Gordon Churchward

The two faces of Janus: virulence gene regulation by CovR/S in group A streptococci.

The group A streptococcus (GAS) causes a variety of human diseases, including toxic shock syndrome and necrotizing fasciitis, which are both associated with significant mortality. Even the superficial self‐limiting diseases caused by GAS, such as pharyngitis, impose a significant economic burden on society. GAS can cause a wide spectrum of diseases because it elaborates virulence factors that enable it to spread and survive in different environmental niches within the human host. The production of many of these virulence factors is directly controlled by the activity of the CovR/S two‐component regulatory system. CovS acts in one direction as a kinase primarily to activate the response regulator CovR and repress the expression of major virulence factors and in the other direction as a phosphatase to permit gene expression in response to environmental changes that mimic conditions found during human infection. This Janus‐like behaviour of the CovR/S system is recapitulated in the binding of CovR to the promoters that it directly regulates. Interactions between different faces of the CovR DNA binding domain appear to depend upon DNA sequence, leading to the potential for differential regulation of virulence gene expression.

Binding of the global response regulator protein CovR to the sag promoter of Streptococcus pyogenes reveals a new mode of CovR-DNA interaction.

CovR (CsrR) is a response regulator of gene expression in Streptococcus pyogenes. It regulates ∼15% of the genome, including the genes encoding several streptococcal virulence factors, and acts primarily as a repressor rather than an activator of transcription. We showed that in vitro, CovR is sufficient to repress transcription from the sag promoter, which directs the expression of streptolysin S, a hemolysin that can damage the membranes of eukaryotic cells and subcellular organelles. Repression was stimulated 10-fold by phosphorylation of CovR with acetyl phosphate. In contrast to binding at the has and cov promoters, which direct the expression of genes involved in capsule biosynthesis and of CovR itself, binding of CovR to Psag was highly cooperative. CovR bound to two extended regions of Psag, an upstream region overlapping the -35 and -10 promoter elements and a downstream region overlapping the translation initiation signals of the sagA gene. Each of these regions contains only a single consensus CovR binding sequence, ATTARA, which at the has promoter defines individual sites to which CovR binds non-cooperatively. At Phas and Pcov the T residues in the sequence ATTARA are important for CovR binding. However, using uracil interference experiments we find that although the ATTARA sequence in the Psag upstream region contains thymine residues important for CovR binding, important thymine residues in the Psag downstream region are located outside this sequence. Furthermore, again in contrast to its behavior at the has and cov promoters where phosphorylation of CovR leads to a 2–3-fold increase in DNA binding affinity, binding of CovR to the sag promoter was stimulated 8–32-fold by phosphorylation. We suggest that these differences in CovR binding mean that individual promoters will be repressed at different intracellular levels of phosphorylated CovR, permitting differences in the response of members of the CovR regulon to environmental and internal metabolic signals.

Phosphorylation of the Group A Streptococcal CovR Response Regulator Causes Dimerization and Promoter-Specific Recruitment by RNA Polymerase

The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR.

Unraveling the Regulatory Network in Streptococcus pyogenes: the Global Response Regulator CovR Represses rivR Directly

The response regulator CovR acts as a master regulator of virulence in Streptococcus pyogenes by repressing transcription of approximately 15% of the group A streptococcus genome directly or indirectly. We demonstrate that phosphorylated CovR represses transcription of rivR directly by binding to conserved sequences located downstream from the promoter to block procession of RNA polymerase. This establishes the first link in a regulatory network where CovR interacts directly with other proteins that modulate gene expression.

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

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.

Specific Binding of Integrase to the Origin of Transfer (oriT) of the Conjugative Transposon Tn916

Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding to oriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.

Xis protein of the conjugative transposon Tn916 plays dual opposing roles in transposon excision

The binding of Tn916 Xis protein to its specific sites at the left and right ends of the transposon was compared using gel mobility shift assays. Xis formed two complexes with different electrophoretic mobilities with both right and left transposon ends. Complex II, with a reduced mobility, formed at higher concentrations of Xis and appeared at an eightfold lower Xis concentration with a DNA fragment from the left end of the transposon rather than with a DNA fragment from the right end of the transposon, indicating that Xis has a higher affinity for the left end of the transposon. Methylation interference was used to identify two G residues that were essential for binding of Xis to the right end of Tn916. Mutations in these residues reduced binding of Xis. In an in vivo assay, these mutations increased the frequency of excision of a minitransposon from a plasmid, indicating that binding of Xis at the right end of Tn916 inhibits transposon excision. A similar mutation in the specific binding site for Xis at the left end of the transposon did not reduce the affinity of Xis for the site but did perturb binding sufficiently to alter the pattern of protection by Xis from nuclease cleavage. This mutation reduced the level of transposon excision, indicating that binding of Xis to the left end of Tn916 is required for transposon excision. Thus, Xis is required for transposon excision and, at elevated concentrations, can also regulate this process.

Back to the future: the new ICE age

The analysis of bacterial genomes has revealed an extraordinary array of conjugal elements (integrative and conjugative element or ICE) that reside in bacterial chromosomes. These elements contribute to the pan‐genomes of individual species and confer a wide variety of properties on their bacterial hosts. ICEBs1 is a conjugal element found in Bacillus subtilis that has a remarkable regulatory mechanism that apparently favours conjugation when there are suitable recipient bacteria at high density or when the bacterial host is facing DNA‐damaging stresses. In the current issue, Bose et al. dissect the mechanism of induction of transfer of this element, and reveal a new, apparently widespread repressor anti‐repressor system and a new mechanism of repressor destruction by proteolysis.