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1.  Genetic Diversity of the Streptococcal Competence (com) Gene Locus 
Journal of Bacteriology  1999;181(10):3144-3154.
The com operon of naturally transformable streptococcal species contains three genes, comC, comD, and comE, involved in the regulation of competence. The comC gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The comD and comE genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the comD-encoded protein being a receptor for CSP. Here we report on the genetic variability of comC and comD within Streptococcus pneumoniae isolates. Comparative analysis of sequence variations of comC and comD shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many S. pneumoniae strains in the laboratory, there is a clear correlation between the presence of a particular comC allele and the cognate comD allele. These findings effectively rule out the possibility that the presence of noncognate comC and comD alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional com pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of S. mitis and S. oralis. The CSPs from these isolates are much more closely related to those from S. pneumoniae than to most CSPs previously reported from S. mitis and S. oralis, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of S. pneumoniae that are under strong selective pressure.
PMCID: PMC93770  PMID: 10322016
2.  Development of Competence for Genetic Transformation of Streptococcus mutans in a Chemically Defined Medium 
Journal of Bacteriology  2012;194(15):3774-3780.
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 (ComS11-17) 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 ComS11-17.
PMCID: PMC3416567  PMID: 22609913
3.  Natural Genetic Transformation of Streptococcus mutans Growing in Biofilms 
Journal of Bacteriology  2001;183(3):897-908.
Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm “lifestyle” for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.
PMCID: PMC94956  PMID: 11208787
4.  A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation 
Journal of Bacteriology  2002;184(10):2699-2708.
In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.
PMCID: PMC135014  PMID: 11976299
5.  A Hydrophobic Patch in the Competence-Stimulating Peptide, a Pneumococcal Competence Pheromone, Is Essential for Specificity and Biological Activity 
Journal of Bacteriology  2006;188(5):1744-1749.
Induction of competence for natural genetic transformation in Streptococcus pneumoniae depends on pheromone-mediated cell-cell communication and a signaling pathway consisting of the competence-stimulating peptide (CSP), its membrane-embedded histidine kinase receptor ComD, and the cognate response regulator ComE. Extensive screening of pneumococcal isolates has revealed that two major CSP variants, CSP1 and CSP2, are found in members of this species. Even though the primary structures of CSP1 and CSP2 are about 50% identical, they are highly specific for their respective receptors, ComD1 and ComD2. In the present work, we have investigated the structural basis of this specificity by determining the three-dimensional structure of CSP1 from nuclear magnetic resonance data and comparing the agonist activity of a number of CSP1/CSP2 hybrid peptides toward the ComD1 and ComD2 receptors. Our results show that upon exposure to membrane-mimicking environments, the 17-amino-acid CSP1 pheromone adopts an amphiphilic α-helical configuration stretching from residue 6 to residue 12. Furthermore, the pattern of agonist activity displayed by the various hybrid peptides revealed that hydrophobic amino acids, some of which are situated on the nonpolar side of the α-helix, strongly contribute to CSP specificity. Together, these data indicate that the identified α-helix is an important structural feature of CSP1 which is essential for effective receptor recognition under natural conditions.
PMCID: PMC1426553  PMID: 16484185
6.  Regulation of Bacteriocin Production in Streptococcus mutans by the Quorum-Sensing System Required for Development of Genetic Competence 
Journal of Bacteriology  2005;187(12):3980-3989.
In Streptococcus mutans, competence for genetic transformation and biofilm formation are dependent on the two-component signal transduction system ComDE together with the inducer peptide pheromone competence-stimulating peptide (CSP) (encoded by comC). Here, it is shown that the same system is also required for expression of the nlmAB genes, which encode a two-peptide nonlantibiotic bacteriocin. Expression from a transcriptional nlmAB′-lacZ fusion was highest at high cell density and was increased up to 60-fold following addition of CSP, but it was abolished when the comDE genes were interrupted. Two more genes, encoding another putative bacteriocin and a putative bacteriocin immunity protein, were also regulated by this system. The regions upstream of these genes and of two further putative bacteriocin-encoding genes and a gene encoding a putative bacteriocin immunity protein contained a conserved 9-bp repeat element just upstream of the transcription start, which suggests that expression of these genes is also dependent on the ComCDE regulatory system. Mutations in the repeat element of the nlmAB promoter region led to a decrease in CSP-dependent expression of nlmAB′-lacZ. In agreement with these results, a comDE mutant and mutants unable to synthesize or export CSP did not produce bacteriocins. It is speculated that, at high cell density, bacteriocin production is induced to liberate DNA from competing streptococci.
PMCID: PMC1151730  PMID: 15937160
7.  CinA is regulated via ComX to modulate genetic transformation and cell viability in Streptococcus mutans 
Fems Microbiology Letters  2012;331(1):44-52.
