0), 140 mM choline GSK126 price Cl, 5 mM MgCl2, 1 mM dithiothreitol and 10% v/v glycerol]. They were passed through a French press (Avanti Products)

to disrupt the cells. Membrane fractions containing the inside-out vesicles were collected by ultracentrifugation. The fluorescence assays of cation/proton antiport activities were conducted on the membrane vesicles using acridine orange, a ΔpH probe which may be used to infer the transmembrane pH gradient. To assess antiport activity, 66 μg of membrane vesicle protein was mixed into 2 mL of assay buffer (10 mM Bis-Tris propane, 140 mM choline Cl, 5 mM MgCl2, 1 μM acridine orange). The assay was initiated by adding Tris-succinate to a final concentration of 5 mM. This induced a quenching of fluorescence intensity as succinate metabolism caused the respiratory chain to establish a pH gradient of ‘acid in’ across the vesicle membrane. Then, for evaluating cation/proton antiport activities, 1 mM of cation (Na+, Li+, K+, or Ca2+) was added to the assay buffer. Finally, 10 mM NH4Cl was added into the assay buffer to re-establish a baseline by collapsing the pH gradient. We have estimated the cation/proton antiport activity as the % dequenching by the observed changes in the acridine orange fluorescence intensity after cation addition. Measurements were conducted PARP inhibitor using a Hitachi High-Technologies

model F-4500 fluorescence spectrophotometer. Genomic analysis showed that the Tr-mrp genes cluster was located on a large plasmid of 0.92 Mb. The cluster is composed of seven separate genes Pyruvate dehydrogenase lipoamide kinase isozyme 1 encoding putative hydrophobic proteins as seen in the mrp operon from Bacillus (Fig. 1). The sodium sensitive phenotype of E. coli KNabc can be complemented by the heterogenous expression of Na+-efflux systems including Mrp, as previously described (Yang et al., 2006; Swartz et al., 2007). As shown in Fig. 2a, expression of Bp-Mrp strongly restored the sodium tolerance of E. coli KNabc. However,

no such recovery of the growth was observed in E. coli KNabc transformed with Tr-Mrp, indicating it was unlikely that Tr-Mrp conferred Na+-efflux in E. coli. In addition, its expression led to a decreased growth level of the transformants even in LBK medium without added NaCl. Cation/proton antiport activities of Tr-Mrp were measured in the inside-out vesicles prepared from E. coli KNabc transformants. The inside-out vesicles expressing Bp-Mrp, which has been characterized previously, exhibited the obvious pH-dependent Na+/H+ antiport activity (Fig. 2b) (Swartz et al., 2007). On the other hand, no Na+/H+ antiport activity was observed in the inside-out vesicles containing Tr-Mrp (Fig. 2b). However, the vesicles containing Tr-Mrp exhibited Ca2+-stimulated dequenching, which was detectable in the broad pH range of 7.0–9.0 (Fig. 3a). As no significant dequenching was observed by added CaCl2 to the vesicles containing Bp-Mrp or the empty vector (Fig.