Lanes C, T, A and G show the PLX4032 solubility dmso dideoxy-AZD1390 purchase terminator sequencing ladder and lane RT the reverse transcription product obtained using primer pe_esxA_2. The TSP is marked by an arrow.

The same TSP was identified using primer pe_esxA_1 (data not shown). Primer extension analysis located the transcriptional start point (TSP) of esxA 74 bp upstream of the start codon of esxA (Figure 1A-C). It was preceded by the predicted -10 and -35 σA promoter elements, and further up by the σB promoter. To verify and compare the function of the putative σA and σB promoter sequences, we cloned the esxA promoter region upstream of the firefly luciferase reporter gene and analyzed the luciferase activity of this construct, pesxAp-luc + , as well as of constructs containing either a deletion of the σA or σB promoter (pesxApΔσA -luc + , pesxApΔσB -luc + ). Whereas the relative luciferase activities of pesxAp-luc + and pesxApΔσB -luc + after 3 h of growth were comparable, pesxApΔσA -luc + showed almost no activity, suggesting that esxA possesses a σA-dependent promoter (Figure 2). We could rule out a direct involvement of σB in the control of the esxA promoter, furthermore, by testing the esxA upstream region in the heterologous two-plasmid system that was established to identify

σB-dependent S. aureus promoters [30]. The upstream region of esxA was cloned into the reporter plasmid pSB40N resulting in plasmid pesxAp which then was introduced into E. coli DH5α containing either pAC7-sigB, expressing the S. aureus sigB gene from an inducible promoter, or the empty LXH254 plasmid pAC7. If the S. aureus σB – E. coli RNA polymerase core enzyme hybrid recognized the esxA promoter, dark blue colonies would be expected on the indicator LBACX-ARA agar [29] in combination with pAC7-sigB, as with the σB-dependent promoters of asp23 or yabJ (positive controls); if not, uncolored colonies

would be expected, as with the σB-independent promoter of capA or the empty next pSB40N (negative controls). In contrast, transformants containing the empty pAC7 vector should produce uncolored colonies. However, both combinations, pesxAp with either pAC7 or pAC7-sigB, developed an identical only light blue color in E. coli DH5α, indicating that the esxA promoter was recognized weakly by an E. coli RNA polymerase, but that the observed transcriptional activity was independent from σB (data not shown). Overall, the results of the esxA promoter and terminator sequence analyses supported a monocistronic transcription of esxA from a σA-dependent promoter. Figure 2 σ A -dependence of the esxA promoter. Luciferase activities of plasmids pesxAp-luc + (wt), pesxApΔσA-luc + (ΔσA) and pesxApΔσB-luc + (ΔσB) in S. aureus Newman. The strains were grown in LB broth at 37°C and 180 rpm for 3 h. Data shown are the means ± SD of four independent experiments. Statistical significances between the different strains were assessed with a paired, two-tailed Student’s t-test (* p < 0.01).