, 2007). Ohki & Tateno (2004) described the increased expression of the bmr3 efflux transporter due to a double mutation at positions −18 and +4 from the transcription start site. Transcriptional lacZ reporter gene fusions with a region upstream of the bmrA SD sequence were constructed and integrated by double crossing

over into the amyE locus of the B. subtilis 168 chromosome. Measurements of β-galactosidase activity determined the putative promoter region (Fig. 2). Subsequently, primer extension was used to identify the transcription start downstream of a potential promoter (Fig. 2a). The wild-type promoter shows a nearly perfect −10 box with TATGAT, a 17-bp spacer, but a weak −35 box with CTGAAA. In mutant 8R, the C of the −35 box was altered to T, making it more similar to the consensus σA−35 box TTGACA (Fig. 2b). The second point mutation was located six bases downstream from the transcription start site (+6) altering PD0325901 supplier an A5 stretch to GAAAA (Fig. 1b). To dissect

the contribution of each single mutation on the elevated expression of bmrA, plasmids carrying transcriptional bmrA–lacZ fusions with fragments of different sizes were constructed designated pACMM (double mutant), pACWW (wild type), pACMW (−35 mutation) and pAWM (+6 mutation) (Fig. 1a). All pAC6 derivatives were integrated into the amyE locus PARP inhibition and the β-galactosidase activities measured (see Fig. 1a). Increased β-galactosidase activities compared with the wild type were found in the double mutant and in the single mutant affecting the −35 box, whereas only marginally different β-galactosidase activities were measured for the +6 mutation (3.5-fold increased). The 157-bp upstream region increased β-galactosidase 10–11-fold in both the double and the MW mutant compared with the wild type. To investigate the impact of the mutations

on bmrA http://www.selleck.co.jp/products/Abiraterone.html expression, total RNA from wild-type strain 168 and mutant 8R was isolated, DNase treated and assayed using real-time PCR. The amount of bmrA mRNA in mutant 8R with the −35 and +6 mutations was 135-fold increased, whereas in strain YH2M with the −35 mutation alone, the amount of bmrA mRNA was about 13-fold increased (Fig. 2(b). 8R-ind). Real-time PCR on total RNA isolated from B. subtilis 8R propagated in the presence of CmC (0.5 μM) corroborated the results of the Jault laboratory on the constitutive expression of bmrA (Steinfels et al., 2004). To analyze the binding of the RNAP to the bmrA promoter region, EMSAs were performed. The four 157-bp fragments used for the lacZ-reporter gene fusions were radioactively labelled and incubated with increasing concentrations of B. subtilis RNAP. As shown in Fig. 3a and b, the −35/+6 mutant MM and the single −35 mutant MW displayed a 30-fold increased affinity for RNAP. Interestingly, the single mutant WM carrying only the +6 mutation behaved like the wild type. The addition of heparin (Fig.