IN-VITRO BINDING OF THE SALMONELLA-DUBLIN VIRULENCE PLASMID REGULATORY PROTEIN SPVR TO THE PROMOTER REGIONS OF SPVA AND SPVR

The spy regulon of Salmonella dublin is essential for virulence in mic e. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial gro wth and in macrophages, We constructed fusion proteins to the maltose- binding protein (MBP) and a His tag peptide (His) to overcome the inso lubility and to facilitate purification of SpvR. We demonstrated that both fusion proteins, MBP-SpvR and His-SpvR, were able to induce spvA expression in vivo. MBP-SpvR was produced as soluble protein, whereas His-SpvR was only marginally present in the soluble cell fraction, Aff inity chromatography resulted in at least 95% pure MBP-SpvR protein an d in an enrichment of His-SpvR. Gel mobility shift assay revealed that the SpvR fusion proteins were able to bind to 125- and 147-bp DNA fra gments of the spvA and spvR promoter regions, respectively. DNase I fo otprint experiments showed that the fusion proteins protected DNA regi ons of 54 and 50 bp within the spvA and spvR promoter regions, respect ively.