The Streptococcus mutans ComX-regulon encompasses >200 mostly uncharacterized genes, including cinA. Here we report that cinA is regulated by ComX in the presence of the competence stimulating peptide (CSP), wherein loss of CinA (strain SmuCinA) results in reduced transformability with or without added CSP by 74- and 15-fold, respectively (p<0.003). In CSP-supplemented cultures, a 2-fold increase in cell viability was noted for SmuCinA relative to UA159 (p<0.002), suggesting CinA’s involvement in the CSP-modulated cell killing response. Relative to UA159, loss of CinA also rendered the mutant hypersensitive to killing by methyl methanesulfonate (MMS), which impairs homologous recombination. Despite our use of a non-polar mutagenesis strategy to knockout cinA, which is the first gene of the multicistronic operon harboring cinA, we noted a drastic reduction in recA expression. By using a CinA-complemented mutant, we were able to partially, but not completely restore all phenotypes to UA159 levels. Complementation results suggested that although cinA participates in modulating competence, viability and MMS tolerance, genes downstream of the cinA transcript may also regulate these phenotypes, a finding that warrants further examination. This is the first report that describes a role for S. mutans’ CinA in contending with DNA damage, genetic transformation and cell survival.
PMCID: PMC3343223  PMID: 22428842
Streptococcus mutans; cinA; comX; CSP; genetic competence; cell death
8.  Phenotypic Heterogeneity of Genomically-Diverse Isolates of Streptococcus mutans 
PLoS ONE  2013;8(4):e61358.
High coverage, whole genome shotgun (WGS) sequencing of 57 geographically- and genetically-diverse isolates of Streptococcus mutans from individuals of known dental caries status was recently completed. Of the 57 sequenced strains, fifteen isolates, were selected based primarily on differences in gene content and phenotypic characteristics known to affect virulence and compared with the reference strain UA159. A high degree of variability in these properties was observed between strains, with a broad spectrum of sensitivities to low pH, oxidative stress (air and paraquat) and exposure to competence stimulating peptide (CSP). Significant differences in autolytic behavior and in biofilm development in glucose or sucrose were also observed. Natural genetic competence varied among isolates, and this was correlated to the presence or absence of competence genes, comCDE and comX, and to bacteriocins. In general strains that lacked the ability to become competent possessed fewer genes for bacteriocins and immunity proteins or contained polymorphic variants of these genes. WGS sequence analysis of the pan-genome revealed, for the first time, components of a Type VII secretion system in several S. mutans strains, as well as two putative ORFs that encode possible collagen binding proteins located upstream of the cnm gene, which is associated with host cell invasiveness. The virulence of these particular strains was assessed in a wax-worm model. This is the first study to combine a comprehensive analysis of key virulence-related phenotypes with extensive genomic analysis of a pathogen that evolved closely with humans. Our analysis highlights the phenotypic diversity of S. mutans isolates and indicates that the species has evolved a variety of adaptive strategies to persist in the human oral cavity and, when conditions are favorable, to initiate disease.
PMCID: PMC3628994  PMID: 23613838
9.  The hdrRM Operon of Streptococcus mutans Encodes a Novel Regulatory System for Coordinated Competence Development and Bacteriocin Production▿  
Journal of Bacteriology  2010;192(7):1844-1852.
The Streptococcus mutans hdrRM operon encodes a novel two-gene regulatory system induced by high cell density. Previous studies identified hdrM as the only known negative regulator of competence development in S. mutans. In the present study, we demonstrated that the HdrRM system bypasses the prototypical competence gene regulators ComC and ComDE in the transcriptional regulation of the competence-specific sigma factor comX and the late competence genes. Similarly, the HdrRM system can abrogate the requirement for ComE to produce the bacteriocin mutacin IV. To further probe the regulatory mechanism of hdrRM, we created an hdrR overexpression strain and showed that it could reproduce each of the hdrM competence and mutacin phenotypes, indicating that HdrM acts as a negative regulator of HdrR activity. Using a mutacin IV-luciferase reporter, we also demonstrated that the hdrRM system utilizes the same promoter elements recognized by ComE and thus appears to comprise a novel regulatory pathway parallel to ComCDE.
PMCID: PMC2838059  PMID: 20118256
10.  Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae 
Journal of Bacteriology  2003;185(1):349-358.
Natural transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration. The period of competence induced by CSP lasts only 15 min (quarter-height peak width). The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions. To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an Escherichia coli overexpression strain and core RNA polymerase was purified from a comX-deficient S. pneumoniae strain. The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes ssbB, cinA, cglA, celA, and dalA and was inhibited by anti-ComX antibody, but not by anti-σ70 antibody. Western blotting using antibodies specific for ComX, σ70, and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and σA remained between 60 and 125% of its normal level. ComX reached a molar ratio to RNA polymerase of at least 1.5. We conclude that ComX is unstable and acts as a competence-specific sigma factor.
