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 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 Tn917 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::Tn917 mutant strains.
McNamara
P J
PJ
Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA. pjmcnamara@facstaff.wisc.edu
Milligan-Monroe
K C
KC
Khalili
S
S
Proctor
R A
RA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012097:Repressor Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000235:genetics* / Q000472:pathogenicity*; D014161:Transduction, Genetic
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D012097:Repressor Proteins; C408643:rot protein, Staphylococcus aureus; C021091:staphylococcal alpha-toxin