IN-VIVO ANALYSIS OF THE INTERACTIONS OF THE LYSR-LIKE REGULATOR SPVR WITH THE OPERATOR SEQUENCES OF THE SPVA AND SPVR VIRULENCE GENES OF SALMONELLA-TYPHIMURIUM

The interaction of the Salmonella typhimurium virulence gene regulator , SpvR, with its operator sites upstream of the spvA and spvR genes wa s analysed in vivo by dimethyl sulphate (DMS) footprinting and site-di rected mutagenesis. DMS methylation protection assays showed that, in vivo, SpvR forms direct protein-DNA contacts with nucleotides clustere d in two regions (+1 to -27 and -51 to -71) of the spvA regulatory reg ion. These regions were subjected to site-directed mutagenesis and the effects on SpvR binding and gene activation assessed. Mutations that prevented occupancy of the promoter distal site (-51 to -71) in vivo a lso prevented occupancy of the promoter proximal site (+1 to -27), whe reas mutations in the proximal site affected binding only at the proxi mal site and not the distal site. SpvR binding at the promoter proxima l site was an essential prerequisite for transcription activation. The se findings demonstrated a hierarchy of SpvR binding in which the prom oter distal site is dominant to the proximal. The spvR gene was found to possess an operator site that resembled closely the distal SpvR bin ding site of the spvA operator. Nonetheless, SpvR interaction with the spvR operator was difficult to detect in vivo. When the nucleotide se quence of the spvR operator was altered at two nucleotides so that it corresponded more precisely to that of the distal site of the spvA ope rator, strong SpvR-DNA interactions were detected, with nucleotides in the region -31 to -67 being protected from DMS methylation in vivo. H owever, despite the improved interaction with the transcriptional acti vator, the altered regulatory region was poorer at promoting spvR gene transcription than the wild type. We describe a two-step model for ac tivation of the spvA promoter and discuss the possibility that a speci fic cofactor in addition to sigma factor RpoS is required far SpvR act ion at this promoter in vivo.