Expression of SarX, a negative regulator of agr and exoprotein synthesis, is activated by MgrA in Staphylococcus aureus

Abstract

The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, saeRS, arlRS, and sarA-like genes. As part of our continuing efforts to understand the regulatory mechanisms that involve sarA-like genes, we describe here the characterization of a novel transcriptional regulator called SarX, a member of the SarA protein family. The transcription of sarX was growth phase dependent and was expressed maximally during the stationary phase of growth, which was significantly decreased in the mgrA mutant. MgrA acted as an activator of sarX expression as confirmed by transcriptional fusion and Northern blot analyses. Purified MgrA protein bound to the upstream region of the sarX promoter as demonstrated by gel shift assay. The expression levels of various potential target genes involved in virulence and regulation, specifically those affected by sarA and mgrA, were analyzed with isogenic sarX mutant strains. Our data indicated that SarX acted as a repressor of the agr locus and consequently target genes regulated by the agr system. We propose that SarX is an important regulator in the SarA protein family and may be part of the common pathway by which agr and members of the sarA gene family control virulence in S. aureus.

FIG. 1.

Transcription and promoter analysis of the sarX gene in S. aureus. (A) Northern analysis of the sarX transcripts in the wild-type strain RN6390 at the different growth phases. The blot was probed with a 450-bp sarX DNA fragment containing the entire open reading frame of the sarX gene and a 723-bp internal region of 16S rRNA genes, respectively. (B) Primer extension of the sarX transcript by using total RNA isolated from the wild-type strain RN6390 at the post-exponential phase of growth. (C) The nucleotide sequence of the 185-bp promoter region of sarX is shown and marked with putative promoter recognition sites (−35 and −10) and regulatory regions in bold. The transcriptional start site (+1) was identified based on a primer extension experiment. The ribosome-binding sequences (RBS) and translational start codon (TTG) are marked in bold. The predicted translational start codon (ATG) in various S. aureus genomes is underlined.

FIG. 2.

Northern analysis of the sarX transcript in the wild-type (wt) strain, various isogenic mutants, and complemented strains at the post-exponential phase (OD₆₅₀ of ∼1.7) of growth (A) and at both the exponential and post-exponential phases (B). A 450-bp DNA fragment containing the sarX gene was used for hybridization. cp s and cp m denote complementation in single copy and multicopy, respectively, with the indicated genes. The 723-bp internal fragment of 16S rRNA was used for hybridization as a loading control (A), or the region of 23S and 16S rRNA of the ethidium bromide-stained gel was used for the hybridization (B).

FIG. 3.

Autoradiogram of an 8.0% polyacrylamide gel showing gel shifts for purified MgrA protein with a γ-³²P-labeled, 185-bp sarX promoter fragment. Mobility of the band was noted in the presence of increasing amounts of MgrA protein, as indicated on the top. Unlabeled, specific 185-bp sarX fragment and nonspecific 200-bp PsarA fragment (nt 1 to 200; GenBank accession no. U46541) competitors with 50-fold excess (molar ratio), as indicated, contained 0.5 μg of the purified MgrA protein.

FIG. 4.

Analysis of expression of agr transcripts in a sarX mutant and various isogenic mutant strains. (A) Northern blots of agr RNAII and RNAIII and 16S rRNA transcripts (a loading control) for the wild-type (wt), sarX mutant, single-copy-complemented (cp s), and trans-complemented (cp m) strains and isogenic sarA, agr, and double mutant strains from the exponential (OD₆₅₀ of ∼0.7) and post-exponential (OD₆₅₀ of ∼1.7) phases of growth. The blots were probed with 3.0-kb agr RNAII, 0.5-kb agr RNAIII, and 0.723-kb internal fragments of 16S rRNA genes (as a loading control). (B) gfp_uvr expression as driven by agr RNAIII in the wild type and the sarX mutant at the 8-h time point (post-exponential phase). To minimize variations in fluorescence attributable to cell density, the data are presented as the averages of triplicate samples in fluorescence unit per absorbance at 650 nm. GFP, green fluorescent protein.

FIG. 5.

Analysis of expression of target gene transcripts in a sarX mutant and its various isogenic strains. (A) Northern blots of α- and β-hemolysins (hla and hlb), V8 protease (sspA), and 16S rRNA transcripts (a loading control) for the wild-type (wt), sarX mutant, single-copy-complemented (cp s), and multicopy-complemented (cp m) sarX mutant strains and isogenic sarA, agr, and double mutant strains from the various growth phases as indicated. (B) Promoter activation of the 235-bp hla promoter fused to a gfp_uvr reporter gene in the wild type and the sarX mutant at the 8-h time point. To minimize variations in fluorescence attributable to cell density, the data are presented as the averages of triplicate samples in fluorescence unit per absorbance at 650 nm. GFP, green fluorescent protein.

FIG. 6.

Autoradiogram of an 8.0% polyacrylamide gel showing gel shifts for purified His fusion SarX protein (119 residue) with a γ-³²P-labeled, 179-bp agr promoter fragment. Mobility of the band was noted in the presence of increasing amounts of SarX protein, as indicated on the top. Cold competition lanes with 50-fold excess (molar ratio) of the unlabeled 179-bp agr fragment and 200 bp P2 sarA DNA fragment (as indicated) contained 0.5 μg of the purified SarX protein.

FIG. 7.

Proposed model for the regulation of the expression of agr transcription in S. aureus. There are mainly two types of pathways involved in agr regulation: autoactivation by the AgrA and SarA family proteins. Among SarA paralogs, two pathways are involved: the SarA-SarR pathway operates in the exponential phase of growth, whereas the SarX-MgrA pathway operates mostly in the post-exponential phase of growth, when SarX is expressed. Other regulatory systems may be involved directly or indirectly. We also speculate that these regulators interact with each other to ensure optimal expression of agr transcription. The letters B, D, C, and A appearing in boxes are the agrB, agrD, agrC, and agrA genes, respectively, encoded within the agr RNAII transcript.