Alternative Sigma Factor SigX Research Articles

The ComRS-SigX Pathway Regulates Natural Transformation in Streptococcus ferus.

The ability to take up and incorporate foreign DNA via natural transformation is a well-known characteristic of some species of Streptococcus, and is a mechanism that rapidly allows for the acquisition of antibacterial resistance. Here, we describe that the understudied species Streptococcus ferus is also capable of natural transformation and uses a system analogous to that identified in Streptococcus mutans. S. mutans natural transformation is under the control of the alternative sigma factor sigX (also known as comX), whose expression is induced by two types of peptide signals: CSP (competence stimulating peptide, encoded by comC) and XIP (sigX-inducing peptide, encoded by comS). These systems induce competence via either the two-component signal-transduction system ComDE or the RRNPP transcriptional regulator ComR, respectively. Protein and nucleotide homology searches identified putative orthologs of comRS and sigX in S. ferus, but not homologs of S. mutans blpRH (also known as comDE). We demonstrate that natural transformation in S. ferus is induced by a small, double-tryptophan containing sigX-inducing peptide (XIP), akin to that of S. mutans, and requires the presence of the comR and sigX orthologs for efficient transformation. Additionally, we find that natural transformation is induced in S. ferus by both the native XIP and the XIP variant of S. mutans, implying that cross talk between the two species is possible. This process has been harnessed to construct gene deletions in S. ferus and provides a method to genetically manipulate this understudied species. IMPORTANCE Natural transformation is the process by which bacteria take up DNA and allows for acquisition of new genetic traits, including those involved in antibiotic resistance. This study demonstrates that the understudied species Streptococcus ferus is capable of natural transformation using a peptide-pheromone system like that previously identified in Streptococcus mutans and provides a framework for future studies concerning this organism.

Conserved Pheromone Production, Response and Degradation by Streptococcus mutans.

Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM but can also stimulate bacteriocin genes as a late response. Interconnections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the activity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was able to degrade CSP, and that such activity was not significantly different in isogenic mutants with deletion of the protease gene htrA or the competence genes sigX, oppD, and comR. CSP cleavage was also detected in all the wild type strains, indicating that this is a conserved feature in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 and 26000 nM. Transformation in the presence of exogenous XIP was detected in all but one of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as the ability to degrade CSP, are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favor diseases.

The conserved mosaic prophage protein paratox inhibits the natural competence regulator ComR in Streptococcus

Horizontal gene transfer is an important means of bacterial evolution. This includes natural genetic transformation, where bacterial cells become “competent” and DNA is acquired from the extracellular environment. Natural competence in many species of Streptococcus, is regulated by quorum sensing via the ComRS receptor-signal pair. The ComR-XIP (mature ComS peptide) complex induces expression of the alternative sigma factor SigX, which targets RNA polymerase to CIN-box promoters to activate genes involved in DNA uptake and recombination. In addition, the widely distributed Streptococcus prophage gene paratox (prx) also contains a CIN-box, and here we demonstrate it to be transcriptionally activated by XIP. In vitro experiments demonstrate that Prx binds ComR directly and prevents the ComR-XIP complex from interacting with DNA. Mutations of prx in vivo caused increased expression of the late competence gene ssb when induced with XIP as compared to wild-type, and Prx orthologues are able to inhibit ComR activation by XIP in a reporter strain which lacks an endogenous prx. Additionally, an X-ray crystal structure of Prx reveals a unique fold that implies a novel molecular mechanism to inhibit ComR. Overall, our results suggest Prx functions to inhibit the acquisition of new DNA by Streptococcus.

Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium.

Like Streptococcus mutans, lactobacilli are commonly isolated from carious sites, although their exact role in caries development remains unclear. This study used mixed-species models to analyze biofilm formation by major groups of oral lactobacilli, including L. casei, L. fermentum, L. rhamnosus, L. salivarius ssp. salivarius, and L. gasseri. The results showed that lactobacilli did not form good biofilms when grown alone, although differences existed between different species. When grown together with S. mutans, biofilm formation by L. gasseri and L. rhamnosus was increased by 2-log (P < 0.001), while biofilms by L. fermentum reduced by >1-log (P < 0.001). L. casei enhanced biofilm formation by ~2-log when grown with S. mutans wild-type, but no such effects were observed with S. mutans deficient of glucosyltransferase GtfB and adhesin P1. Both S. mutans and L. casei in dual-species enhanced resistance to acid killing with increases of survival rate by >1-log (P < 0.001), but drastically reduced the survival rates following exposure to hydrogen peroxide (P < 0.001), as compared to the respective mono-species cultures. When analyzed by RNA-seq, more than 134 genes were identified in S. mutans in dual-species with L. casei as either up- or down-regulated when compared to those grown alone. The up-regulated genes include those for superoxide dismutase, NADH oxidase, and members of the mutanobactin biosynthesis cluster. Among the down-regulated genes were those for GtfB and alternative sigma factor SigX. These results further suggest that interactions between S. mutans and oral lactobacilli are species-specific and may have significant impact on cariogenic potential of the community.

