Identification, Cloning, and Initial Characterization of rot , a Locus Encoding a Regulator of Virulence Factor Expression in Staphylococcus aureus

Abstract

A chromosomal insertion of transposon Tn 917 partially restores the expression of protease and alpha-toxin activities to PM466, a genetically defined agr -null derivative of the wild-type Staphylococcus aureus strain RN6390. In co-transduction experiments, transposon-encoded erythromycin resistance and a protease- and alpha-toxin-positive phenotype are transferred at high frequency from mutant strains to agr -null strains of S. aureus . Southern analysis of chromosomal DNA and sequence analysis of DNA flanking the Tn 917 insertion site in mutant strains revealed that the transposon interrupted a 498-bp open reading frame (ORF). Similarity searches using a conceptual translation of the ORF identified a region of homology to the known staphylococcal global regulators AgrA and SarA. To verify that the mutant allele conferred the observed phenotype, a wild-type allele of the mutant gene was introduced into the genome of a mutant strain by homologous recombination. The resulting isolates had a restored agr -null phenotype. Virulence factor gene expression in mutant, restored mutant, and wild-type strains was quantified by measuring alpha-toxin activity in culture supernatant fluids and by Northern analysis of the alpha-toxin transcript. We named this ORF rot (for repressor of toxins) (GenBank accession no. AF189239 ) because of the activity associated with rot ::Tn 917 mutant strains.