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SummaryThe 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 Januslike 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.

Conjugative transposons are important determinants of antibiotic resistance, especially in gram-positive bacteria. They are remarkably promiscuous and can conjugate between bacteria belonging to different species and genera. Transposon-promoted conjugation may be similar to F plasmid-promoted conjugation, as it appears that only one strand of the transposon DNA is transferred from donor to recipient. The recent determination of the entire nucleotide sequence of Tn916 allowed us to make specific predictions about the possible function of different open reading frames and the position of a (hypothetical) origin of transfer. The mechanism of recombination during conjugative transposition differs from that of other transposons, as shown by the absence of a duplication of the target sequence upon integration. The current model for recombination postulates that staggered double-stranded cleavages occur at each end of the transposon. One DNA strand is cut six bases from the end of the transposon, and the other strand is cut immediately adjacent to the end. The ends of the excised transposon are then ligated to form a circular intermediate with a six-base heteroduplex. Staggered cleavages of the circular intermediate and the target DNA allow the transposon to insert into the target, where it is flanked by heteroduplex regions that are resolved by replication. All hosts examined contain preferential target sites: these are not specific sequences but apparently consist of bent DNA. The site-specific recombinases encoded by conjugative transposons belong to the integrase family. Like phage lambda integrase, the integrase of Tn916 has two DNA-binding domains that recognize different sequences, one within the ends of the element and one that includes target DNA. The affinity of Tn916 integrase for target sites correlates with the frequency of integration into a particular site. The similarity between conjugative transposons and phage lambda is striking and suggests that both use the same mechanism of recombination. In lambda, however, recombining sites must be homologous. Homology may be necessary because of branch migration, which is thought to occur during recombination. In conjugative transposition, the recombining sites are nearly always different, and therefore branch migration probably does not occur. This review presents a speculative model for the alignment of the ends of Tn916 during excision that was adapted from one recently proposed for lambda.

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.