Control of the Staphylococcus aureus toxic shock tst promoter by the global regulator SarA

Abstract

The Staphylococcus aureus SarA global regulator controls the expression of numerous virulence genes, often in conjunction with the agr quorum-sensing system and its effector RNA, RNAIII. In the present study, we have examined the role of both SarA and RNAIII on the regulation of the promoter of tst, encoding staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1). In vitro DNA-protein interaction studies with purified SarA using gel shift and DNase I protection assays revealed one strong SarA binding site and evidence for a weaker site nearby within the minimal 400-bp promoter region upstream of tst. In vivo analysis of tst promoter activation using a p(tst)-luxAB reporter inserted in the chromosome revealed partial but not complete loss of tst expression in a Δhld-RNAIII strain. In contrast, disruption of sarA abrogated tst expression. No significant tst expression was found for the double Δhld-RNAIII-ΔsarA mutant. Introduction of a plasmid containing cloned hld-RNAIII driven by a non-agr-dependent promoter, p(HU), into isogenic parental wild-type or ΔsarA strains showed comparable levels of RNAIII detected by quantitative reverse transcription-PCR (qRT-PCR) but a two-log(10) reduction in p(tst)-luxAB reporter expression in the ΔsarA strain, arguing that RNAIII levels alone are not strictly determinant for tst expression. Collectively, our results indicate that SarA binds directly to the tst promoter and that SarA plays a significant and direct role in the expression of tst.

FIG. 1.

Gel shift showing binding of SarA on the tst promoter. Increasing amounts of recombinant SarA were incubated with 10 fmol of a radiolabeled 250-bp DNA probe containing the tst promoter. As a nonspecific competitor, 2 pmol of an unlabeled 100-bp DNA fragment, situated far upstream of tst, was added to SarA (at 111 nM). The same molar ratio was used for the addition of the 250-bp cold specific competitor. S, specific; NS, nonspecific.

FIG. 2.

DNase I footprint assay of SarA on the tst promoter. Increasing amounts of recombinant SarA, 62 nM, 111 nM, and 145 nM, were incubated with an asymmetrically radiolabeled 381-bp DNA probe containing the tst promoter. Nucleotide coordinates are defined relative to the transcriptional start site at +1. The continuous lines show strong protection by SarA, while the dashed lines show milder protection. The positions of predicted binding boxes are indicated. For the antisense panel, adjacent sequencing reactions were carried out using the same primers as those used for the probe. For the sense panel, the sequencing marker ladder corresponds to an unrelated sequence.

FIG. 3.

(A) Nucleotide sequences and schematic diagram of the tst promoter region. The bracketed dashed lines indicate the limits of the SarA DNase I protected region. The dotted line refers to a previously proposed CRE site for CcpA-mediated catabolite repression (48). Box1 and Box2 correspond to consensus sites for SarA binding. The arrow marked with “+1” denotes the tst transcription start site determined by 5′RACE. The start codon of the TSST-1 open reading frame is indicated. (B) Comparison and nucleotide conservation between the published SarA consensus sequence (7) and both SarA binding sites proposed on this promoter. Both SarA sites contain incomplete 8-bp inverted repeats: Box1 TTTAATTA-TATTTAAA with a 5-bp spacer region and Box2 TATAAATA-TATTTAAA with a 1-bp spacer region.

FIG. 4.

Gel shift experiments showing binding of SarA with segments of the tst promoter encompassing either binding Box1 (upper) or binding Box2 (bottom). Increasing amounts of recombinant SarA were incubated with 10 fmol of a radiolabeled DNA probe.

FIG. 5.

(A) Luciferase reporter assay for the tst promoter. The histogram shows measured luminometric activity of different strains in stationary growth phase. All cultures were first normalized to an OD₆₀₀ of 0.5 and then assayed. Bars show ± standard deviations. The asterisk indicates significant difference between strains DA102 and DA103 calculated by Student's two-tailed t test (P < 0.05). (B) Luciferase reporter assay for the tst promoter in the indicated strains harboring the plasmid pDA201, which drives hld-RNAIII transcript expression by pHU independent of sarA and agr regulation (left). Quantitative qRT-PCR measurements of RNAIII expression in both strains, setting DA104 as 100% (right). Bars show ± standard deviations. All data are compiled from three independent experiments. RLU, relative light units.

FIG. 6.

Scheme of tst transcriptional regulation. Positive regulation is represented by the arrows, negative regulation by the bars.