The cryptic competence pathway in Streptococcus pyogenes is controlled by a peptide pheromone

Abstract

Horizontal gene transfer is an important means of bacterial evolution that is facilitated by transduction, conjugation, and natural genetic transformation. Transformation occurs after bacterial cells enter a state of competence, where naked DNA is acquired from the extracellular environment. Induction of the competent state relies on signals that activate master regulators, causing the expression of genes involved in DNA uptake, processing, and recombination. All streptococcal species contain the master regulator SigX and SigX-dependent effector genes required for natural genetic transformation; however, not all streptococcal species have been shown to be naturally competent. We recently demonstrated that competence development in Streptococcus mutans requires the type II ComRS quorum-sensing circuit, comprising an Rgg transcriptional activator and a novel peptide pheromone (L. Mashburn-Warren, D. A. Morrison, and M. J. Federle, Mol. Microbiol. 78:589-606, 2010). The type II ComRS system is shared by the pyogenic, mutans, and bovis streptococci, including the clinically relevant pathogen Streptococcus pyogenes. Here, we describe the activation of sigX by a small-peptide pheromone and an Rgg regulator of the type II ComRS class. We confirm previous reports that SigX is functional and able to activate sigX-dependent gene expression within the competence regulon, and that SigX stability is influenced by the cytoplasmic protease ClpP. Genomic analyses of available S. pyogenes genomes revealed the presence of intact genes within the competence regulon. While this is the first report to show natural induction of sigX, S. pyogenes remained nontransformable under laboratory conditions. Using radiolabeled DNA, we demonstrate that transformation is blocked at the stage of DNA uptake.

Fig 1

Proposed model for competence gene regulation in S. pyogenes. Based on previous findings in S. mutans (34), we hypothesize that ComRS regulates the expression of sigX in GAS. ComS is secreted from the bacterial cell and processed by an unknown mechanism to produce the mature sigX-inducing peptide (XIP). Importation of XIP depends on the Opp transporter, and once in the cytosol, XIP interacts with ComR, proposed here to be a dimer. Together, ComR and XIP bind the P1 promoter regions upstream of comS and sigX, activating their transcription. SigX accumulation is dependent on the ClpP protease, but once produced, SigX and RNA polymerase recognize and bind to CIN box-containing promoter regions upstream of late competence genes to activate transcription.

Fig 2

Characterization of the early and late competence genes in S. pyogenes. The early genes comprise the quorum-sensing system comRS and the alternative sigma factor sigX, which are required for activation of late competence genes. The P1 promoter pattern to which ComR binds is designated P1. The P1 consensus sequence is AACAN GACA N₄ TGTCN TGTT N₁₉ TATAAT (34). The genes listed as late genes are those known to be induced by ComX and that are required for transformation in S. pneumoniae. These consist of genes involved in DNA uptake and transport and those required for DNA processing and recombination. Binding sites for SigX, known as CIN boxes (C), have the consensus sequence TACGAATA. An asterisk indicates that gene designations and spy numbers are based on the M1 SF370 genome. Solid lines within genes represent stop codons, dashed lines represent frameshift mutations, and the double line in comR indicates a 9-bp insertion. Mutations are based on publically available sequenced genomes but have not been substantiated, except for those found in NZ131. Genomes include NZ131 (a), MGAS2096 (b), MGAS6180 (c), MGAS10394 (d), MGAS10750 (e), MGAS315 (f), and SSI-1 (g). ≠, Synonym for sigX in S. pneumoniae, comX; +, synonyms for the comY operon in S. pneumoniae and Bacillus subtilis, cgl and comG; ‡, synonym for the comE operon in S. pneumoniae, cel.

Fig 3

Induction of sigX is XIP dose and allele dependent. The dose-dependent effects of XIP on growth and sigX expression were determined in wild-type strains containing the P_sigX::luxAB reporter construct. (A) Growth of MW134 (MGAS315 plus P_sigX::luxAB). Strains were diluted from mid-logarithmic phase to an OD₆₀₀ of 0.01 and grown at 37°C until an OD₆₀₀ of 0.1, at which time the indicated concentrations of XIP (nM) or the DMSO vehicle (V) were added (indicated by the arrow). (B and C) Dose-dependent induction of sigX by XIP. MW134 (MGAS315 plus P_sigX::luxAB) and MW200 (MGAS8232 plus P_sigX::luxAB) were grown as described above. After XIP addition, cultures were incubated at 37°C for 60 min, at which time samples were taken to measure luciferase activity. RLU (relative light units) measure counts per minute/OD₆₀₀. Error bars represent the standard deviations from three separate experiments.

Fig 4

Induction of sigX and late gene expression requires exogenous peptide pheromone and comR. Activity of the P_sigX::luxAB and P_ssbB::luxAB reporter constructs in various genetic backgrounds was determined in CDM after addition of exogenous synthetic signaling peptides. S. pyogenes strains were diluted to an OD₆₀₀ of 0.01 and grown at 37°C until an OD₆₀₀ of 0.1, at which time synthetic peptide (XIP or SilCR) or the DMSO vehicle (V) was added to a final concentration of 200 nM (XIP) and/or 1 μM (SilCR). Samples were taken every 30 min. Data shown are representative of three similar experiments. RLU (relative light units) measure counts per minute/OD₆₀₀. (A) MGAS315, M3 XIP; (B) MGAS315, M3 XIP; (C) MGAS8232, M1 XIP and SilCR.

Fig 5

SigX accumulation is dependent on XIP and ClpP. Cultures of the wild-type and the clpP deletion strain were grown at 37°C in CDM to mid-logarithmic phase and supplemented with DMSO (V) or XIP for 30 or 60 min. Whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-SigX antiserum. Molecular mass standards (in kDa) are shown to the left. The calculated molecular mass of SigX is 19.6 kDa.

Fig 6

Natural transformation in S. pyogenes is blocked at DNA uptake. DNA uptake by S. mutans UA159 and S. pyogenes MGAS315 wild-type and ΔcomR strains was determined by incubation with radiolabeled DNA. Cultures were diluted to an OD₆₀₀ of 0.01 and grown in CDM at 37°C until an OD₆₀₀ of 0.1, at which time cells were supplemented with 1 μM XIP for 60 min. Cells were then washed and suspended in CDM containing 1 μM XIP for 15 min. Radiolabeled DNA was added to cell cultures and allowed to incubate. At the indicated times, cells were harvested and the amount of radiolabeled DNA was measured. Error bars represent standard deviations from three separate experiments.