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We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [3H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [3H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [3H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.

Despite the major biological and pharmacological significance, structural and functional study of membrane proteins remains a significant challenge. A main issue is the isolation of these proteins in a stable and functional state from native lipid membranes. Detergents are amphiphilic compounds widely used to extract membrane proteins from the native membranes and maintain them in a stable form during downstream analysis. However, due to limitations of conventional detergents it is essential to develop novel amphiphiles with optimal properties for protein stability in order to advance membrane protein research. Here we designed and synthesized 1,3,5-triazinecored dimaltoside amphiphiles derived from cyanuric chloride. By introducing variations in the alkyl chain linkage (ether/thioether) and an amine-functionalized diol linker (serinol/ diethanolamine), we prepared two sets of 1,3,5-triazine-based detergents. When tested with several model membrane proteins, these agents showed remarkable efficacy in stabilizing three

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Escherichia coli has an L-alanine export system that protects the cells from toxic accumulation of intracellular L-alanine in the presence of L-alanyl-L-alanine (L-Ala-L-Ala). When a DadA-deficient strain was incubated with 6.0 mM L-Ala-L-Ala, we detected L-alanine and D-alanine using highperformance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with D-amino acid oxidase resulted in the disappearance of a signal corresponding to D-alanine. Additionally, the culture supernatant enabled a D-alanine auxotroph to grow without D-alanine supplementation, confirming that the signal detected by HPLC was authentic D-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of Dalanine increased to 11.5 mM and 8. indicate that E. coli has a transport system(s) that exports D-alanine and that this function is most likely modulated by proton electrochemical potential.