MUTATIONAL CHARACTERIZATION OF PROMOTER REGIONS RECOGNIZED BY THE SALMONELLA-DUBLIN VIRULENCE PLASMID REGULATORY PROTEIN SPVR

The virulence plasmid-encoded spy regulon is essential for virulence o f Salmonella dublin in mice. The spvR gene product belongs to the LysR family of transcriptional regulator proteins. SpvR induces the expres sion of the spvABCD operon and positively regulates its own expression . DNase I protection analysis with purified SpvR fusion proteins ident ified SpvR binding sites within the spvA and spvR promoters (P. Grob a nd D. G. Guiney, J. Bacteriol. 178:1813-1820, 1996). We have used PCR mutagenesis, combined with functional selection for reduced SpvR affin ity, to define the DNA elements essential for SpvR binding. For the sp vR promoter fragment, a screen for reduced expression was also applied . Sequence analysis of the resulting mutant fragments reveals that the base pair changes are clustered in distinct regions. Determination of the apparent dissociation constants of SpvR for the mutant promoters showed that the spvA LysR-type motif and the upstream palindromic sequ ences of both promoters play an important role in SpvR recognition.