Quorum sensing of Streptococcus mutans is activated by Aggregatibacter actinomycetemcomitans and by the periodontal microbiome

BackgroundThe oral cavity is inhabited by complex microbial communities forming biofilms that can cause caries and periodontitis. Cell-cell communication might play an important role in modulating the physiologies of individual species, but evidence so far is limited.ResultsHere we demonstrate that a pathogen of the oral cavity, Aggregatibacter actinomycetemcomitans (A. act.), triggers expression of the quorum sensing (QS) regulon of Streptococcus mutans, a well-studied model organism for cariogenic streptococci, in dual-species biofilms grown on artificial saliva. The gene for the synthesis of the QS signal XIP is essential for this interaction. Transcriptome sequencing of biofilms revealed that S. mutans up-regulated the complete QS regulon (transformasome and mutacins) in the presence of A. act. and down-regulated oxidative stress related genes. A.act. required the presence of S. mutans for growth. Fimbriae and toxins were its most highly expressed genes and up-regulation of anaerobic metabolism, chaperones and iron acquisition genes was observed in co-culture. Metatranscriptomes from periodontal pockets showed highly variable levels of S. mutans and low levels of A. act.. Transcripts of the alternative sigma-factor SigX, the key regulator of QS in S. mutans, were significantly enriched in periodontal pockets compared to single cultures (log2 4.159, FDR ≤0.001, and expression of mutacin related genes and transformasome components could be detected.ConclusionThe data show that the complete QS regulon of S. mutans can be induced by an unrelated oral pathogen and S. mutans may be competent in oral biofilms in vivo.

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species.

Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a ‘test-bed’ assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides. Together, our results not only provide a model for XIP recognition and specificity, but also allow for the prediction of novel XIP peptides that induce ComR activity.

The Alternative Sigma Factor SigX Controls Bacteriocin Synthesis and Competence, the Two Quorum Sensing Regulated Traits in Streptococcus mutans.

Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S. mutans cells can split into a competent and non-competent subpopulation. The origin of this population heterogeneity has not been experimentally determined and it is unknown how the two QS systems are connected. We developed a toolbox of single and dual fluorescent reporter strains and systematically knocked out key genes of the competence signaling cascade in the reporter strain backgrounds. By following signal propagation on the single cell level we discovered that the master regulator of competence, the alternative sigma factor SigX, directly controls expression of the response regulator for bacteriocin synthesis ComE. Consequently, a SigX binding motif (cin-box) was identified in the promoter region of comE. Overexpressing the genetic components involved in competence development demonstrated that ComRS represents the origin of bimodality and determines the modality of the downstream regulators SigX and ComE. Moreover these analysis showed that there is no direct regulatory link between the two QS signaling cascades. Competence is induced through a hierarchical XIP signaling cascade, which has no regulatory input from the CSP cascade. CSP exclusively regulates bacteriocin synthesis. We suggest renaming it mutacin inducing peptide (MIP). Finally, using phosphomimetic comE mutants we show that unimodal bacteriocin production is controlled posttranslationally, thus solving the puzzling observation that in complex media competence is observed in a subpopulation only, while at the same time all cells produce bacteriocins. The control of both bacteriocin synthesis and competence through the alternative sigma-factor SigX suggests that S. mutans increases its genetic repertoire via QS controlled predation on neighboring species in its natural habitat.

Regulated proteolysis of the alternative sigma factor SigX in Streptococcus mutans: implication in the escape from competence.

BackgroundSigX (σX), the alternative sigma factor of Streptococcus mutans, is the key regulator for transcriptional activation of late competence genes essential for taking up exogenous DNA. Recent studies reveal that adaptor protein MecA and the protease ClpC act as negative regulators of competence by a mechanism that involves MecA-mediated proteolysis of SigX by the ClpC in S. mutans. However, the molecular detail how MecA and ClpC negatively regulate competence in this species remains to be determined. Here, we provide evidence that adaptor protein MecA targets SigX for degradation by the protease complex ClpC/ClpP when S. mutans is grown in a complex medium.ResultsBy analyzing the cellular levels of SigX, we demonstrate that the synthesis of SigX is transiently induced by competence-stimulating peptide (CSP), but the SigX is rapidly degraded during the escape from competence. A deletion of MecA, ClpC or ClpP results in the cellular accumulation of SigX and a prolonged competence state, while an overexpression of MecA enhances proteolysis of SigX and accelerates the escape from competence. In vitro protein-protein interaction assays confirm that MecA interacts with SigX via its N-terminal domain (NTD1–82) and with ClpC via its C-terminal domain (CTD123–240). Such an interaction mediates the formation of a ternary SigX-MecA-ClpC complex, triggering the ATP-dependent degradation of SigX in the presence of ClpP. A deletion of the N-terminal or C-terminal domain of MecA abolishes its binding to SigX or ClpC. We have also found that MecA-regulated proteolysis of SigX appears to be ineffective when S. mutans is grown in a chemically defined medium (CDM), suggesting the possibility that an unknown mechanism may be involved in negative regulation of MecA-mediated proteolysis of SigX under this condition.ConclusionAdaptor protein MecA in S. mutans plays a crucial role in recognizing and targeting SigX for degradation by the protease ClpC/ClpP. Thus, MecA actually acts as an anti-sigma factor to regulate the stability of SigX during competence development.

