Mutational analysis of SpvR binding to DNA in the regulation of the Salmonella plasmid virulence operon.
The Salmonella plasmid-borne spvR gene encodes a 33-kDa regulatory protein that activates transcription of the spvABCD operon during the stationary phase of bacterial growth. We used gel mobility shift assays to demonstrate that SpvR recognizes a specific target DNA sequence within a 318-bp EcoRI-ApaI fragment upstream of spvA. The addition of unlabeled target DNA to the radioactive labeled DNA-SpvR complex resulted in competitive inhibition of band retardation confirming the specificity of SpvR binding. Introduction of target DNA on a high copy number plasmid into wild-type Salmonella dublin Lane resulted in a substantial decrease of SpvB synthesis, confirming the binding properties of this DNA segment in vivo. Three SpvR mutants were constructed and were shown to abolish the positive regulatory function of SpvR. By site-specific mutagenesis of spvR, three single amino acids within the putative SpvR N-terminal alpha-helix domains were substituted by prolines. This resulted in loss of binding to the spvA promoter sequence and in loss of activation of the spvABCD genes. This study demonstrates that the regulatory function of SpvR is mediated by specific binding to the promoter region of the spvABCD operon.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011401:Promoter Regions, Genetic; D012475:Salmonella / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D014774:Virulence / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial