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A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator.

ABSTRACT: All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX-dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX. We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double-tryptophan-containing sigX-inducing peptide (XIP) pheromone. In all three groups, the XIP gene (comS), and sigX have identical, non-canonical promoters consisting of 9 bp inverted repeats separated from a -10 hexamer by 19 bp. comS is adjacent to a gene encoding a putative transcription factor of the Rgg family and is regulated by its product, which we designate ComR. Deletion of comR or comS in Streptococcus mutans abolished transformability, as did deletion of the oligopeptide permease subunit oppD, suggesting that XIP is imported. Providing S. mutans with synthetic fragments of ComS revealed that seven C-terminal residues, including the WW motif, cause robust induction of both sigX and the competent state. We propose that this circuit is the proximal regulator of sigX in S. mutans, and we infer that it controls competence in a parallel way in all pyogenic and bovis streptococci.

SUBMITTER: Mashburn-Warren L

PROVIDER: S-EPMC3058796 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator.

Mashburn-Warren Lauren L Morrison Donald A DA Federle Michael J MJ

Molecular microbiology 20100914 3

All streptococcal genomes encode the alternative sigma factor SigX and 21 SigX-dependent proteins required for genetic transformation, yet no pyogenic streptococci are known to develop competence. Resolving this paradox may depend on understanding the regulation of sigX. We report the identification of a regulatory circuit linked to the sigX genes of mutans, pyogenic, and bovis streptococci that uses a novel small, double-tryptophan-containing sigX-inducing peptide (XIP) pheromone. In all three ...[more]

PMID: 20969646

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Project description:Streptococcus gallolyticus subsp. gallolyticus, a member of the group D streptococci, is normally found in the bovine rumen and human gut. It is an opportunistic pathogen that was recently determined to be a bacterial driver of colorectal cancer, in addition to causing other diseases, such as infective endocarditis, bacteremia, neonatal meningitis, and septicemia. As an emerging pathogen, not much is known about this bacterium, its virulence mechanisms, or its virulence regulatory pathways. Previous studies suggest that S. gallolyticus subsp. gallolyticus uses a ComRS pathway, one of many Streptococcus quorum-sensing circuitries, for competence. However, thus far, the ubiquitous ComABCDE pathway has not been studied, nor has its regulatory role in S. gallolyticus subsp. gallolyticus We therefore sought to study the S. gallolyticus subsp. gallolyticus ComABCDE quorum-sensing pathway and have identified its peptide pheromone, which is termed the competence-stimulating peptide (CSP). We further determined that this peptide regulates the production of bacteriocin-like inhibitory substances (BLISs), a phenotype that has been linked with the ComABCDE pathway in both Streptococcus pneumoniae and Streptococcus mutans Our data show that S. gallolyticus subsp. gallolyticus TX20005 produces a 21-mer CSP signal, which differs from CSP signals of other Streptococcus species in that its active form begins three residues after the double-glycine leader signal of the ComC precursor peptide. Additionally, our data suggest that this peptide might not be related to competence induction, as opposed to CSP signaling peptides in other Streptococcus species. This study provides the first evidence that S. gallolyticus subsp. gallolyticus utilizes quorum sensing to eliminate competitors, presenting a potential pathway to target this emerging human pathogen.IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus is an emerging human pathogen known as a causative agent of infective endocarditis, and recently, of colorectal cancer. In this work, we revealed a functional quorum-sensing circuitry in S. gallolyticus subsp. gallolyticus, including the identification of the central signaling peptide pheromone, competence-stimulating peptide (CSP), and the regulatory role of this circuitry in the production of bacteriocin-like inhibitory substances (BLISs). This work uncovered a mechanism by which this bacterium outcompetes other bacterial species and thus provides a potential tool to study this opportunistic pathogen.

Project description:BACKGROUND: Streptococcus mutans, the etiological agent of human dental caries, displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (comX inducing peptide, XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. ComR-XIP controls the expression of both PcomX and PcomS, with the latter creating a positive feedback loop. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we began to dissect how XrpA exerts control over competence development in S. mutans. METHODS: RNA-Seq was utilized to compare the transcriptomes of S. mutans wild-type strain UA159 (3 replicates), UA159 with addition of 2 uM sXIP (3 replicates) and with a mutant of xrpA (comX::T162C; 3 replicates). Strains were grown to OD600 nm = 0.5 in FMC medium before harvest. sXIP was added at OD600 nm = 0.2. Deep sequencing was performed at the University of Florida ICBR facilities (Gainesville, FL). Approximately 20 million short-reads were obtained for each sample. After removing adapter sequences from each short-read and trimming of the 3’-ends by quality scores (59), the resulting sequences were mapped onto the reference genome of strain UA159 (GenBank accession no. AE014133) using the short-read aligner. Mapped short-read alignments were then converted into readable formats using SAMTOOLS. RESULTS: Using an optimzed data analysis workflow, we mapped 14-19 million reads per sample to the genome of UA159. For viewing of the mapped reads aligned to the genome, .bam files were uploaded into the Integrative Genomics Viewer (IGV – version 2.3.55) (61). A .csv file containing raw read counts for each replicate (3) was then uploaded to Degust (http://degust.erc.monash.edu/) and edgeR analysis performed to determine Log2 fold change and a false discovery rate (FDR). Comparison of the transcriptome by RNA-Seq of the wild-type with the mutant lacking XrpA revealed 56 differentially expressed genes, with 34 upregulated in xrpA and 22 downregulated. Many of the upregulated genes were competence-related genes, including comX and genes that are a part of the ComX regulon of S. mutans . Since loss of xrpA caused upregulation of competence genes, we also analyzed the transcriptome of UA159 treated with 2 uM sXIP to induce competence, with UA159 treated with vehicle (DMSO) as a control. Cells treated with sXIP had 137 genes differentially expressed compared to the DMSO control. CONCLUSIONS: Collectively, these data reveal that the novel regulator XrpA exerts its influence over competence development by impacting ComRS functions, resulting in decreased expression of comX and late com gene expression that negatively impact transformability. These results highlight XrpA as a new negative regulator of competence signaling as well as broaden our understanding of the complex regulatory mechanisms that modulate competence and virulence in S. mutans.

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A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator.

Publications

A novel double-tryptophan peptide pheromone controls competence in Streptococcus spp. via an Rgg regulator.

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