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.

Fig. 1.

Rgg binds to PspeB in vivo only in the post-exponential phase of growth. Quantitative PCR was used to measure the amounts of PspeB and, as a negative control, groEL in ChIP samples obtained from the rgg mutant and strain SA5 in either the exponential or the post-exponential phase of growth.

Fig. 2.

Genomic Rgg-binding sites. The enrichment of DNA (y axis) in ChIP samples is plotted against the location of the DNA in the 1.8-Mb S. pyogenes genome (x axis).

Fig. 3.

Functions of genes associated with Rgg binding sites. The x axis indicates the number of ORFs (shaded bars) or noncoding DNAs proximal and upstream (filled bars) of genes bound by Rgg and the respective functions (y axis).

Fig. 4.

Gel shift assays of selected target DNAs. The binding of Rgg to PspeB and the noncoding DNAs upstream of Spy49_1113, Spy49_0746, spd-3, pulA, and Spy49_0396 that bound to Rgg in vivo was assessed by incubation without Rgg or with 6.5, 26, 39, and 52 pmol of purified Rgg with radiolabeled DNA. Cold nonspecific DNA refers to unlabeled groEL, and the cold specific probe is unlabeled competing DNA.

Fig. 5.

Promoter activity of DNA bound by Rgg. Transcriptional fusions were constructed between the DNA upstream of speB (A), pulA (B), spy49_1113 (C), spy49_396 (D), spy49_746 (E), and spd-3 (F) and the firefly luciferase gene (luc). The plasmids were transformed into the wild type (wt) and the rgg mutant, which were then grown to the post-exponential phase of growth, and promoter activity was determined by measuring luciferase. The data shown are representative results from three independent biological replicates.