title: In vitro binding of the Salmonella dublin virulence plasmid regulatory protein SpvR to the promoter regions of spvA and spvR.
abstract: The spv regulon of Salmonella dublin is essential for virulence in mice. SpvR, a LysR-type regulator, induces the expression of the spvABCD operon and its own expression in the stationary phase of bacterial growth and in macrophages. We constructed fusion proteins to the maltose-binding protein (MBP) and a His tag peptide (His) to overcome the insolubility 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. Affinity chromatography resulted in at least 95% pure MBP-SpvR protein and 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 fragments of the spvA and spvR promoter regions, respectively. DNase I footprint experiments showed that the fusion proteins protected DNA regions of 54 and 50 bp within the spvA and spvR promoter regions, respectively.
authors.affiliation: Department of Medicine, School of Medicine, University of California at San Diego, La Jolla, California 92093-0640, USA.
authors.forename: P
authors.initials: P
authors.lastname: Grob
authors.forename: D G
authors.initials: DG
authors.lastname: Guiney
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012475:Salmonella / Q000235:genetics* / Q000472:pathogenicity; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D003850:Deoxyribonuclease I