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 pro tein that activates transcription of the spvABCD operon during the sta tionary phase of bacterial growth. We used gel mobility shift assays t o demonstrate that SpvR recognizes a specific target DNA sequence with in a 318-bp EcoRI-ApaI fragment upstream of spvA. The addition of unla beled target DNA to the radioactive labeled DNA-SpvR complex resulted in competitive inhibition of band retardation confirming the specifici ty of SpvR binding. Introduction of target DNA on a high copy number p lasmid 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 sho wn to abolish the positive regulatory function of SpvR: By site-specif ic mutagenesis of spvR, three single amino acids within the putative S pvR N-terminal alpha-helix domains were substituted by prolines. This resulted in loss of binding to the spvA promotor sequence and in loss of activation of the spvABCD genes. This study demonstrates that the r egulatory function of SpvR is mediated by specific binding to the prom otor region of the spvABCD operon. (C) 1995 Academic Press, Inc.