Regulation of streptokinase expression by CovR/S in Streptococcus pyogenes: CovR acts through a single high-affinity binding site

Abstract

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the -10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.

Figure 1. CovR binding sites at different promoters

The interaction between CovR and different promoters is represented by regions protected by CovR from nuclease digestion (heavy black lines) and consensus or near-consensus CovR binding sequences (open arrows). (For clarity, not all near-consensus sequences are shown.) The promoters (indicated by the solid arrows) are aligned by their start points of transcription. Only one of the two closely overlapping promoters of Pska is shown.

Figure 2. The ska promoter/operator region

JRS4 sequence of the region upstream of the translation initiation site of ska which is indicated by the arrow labeled Ska. Possible transcription initiation sites are indicated by arrows labeled P1 and P2. The putative −35 and −10 regions associated with P1 and P2 are indicated. Consensus ATTARA CovR binding sites are indicated (CB-1, CB-2 and CB-4), as is a related non consensus sequence, ATTAR (CB-3). A 13 base pair sequence that is not present in group C streptococci is indicated by Δ. The vertical double-headed arrow indicates the center of bending of this region. The site of a 21 base pair insertion present in M1 and M3 strains is indicated by Ω. Regions protected from nuclease digestion by CovR are indicated by boxes. The vertical line upstream of the arrow labeled CB-3 indicates the 5′ end of the truncated promoter fragment used in the invitro transcription experiment described in figure 5B.

Figure 3. Effect of mutations on the binding of CovR and CovR-P to Pska

A: mutation in CB-4. Lanes 1–6 and 13–18: wild type DNA; lanes 7–12 and 19–24: mutant DNA with a TT to GG transversion in site CB-4 (denoted CB-4*). Lanes 1–6 and lanes 7–12: unphosphorylated CovR at 0, 0.25, 0.5, 1, 2 and 3 μM respectively. Lanes 13–18 and lanes 19–24: phosphorylated CovR at 0, 0.25, 0.5, 1, 2 and 3 μM respectively. The locations of the CovR binding sites CB-1 throgh CB-4 are indicated at the right of the figure. The solid lines indicate regions of DNaseI protection and the dashed line indicates partial DNaseI protection. The positions of the −10 and −35 regions of the promoter are indicated, and the arrow indicates the start point and direction of transcription. B: mutation in CB-3. Lanes 1–3: wild type DNA. Lanes 4–6: Mutant DNA with a TT to GG transversion in site CB-3 (denoted CB-3*). Lanes 1–3 and lanes 4–6; phosphorylated CovR at 0, 3, and 6 μM respectively. The location of the CovR binding sites CB1–CB4 are indicated at the right of the figure. The solid lines indicate regions of DNaseI protection. The positions of the −10 and −35 regions of the promoter are indicated, and the arrow indicates the start point and direction of transcription.

Figure 4. Effect of mutations on the binding of CovR and CovR-P to Pska: mutations in CB-1 and CB2

Lanes 1–4:wild type DNA. Lanes 5–8: Mutant DNA with a TT to GG transversion in site CB-1 (CB-1*). Lanes 9–12: Mutant DNA with a TT to GG transversion in site CB-2 (CB-2*). Lanes 1–4, lanes 5–8 and lanes 9–12; phosphorylated CovR at 0, 0.25, 0.5, 1 and 3 μM respectively. The location of the CovR binding sites CB1 and CB2 are indicated at the right of the figure. The solid line indicates a region of DNaseI protection. The arrow indicates the direction of transcription.

Figure 5. Repression of Pska by CovR and CovR-P in vitro

A. Run-off transcriptions using a wild-type template. Upper panel: Lanes 1–9 ; CovR-P at concentrations of 0, 0.003, 0.006, 0.012, 0.025, 0.050, 0.1, 0.2 and 0.4 μM respectively. Lanes 10–18: CovR at concentrations of 0, 0.05, 0.09, 0.19, 0.38, 0.75, 1.5, 3.0 and 6.0 μM respectively. Lane M: molecular weight markers. The transcripts originating from Pska and Pkan are indicated by the arrows. Lower panel. Densitometric analysis of the median transcript volume. Data is plotted as the ratio of ska/kanR transcript in the presence of CovR-P (triangles) or CovR (squares). The amount of transcript generated in the absence of CovR was defined as 100% and relative transcription was determined by the amount of transcript generated at a given CovR or CovR~P concentration divided by the amount of transcript with no CovR or CovR~P present. B. Run-off transcriptions using a truncated template. Upper panel: Lanes 1–7; CovR-P at concentrations of 0, 0.009, 0.2, 0.008, 0.026, 0.8, and 2.4 μM respectively. Lanes 8–14: CovR at concentrations of 0, 0.02, 0.06, 0.18, 0.56, 1.7 and 5.1 μM respectively. The transcripts originating from Pska and Pkan are indicated by the arrows. Lower panel. Densitometric analysis of the median transcript volume as described in A above.