Identification of the Alternative Sigma Factor SigX Regulon and Its Implications for Pseudomonas aeruginosa Pathogenicity

Pseudomonas aeruginosa is distinguished by its broad metabolic diversity and its remarkable capability for adaptation, which relies on a large collection of transcriptional regulators and alternative sigma (σ) factors. The largest group of alternative σ factors is that of the extracytoplasmic function (ECF) σ factors, which control key transduction pathways for maintenance of envelope homeostasis in response to external stress and cell growth. In addition, there are specific roles of alternative σ factors in regulating the expression of virulence and virulence-associated genes. Here, we analyzed a deletion mutant of the ECF σ factor SigX and applied mRNA profiling to define the SigX-dependent regulon in P. aeruginosa in response to low-osmolarity-medium conditions. Furthermore, the combination of transcriptional data with chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) led to the identification of the DNA binding motif of SigX. Genome-wide mapping of SigX-binding regions revealed enrichment of downstream genes involved in fatty acid biosynthesis, type III secretion, swarming and cyclic di-GMP (c-di-GMP) signaling. In accordance, a sigX deletion mutant exhibited altered fatty acid composition of the cell membrane, reduced cytotoxicity, impaired swarming activity, elevated c-di-GMP levels, and increased biofilm formation. In conclusion, a combination of ChIP-seq with transcriptional profiling and bioinformatic approaches to define consensus DNA binding sequences proved to be effective for the elucidation of the regulon of the alternative σ factor SigX, revealing its role in complex virulence-associated phenotypes in P. aeruginosa.

Competence for Natural Genetic Transformation in the Streptococcus bovis Group Streptococci S. infantarius and S. macedonicus

Natural genetic transformation is common among many species of the genus Streptococcus, but it has never, or rarely, been reported for the Streptococcus pyogenes and S. bovis groups of species, even though many streptococcal competence genes and the competence regulators SigX, ComR, and ComS are well conserved in both groups. To explore the incidence of competence in the S. bovis group, 25 isolates of S. infantarius and S. macedonicus were surveyed by employing culture in chemically defined media devoid of peptide nutrients and treatment with synthetic candidate pheromone peptides predicted from the sequence of the gene comS. Approximately half of strains examined were transformable, many transforming at high rates comparable to those for the well-characterized streptococcal natural transformation systems. In S. infantarius, nanomolar amounts of the synthetic pheromone LTAWWGL induced robust but transient competence in high-density cultures, but mutation of the ComRS locus abolished transformation. We conclude that at least these two species of the S. bovis group retain a robust system of natural transformation regulated by a ComRS pheromone circuit and the alternative sigma factor SigX and infer that transformation is even more common among the streptococci than has been recognized. The tools presented here will facilitate targeted genetic manipulation in this group of streptococci.

Development of Competence for Genetic Transformation of Streptococcus mutans in a Chemically Defined Medium

Streptococcus mutans develops competence for genetic transformation in response to regulatory circuits that sense at least two peptide pheromones. One peptide, known as CSP, is sensed by a two-component signal transduction system through a membrane receptor, ComD. The other, derived from the primary translation product ComS, is thought to be sensed by an intracellular receptor, ComR, after uptake by oligopeptide permease. To allow study of this process in a medium that does not itself contain peptides, development of competence was examined in the chemically defined medium (CDM) described by van de Rijn and Kessler (Infect. Immun. 27:444, 1980). We confirmed a previous report that in this medium comS mutants of strain UA159 respond to a synthetic peptide comprising the seven C-terminal residues of ComS (ComS(11-17)) by increasing expression of the alternative sigma factor SigX, which in turn allows expression of competence effector genes. This response provided the basis for a bioassay for the ComS pheromone in the 100 to 1,000 nM range. It was further observed that comS(+) (but not comS mutant) cultures developed a high level of competence in the late log and transition phases of growth in this CDM without the introduction of any synthetic stimulatory peptide. This endogenous competence development was accompanied by extracellular release of one or more signals that complemented a comS mutation at levels equivalent to 1 μM synthetic ComS(11-17).

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

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.