The three promoters were all induced in a concentration dependent manner, with induction lag times becoming shorter and induction rates steeper as oxacillin concentrations increased. This was mirrored by a corresponding stepwise decrease in growth rates. Induction rates generally began to slow after 60 min, upon the onset of oxacillin induced lysis [28], but again this was concentration dependent with induction rates beginning to decrease earlier in cultures with higher oxacillin concentrations. Figure 2 Induction kinetics

of three CWSS promoters in response to Dactolisib nmr varying concentrations of oxacillin. Luciferase Entospletinib price activities and growth curves of strain BB255 containing: A, p sas016-luc+; B, p sa0908-luc+; and C, p tcaA-luc+; after addition of 0, 0.5, 1, 2 or 5-fold the MIC of oxacillin at time point zero. Previous findings, using Northern blots to measure oxacillin induction levels of sas016 after 30 min, indicated that inhibitory concentrations of oxacillin

were required for induction [20]. Figure 2 confirmed that the sub-inhibitory concentration of 0.5x MIC did not noticeably induce promoter activity after 30 min, however, luciferase activity from all three promoters began to increase sharply after 60 min and find more continued to rise up to the final sampling point of 120 min. Although all three promoters displayed similar relative concentration- and time-dependent induction kinetics,

the sas016 promoter produced the highest levels of luciferase activity, resulting in greater fold-changes between samples and making it the most sensitive of the three reporters. Therefore we chose the sas016 promoter-luciferase fusion construct as the best indicator to compare induction characteristics of different cell wall active antibiotics. Correlation between sas016 transcript induction and luciferase activity from p sas016 p -luc+ To confirm that levels of luciferase www.selleck.co.jp/products/azd9291.html activity from p sas016 p- luc + accurately represented levels of sas016 gene expression, Northern blots were performed on BB255 p sas016 p- luc + RNA samples extracted from cultures grown using the same conditions and oxacillin concentrations used for luciferase assays. Samples were harvested 20 min and 60 min after antibiotic induction and hybridized with sas016 and luc + specific DIG probes (Figure 3). Northern blots showed identical patterns of transcriptional induction for both the chromosomal sas016 gene and the luciferase gene under the control of the sas016 promoter in p sas016 p- luc +. Induction of both transcripts was highly oxacillin-concentration dependent and transcript intensities increased over time becoming stronger after 60 min than after 20 min, correlating very well with concentration-specific induction curves from luciferase assays (Figure 2).