepam of pTH18cs1 (Hashimoto-Gotoh et al., 2000) possessing the disrupted yfdZ gene with the KMr cassette possessing the FRT (FLP recombination target) sequences at the SacII site of the yfdZ gene (unpublished data), and pCP20 (FLP+, λcI857−, λpRRepts, APr, CPr) (Cherepanov & Wackernagel, 1995). Cells were grown aerobically at 37 °C in Luria broth containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl (pH 7.2) or minimal medium (Fisher et al., 1981) containing 22 mM glucose, 7.5 mM (NH4)2SO4, 1.7 mM MgSO4, 7 mM K2SO4, 22 mM NaCl and 100 mM sodium phosphate (pH 7.1). When necessary, d-alanine (50 μg mL−1), l-alanine (50 μg mL−1), gentamicin (GM, 6.25 μg mL−1), kanamycin (KM, 6.25 μg mL−1), chloramphenicol (CP, 12.5 μg mL−1) and ampicillin (AP, 100 μg mL−1) were added to the medium. Growth was monitored by measuring the OD660 nm. To isolate an l-alanine-exporterless mutant, we used a peptide-feeding method, in which excessive intracellular l-alanine accumulation could occur in the presence of Ala–Ala provided the l-alanine-related metabolic pathways are blocked.