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.

Regulation and characterization of rot transcription in Staphylococcus aureus

“…We found the rot gene to be poorly transcribed in the early (lag) and post-exponential phases of growth, whereas transcription was significantly higher in the exponential phase in the wild-type strain. This finding is consistent with the earlier observation that the rot gene is transcribed maximally in the exponential phase of growth (McNamara et al, 2000). Northern analysis of rot transcription in mutants of agr, sae, sigB and nine sarA family genes revealed that transcription of rot remains unaltered in these mutants at the post-exponential phase of growth, with the exception of sarA and sarS.…”

“…Although the phenotypic characterization of the rot gene has been performed thoroughly, the regulation and characterization of rot transcription are not well defined (McNamara et al, 2000;McNamara & Bayer, 2005;Said-Salim et al, 2003;Tseng & Stewart, 2005). Therefore, in order to determine the transcriptional start site and the promoter structure of the rot transcript and the probable length of the rot open reading frame, primer extension analysis was performed with total RNA isolated from the wild-type RN6390 and sarA mutant strains (Fig.…”

“…The expression of many of these virulence genes is coordinately controlled by regulatory loci, such as two-component regulatory systems (agr, saeRS, srrAB and arlSR) and global transcriptional regulators (sarA, sigB, sarA paralogues, tcaRA, etc.) (Arvidson & Tegmark, 2001;Cheung et al, 1992;Cheung & Zhang, 2002;Luong et al, 2003;, 2006aManna et al, 2004;McNamara et al, 2000;McNamara & Bayer, 2005;Novick, 2003;Projan & Novick, 1997;Truong-Bolduc et al, 2003).…”

“…Rot, a 166-residue SarA paralogue (or 153-residue as predicted in published S. aureus genomes) represses haemolysins (hla and hld), toxin and lipase (geh), and positively regulates cell-surface-associated protein synthesis [clumping factor, protein A, putative cell-surface adhesin (sdrC)] (McNamara et al, 2000;McNamara & Bayer, 2005;Said-Salim et al, 2003). Transcriptional profiling suggests that inactivation of rot affects the transcription of 168 genes in S. aureus including sarS, another member of the sarA family of genes (Said-Salim et al, 2003).…”

Regulation of Staphylococcal Superantigen-Like Gene, ssl8, Expression in Staphylococcus aureus strain, RN6390

“…15 The success of S. aureus as a pathogen lies in its ability to exploit multiple virulence factors regulated by complex network of global gene regulators that includes but not limited to Agr (accessory gene regulator), SarA (staphylococcal accessory regulator), Sae (S. aureus exoprotein expression), SigB (sigma factor B) and Rot (repressor of toxins). [2][3][4]9,16,17 Recently, we reported the differential expression of ssl8 in seven clinically relevant S. aureus isolates and showed that ssl8 expression in the Newman strain is regulated by Sae and Agr. 16 In this brief communication, we show that the ssl8 expression in the common laboratory strain, RN6390 is regulated differently than the Newman strain suggesting ssl8 regulation is strain dependent.…”