Transcription of the surface-associated virulence factors of the group A streptococcus (GAS) Streptococcus pyogenes, M protein (emm) and the C5a peptidase (scpA), is activated by a protein called Mga (formerly Mry or VirR). To determine whether Mga binds directly to the promoters of the genes it regulates, a protein resulting from the fusion of Mga to the C-terminal end of maltose-binding protein was purified from Escherichia coli. Specific binding to the promoter regions of the scpA and emm alleles of the type M6 GAS strain JRS4 was demonstrated by electrophoresis of the DNA-protein complex. Competition studies showed that the region upstream of scpA bound MBP-Mga with a slightly higher affinity than did the region upstream of emm. DNase I protection experiments identified a single 45-bp binding site immediately upstream of and overlapping the ؊35 region of both promoters. Sequences homologous to the protected regions were found in the promoters of many emm, scp, and emm-like genes from strains of different serotypes of GAS, and a consensus Mga binding site was deduced.

Activation of the spoIIG promoter at the onset of sporulation in Bacillus subtilis requires the regulatory protein, SpoOA, which binds to two sites in the promoter, sites 1 and 2. Phosphorylation of SpoOA is essential for the initiation of sporulation. Therefore, we examined the role of SpoOA phosphorylation in spoHG promoter activation. Phosphorylation of SpoOA stimulated transcription from the spoIIG promoter in vitro. In DNAse I footprinting experiments with the spoIIG promoter, we found that phosphorylation of SpoOA increased its afinity for site 2 more than for site 1, which is the site to which nonphosphorylated SpoOA binds most avidly.This result could not be explained by increased cooperativity between SpoOA bound at sites 1 and 2 because the increased alfinity for site 2 by phosphorylated SpoOA was also observed with a deletion derivative of the spoIIG promoter containing only site 2. We have located SpoOA-binding sequences in the spolHG promoter by DMS protection assays and mutational analysis, and found that site 1 contains one higher-afinity binding sequence whereas site 2 contains two weaker-binding sites. Two substitutions in site 2 of the spolIG promoter that change the sequence to be more like an optimal SpoOA-binding site were found to increase promoter activity. Moreover, phosphorylation of SpoOA was not required in vivo for activation of the spoIIG promoter containing these strong binding sites. The results suggest that the primary role for phosphorylation of SpoOA is to increase its affinity for specific sites rather than to activate an activity of SpoOA that acts on RNA polymerase at promoters.During endospore formation in Bacillus subtilis, transcription of sporulation-specific genes is regulated both temporally and spatially. Particularly at later times, this regulation is achieved largely by the sequential and, in some cases, compartment-specific appearance or activation of a series of secondary sigma factors that direct core RNA polymerase to different sets of promoters (reviewed in reference 6). However, at early times in the

In many pathogenic bacteria, iron starvation serves as an environmental signal that triggers the expression of virulence factors, many of which are found on the cell surface or secreted into the culture supernatant. Using the chelating agent nitrilotriacetic acid, we have established conditions for iron starvation of the important human pathogen Streptococcus pyogenes (the group A streptococcus) and determined that iron limitation results in the specific appearance of several new proteins in the culture supernatant. One of these supernatant proteins is the ADP-ribosylating protein known as streptococcal plasmin receptor (Plr) or as the streptococcal surface glyceraldehyde-3-phosphate-dehydrogenase because of its other activities. Upon iron starvation, Plr is specifically released into the culture supernatant in a time-dependent manner, and its appearance in the supernatant is not accompanied by induction of plr mRNA synthesis. Release of Plr from the bacteria may be important for the virulence of group A streptococci and the manifestation of diseases.