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Journal of Bacteriology, June 2000, p. 3197-3203, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Peter J. McNamara,* Kathy C. Milligan-Monroe, Shirin Khalili, and Richard A. Proctor

Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received 30 December 1999/Accepted 6 March 2000

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.

* Corresponding author. Mailing address: Medical Microbiology and Immunology, 407 Service Memorial Institute, University of Wisconsin Medical School, Madison, WI 53706. Phone: (608) 263-2188. Fax: (608) 262-8418. E-mail: pjmcnamara{at}facstaff.wisc.edu.

Journal of Bacteriology, June 2000, p. 3197-3203, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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