Identification of Rgg Binding Sites in the Streptococcus pyogenes Chromosome

Abstract

Streptococcus pyogenes Rgg is a regulatory protein that controls the transcription of 588 genes in strain NZ131 during the post-exponential phase of growth, including the virulence-associated genes encoding the extracellular SpeB protease, pullulanase A (PulA), and two extracellular nucleases (SdaB and Spd-3). Rgg binds to DNA proximally to the speB promoter (PspeB) to activate transcription; however, it is not known if Rgg binds to the promoters of other genes to influence expression, or if the perturbation of other global regulons accounts for the genome-wide changes in expression associated with the mutant. To address this issue, chromatin immunoprecipitation followed by DNA microarray analysis (ChIP-chip) was used to identify the DNA binding sites of Rgg. Rgg bound to 65 sites in the chromosome. Thirty-five were within noncoding DNA, and 43% of these were adjacent to genes previously identified as regulated by Rgg. Electrophoretic mobility shift assays were used to assess the binding of Rgg to a subset of sites bound in vivo, including the noncoding DNA upstream of speB, the genes encoding PulA, Spd-3, and a transcriptional regulator (SPY49_1113), and prophage-associated genes encoding a putative integrase (SPY49_0746) and a surface antigen (SPY49_0396). Rgg bound to all target DNAs in vitro, consistent with the in vivo results. Finally, analyses with a transcriptional reporter system showed that the DNA bound by Rgg contained an active promoter that was regulated by Rgg. Overall, the results indicate that Rgg binds specifically to multiple sites in the chromosome, including prophage DNA, to influence gene expression.

Growth Phase-Dependent Modulation of Rgg Binding Specificity in Streptococcus pyogenes

“…Rgg can then bind to the promoter region of speB to activate transcription (36). We previously showed that Rgg binds to the speB promoter only during the post-exponential phase of growth, consistent with the model described by Loughman and Caparon (2,36).…”

“…A ChIP assay was used to identify the DNA binding sites of Rgg in the exponential phase of growth using a previously described experimental protocol (2). Briefly, overnight cultures of S. pyogenes strains rgg (15) and SA5 (encoding the Rgg-Myc fusion protein) (2) were inoculated into 40 ml of THY broth in 50-ml tubes to an A 600 of 0.08.…”

“…An Rgg-maltosebinding protein (MBP) fusion protein was expressed and purified as previously described (2). The putative promoter regions upstream of emm49, sof, sfbX49, and speH were amplified using NZ131 gDNA as the template DNA and the primer sets emm49fwd and emm49rev, soffwd and sofrev, sfbx_BamHIfwd and sfbx_XhoIrev, and speHfwd and speHrev, respectively ( Table 2).…”

“…Inactivation of rgg in strain NZ131 (a serotype M49 strain isolated from a patient with poststreptococcal acute glomerulonephritis) alters the expression of 293 and 588 genes in the exponential and post-exponential phases of growth, respectively (26), hindering functional predictions. To address this issue, we recently identified the DNA binding profile of Rgg during the post-exponential phase of growth by using chromatin immunoprecipitation (ChIP) coupled to DNA microarrays (ChIP-chip) as an initial step to identify genes that are directly regulated by Rgg (2). Rgg bound to 35 noncoding DNA regions, 70% of which were adjacent to genes previously reported to be regulated by Rgg.…”

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

“…speB expression is controlled by various transcriptional regulators, including the well-characterized regulator of protease B (RopB) [21–23]. RopB binds at direct and inverted repeats located upstream of the speB promoter to induce speB expression [23,24]. The speB and ropB genes are adjacent on the chromosome and transcribed in opposite directions.…”