Antagonistic Rgg regulators mediate quorum sensing via competitive DNA binding in Streptococcus pyogenes

Abstract

Recent studies have established the fact that multiple members of the Rgg family of transcriptional regulators serve as key components of quorum sensing (QS) pathways that utilize peptides as intercellular signaling molecules. We previously described a novel QS system in Streptococcus pyogenes which utilizes two Rgg-family regulators (Rgg2 and Rgg3) that respond to neighboring signaling peptides (SHP2 and SHP3) to control gene expression and biofilm formation. We have shown that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes, and that SHPs function to activate the QS system. The mechanisms by which Rgg proteins regulate both QS-dependent and QS-independent processes remain poorly defined; thus, we sought to further elucidate how Rgg2 and Rgg3 mediate gene regulation. Here we provide evidence that S. pyogenes employs a unique mechanism of direct competition between the antagonistic, peptide-responsive proteins Rgg2 and Rgg3 for binding at target promoters. The highly conserved, shared binding sites for Rgg2 and Rgg3 are located proximal to the -35 nucleotide in the target promoters, and the direct competition between the two regulators results in concentration-dependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP. These results suggest that exclusionary binding of target promoters by Rgg3 may prevent Rgg2 binding under SHP-limiting conditions, thereby preventing premature induction of the quorum sensing circuit.

Importance: Rgg-family transcriptional regulators are widespread among low-G+C Gram-positive bacteria and in many cases contribute to bacterial physiology and virulence. Only recently was it discovered that several Rgg proteins function in cell-to-cell communication (quorum sensing [QS]) via direct interaction with signaling peptides. The mechanism(s) by which Rgg proteins mediate regulation is poorly understood, and further insight into Rgg function is anticipated to be of great importance for the understanding of both regulatory-network architecture and intercellular communication in Rgg-containing species. The results of this study on the Rgg2/3 QS circuit of S. pyogenes demonstrate that DNA binding of target promoters by the activator Rgg2 is directly inhibited by competitive binding by the repressor Rgg3, thereby preventing transcriptional activation of the target genes and premature induction of the QS circuit. This is a unique regulatory mechanism among Rgg proteins and other peptide-responsive QS regulators.

FIG 1

Rgg/SHP pairs in the NZ131 genome (A) and intergenic region maps for shp2 and rgg2 (B) and rgg3 and shp3 (C) gene pairs. (B and C) Sense DNA strand for the promoter region of each shp gene. Translation start codons for Rgg proteins and SHPs are colored to match the gene colors in panel A. Transcription start sites determined by 5′ RACE are in bold, with corresponding bent arrows and gene designations. Predicted −10 promoter elements for the shp genes are underlined, and the −35 nucleotides are indicated by asterisks. The conserved region shared between the shp promoters is highlighted in gray, with the single nucleotide difference indicated by vertical arrows.

FIG 2

(A) EMSA analysis of Rgg binding to the promoters of shp3 (top) and shp2 (bottom) at increasing protein concentrations. (B) EMSA analysis of Rgg2 binding to a control PrRNA probe (right) or the shp3 promoter in the presence of a 5-fold molar excess of unlabeled specific (P_shp3) or nonspecific (PrRNA) competitor DNA (left).

FIG 3

(A) Footprinting electrophoretograms following DNase I digestion of the sense strand of the shp2 promoter region in the presence of BSA (top), Rgg2 (middle), or Rgg3 (bottom). The box indicates the region of DNA protected by both Rgg proteins, and the corresponding nucleotide sequence is included at the bottom of the panel. Nucleotides possibly protected by Rgg binding are indicated by the double underline. The arrowhead in the Rgg2 panel denotes a hypersensitive site. (B and C) Promoter maps depicting the elucidated Rgg binding sites in the shp2 (B) and shp3 (C) promoter regions. Translation start codons for Rgg proteins are colored to match the gene colors in Fig. 1A. Transcription start sites are in bold, with corresponding bent arrows and gene designations. Predicted −10 promoter elements for the shp and rgg genes are underlined with corresponding designations. The conserved region shared between the shp promoters is highlighted in gray, and nucleotides protected during DNase I digestion by Rgg2 and Rgg3 are indicated by horizontal black bars above and below the sense and antisense DNA strand, respectively. The thin double line above nucleotides in the shp2 promoter corresponds to the possible region of protection indicated by the double underline in panel A.

FIG 4

Schematic diagrams of the shp2 (A) and shp3 (B) promoter region fragments used as probes in EMSA analyses. Plus and minus signs to the right of each probe indicate the presence or absence of detectable binding of the corresponding probe by Rgg2 and Rgg3. EMSA gels illustrating binding are included below each diagram. (C) Schematic diagram of the shp2 promoter region and DNA fragments containing the WT (Pshp2-C1) and Mut (Pshp2-C2) conserved region used as probes in EMSA reactions. Nucleotide sequences of the conserved region of each probe are included below, with the two nucleotides changed by site-directed mutagenesis underlined. (D) EMSA analysis of Rgg2 and Rgg3 binding to the Pshp2-C1 and Pshp2-C2 probes. All EMSA reactions were performed using 200 nM Rgg2 or Rgg3.

FIG 5

(A) Schematic diagram of DNA fragments containing various segments of the shp2 promoter used in transcriptional fusions with luxAB (P_shp2, pBL111; P_shp2 T1, pBL116; P_shp2 mutT1, pBL118; P_shp2 T2, pBL117). P_shp2 mutT1 is identical to the P_shp2 T1 reporter except that two nucleotides in the conserved region have been mutated as shown in Fig. 4C. (B) Luciferase expression from P_shp2, P_shp2 T1, and P_shp2 T2 reporters integrated into WT (BNL148, BNL172, and BNL179), Δrgg3 (BNL149, BNL173, and BNL180), and Δrgg3 Δrgg2 (BNL153, BNL189, and BNL190) strains grown in CDM (chemically defined medium). (C) Luciferase expression from P_shp2 T1 and P_shp2 mutT1 reporters in WT (BNL148 and BNL181), Δrgg3 (BNL149 and BNL182), and Δrgg3 Δrgg2 (BNL153 and BNL183) strains grown in CDM. All luminescence data shown are representative of at least 3 independent experiments.

FIG 6

(A) Co-EMSA analysis of competitive DNA binding by MBP-Rgg2 and Rgg3 using various protein concentrations. (B) Co-EMSA analysis of competitive DNA binding by Rgg proteins in the presence of pure (>95%) sSHP-C8 peptides. sSHP3-revC8 was included as a control. All reaction mixtures contained 10 nM Pshp3 probe.