Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus

A chromosomal insertion of transposon Tn917 partially restores the expressi on of protease and alpha-toxin activities to PM466, a genetically defined a gr-null derivative of the wild-type Staphylococcus aureus strain RN6390. In co-transduction experiments, transposon-encoded erythromycin resistance an d a protease- and alpha-toxin-positive phenotype are transferred at high fr equency from mutant strains to agr-null strains of S. aureus. Southern anal ysis of chromosomal DNA and sequence analysis of DNA flanking the Tn917 ins ertion site in mutant strains revealed that the transposon interrupted a 49 8-bp open reading frame (ORF). Similarity searches using a conceptual trans lation of the ORF identified a region of homology to the known staphylococc al global regulators AgrA and SarA. To verify that the mutant allele confer red the observed phenotype, a wild-type allele of the mutant gene was intro duced into the genome of a mutant strain by homologous recombination. The r esulting 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 Nor thern analysis of the alpha-toxin transcript. We named this ORF rot (for re pressor of toxins) (GenBank accession no. AF189239) because of the activity associated with rot::Tn917 mutant strains.