, 2012). They are involved in the fine tuning of the VraR-dependent expression of the CWSS and have different affinities for VraR or phosphorylated VraR (Belcheva & Golemi-Kotra, 2008; Belcheva et al., 2012). VraR-binding sites in other CWSS promoters have so far only been studied in silico. A 16-bp palindromic sequence TCAGHCTnnAGDCTGA (H = A, T, C; D = A, T, G), deduced from the VraR homologue CesR in L. lactis (Martinez et al., 2007) buy GDC-0199 and partially overlapping the motif identified by Belcheva et al., is present in the promoters of 26 VraSR-dependent genes of the S. aureus N315 genome

(Martinez et al., 2007). As we found the induction levels of the three LCP genes and of the highly induced CWSS gene sas016 to vary over a wide range, we analysed their specific VraR-binding motifs. The transcriptional start sites of msrR, sa0908 and sa2103 are known (Rossi et al., 2003; Over et al., 2011), and the transcriptional start site of sas016 was determined by primer extension to be 29-nt upstream of the ATG (data not shown). Potential VraR-binding sites were predicted in all four promoters investigated in this study, based on previously published motifs (Martinez et al., 2007; Belcheva & Golemi-Kotra, 2008; Belcheva et al., 2012). These sequences were then disrupted and/or deleted in the promoter regions of luciferase reporter gene constructs (Fig. 2). Disruption of the predicted motifs

decreased basal expression before levels and largely abolished induction by oxacillin (Fig. 2). In all four promoters, the regions essential Selleckchem PD0325901 for induction were located close to the −35 boxes. The promoter of sas016 contained a second region that was found to be essential for full induction. The presence of two VraR-binding sites could contribute to the extremely high induction levels of sas016. Alignment of the nucleotide sequences from the VraR-binding regions identified here revealed no obvious consensus sequence. The high-affinity VraR-binding region in the vraSR operon promoter (Belcheva et al., 2012) and the tcaA promoter region required for induction (McCallum et al., 2007)

were both also in close proximity to their respective −35 box. The msrR promoter region needed for induction corresponded to the CesR-like motif identified in silico by Martinez et al. (2007; Fig. 2); however, deletion of the suggested CesR-binding region for sa0908 did not affect transcription (data not shown). For the promoters of sas016 and sa2103, no CesR-like binding sites were previously predicted (Martinez et al., 2007); however, the VraR-binding sites identified here both contained potential CesR-like sequences. To create a reliable VraR-binding consensus for S. aureus CWSS gene induction, detailed promoter analysis of more VraSR-dependent genes is required. The trend, however, seems to involve sequences with a close proximity to the −35 box of the CWSS gene promoter.