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 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.

FIG. 1

(A) Model of the function of the agr-sar system; (B) hypothetical model of the function of the rot gene product. Details can be found in the text. Chromosomal DNA is depicted as a thick black line. Promoters (P) are numbered. Genes (boxes) and their translation product are identically shaded. Phosphorylated proteins are associated with a circled letter P. The straight arrows and squiggly lines represent mRNA. Translation of hla mRNA is illustrated by the addition of a ribosome (black circles) to the message. The curved arrows show the relationships among the components of the system. Positive and negative effects are marked with (+) and (−), respectively.

FIG. 2

(A) Restriction map of the wild-type allele of agr in RN6390 (top) and the agr-null allele (Δagr) in PM466 (bottom). The agr P2 operon and P3 transcript are represented by the striped and shaded boxes, respectively. Chromosomal DNA on the 3′ end of the P2 operon and 3′ end of the P3 transcript are represented by black and gray lines, respectively. Abbreviations: C, ClaI; E, EcoRI; H, HincII; H/C, ligation of a HincII site with a T₄ polymerase blunt-ended ClaI site. (B) Chromosomal DNA from RN6390 (lane 1) and PM466 (lane 2) digested with EcoRI and analyzed by Southern hybridization using agr and flanking single-stranded DNA as the probe. DNA hybridizing to agr-flanking regions is marked with arrows.

FIG. 3

BLASTP alignment of the rot gene product (Rot) and the S. aureus regulatory proteins AgrA (AgrA e value = 1.9; SarA e value = 5.9) and S. epidermidis AgrA (e value = 0.49). Rot was used as the query sequence against the nonredundant database at the National Center for Biotechnology Information. Identities are shown, + denotes similarity, and numbers at right refer to amino acids in the respective proteins.

FIG. 4

Quantitative measurements of alpha-toxin activity in supernatant fluids from post-exponential-phase (10-h) cultures of S. aureus strains RN6390 (wild-type, gray bar), PM466 (vertically striped bar), PM614 (white bar), PM720 (diagonally striped bar), and PM702 (black bar). Relevant genotypes are shown (wt, wild-type; Δagr, agr-null allele, rot⁺, wild-type rot allele; rot::Tn917, insertionally inactivated rot).

FIG. 5

Northern analysis of the alpha-toxin message in RNA from post-exponential-phase cultures of S. aureus strains (A) PM466, (B) PM614, and (C) PM720. The leftmost lane contains 30 μg of total RNA serially diluted (1:2) in subsequent lanes. The transcript was identified by hybridization using digoxigenin-labeled probe specific for the gene encoding alpha-toxin with chemiluminescent chemistry.