PMCID: PMC141820  PMID: 12486073
11.  Natural competence in the genus Streptococcus: evidence that streptococci can change pherotype by interspecies recombinational exchanges. 
Journal of Bacteriology  1997;179(21):6589-6594.
To map the incidence of natural competence in the genus Streptococcus, we used PCR to screen a number of streptococcal strains for the presence of the recently identified competence regulation operon, containing the comC, -D, and -E genes. This approach established that the operon is present in strains belonging to the S. mitis and S. anginosus groups, but it was not detected in the other strains examined. Competence is induced in S. pneumoniae and S. gordonii by strain-specific peptide pheromones, competence-stimulating peptides (CSPs). With its unique primary structure, each CSP represents a separate pheromone type (pherotype), which is recognized by the signalling domain of the downstream histidine kinase, ComD. Thus, all bacteria induced to competence by a particular CSP belong to the same pherotype. In this study, we identified a number of new pherotypes by sequencing the genes encoding the CSP and its receptor from different streptococcal species. We found that in several cases, these genes have a mosaic structure which must have arisen as the result of recombination between two distinct allelic variants. The observed mosaic blocks encompass the region encoding the CSP and the CSP-binding domain of the histidine kinase. Consequently, the recombination events have led to switches in pherotype for the strains involved. This suggests a novel mechanism for the adaptation of naturally competent streptococci to new environmental conditions.
PMCID: PMC179583  PMID: 9352904
12.  Subpopulation-Specific Transcriptome Analysis of Competence-Stimulating-Peptide-Induced Streptococcus mutans▿† 
Journal of Bacteriology  2011;193(8):1863-1877.
Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ∼3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity.
PMCID: PMC3133041  PMID: 21317319
13.  The Streptococcus pneumoniae cia Regulon: CiaR Target Sites and Transcription Profile Analysis 
Journal of Bacteriology  2003;185(1):60-70.
The ciaR-ciaH system is one of 13 two-component signal-transducing systems of the human pathogen Streptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by the cia system, the cia regulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing the S. pneumoniae genome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimal cia regulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and the htrA-spo0J region. In addition, the transcription profile of cia loss-of-function mutants and those with an apparent activated cia system representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed the cia-dependent expression of seven putative target loci and revealed three additional cia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, no cia-dependent expression could be detected. Furthermore, the competence regulon, including the comCDE operon required for induction of competence, was completely repressed by the cia system.
PMCID: PMC141814  PMID: 12486041
14.  A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in Streptococcus thermophilus and Streptococcus salivarius▿ †  
Journal of Bacteriology  2009;192(5):1444-1454.
In streptococcal species, the key step of competence development is the transcriptional induction of comX, which encodes the alternative sigma factor σX, which positively regulates genes necessary for DNA transformation. In Streptococcus species belonging to the mitis and mutans groups, induction of comX relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In Streptococcus thermophilus, a species belonging to the salivarius group, the oligopeptide transporter Ami is essential for comX expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of comX induction in S. thermophilus LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for comX induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored comX induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in S. thermophilus and Streptococcus salivarius strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for comX induction in species from the salivarius group, which differs from the classical phosphorelay three-component system identified previously in streptococci.
PMCID: PMC2820839  PMID: 20023010
15.  The Streptococcus pneumoniae Competence Regulatory System Influences Respiratory Tract Colonization ▿  
Infection and Immunity  2008;76(7):3131-3140.
The Streptococcus pneumoniae ComDE two-component signaling system controls the development of genetic competence in the bacterium and affects virulence in models of pneumonia and bacteremia. We have investigated the impact of the competence pathway during colonization of the nasopharynx, the principal ecological niche of the pneumococcus. Previous work showed that deletion of the pneumococcal CiaRH signaling system inhibited colonization and increased expression of genes required for competence. We anticipated that signaling by the competence pathway might similarly reduce carriage. Consistent with this expectation, a comE deletion that blocked transformation increased colonization fitness such that the mutant outcompeted the wild type in an infant rat model of asymptomatic carriage. Deletion of comD—immediately upstream of comE and likewise required for competence—similarly increased colonization fitness if the orientation of the antibiotic resistance cassette inserted into the comD locus was such that it reduced transcription of comE. However, an alternative comD deletion mutation that caused an increase in comE transcription impaired colonization instead. Activation of the competence system through a comE(D143Y) mutation did not affect colonization, but an inability to secrete the competence-stimulating peptide due to deletion of comAB produced a density-dependent reduction in colonization fitness. These results suggest a model in which signaling by the unactivated form of ComE reduces colonization fitness compared to that of bacteria in which it is either activated or absent entirely, with the most substantial fitness gain accompanying deletion of comE. This observation demonstrates that the pneumococcus incurs a substantial fitness cost in order to retain a functional competence regulatory system.
PMCID: PMC2446691  PMID: 18443092
16.  Regulation of ciaXRH Operon Expression and Identification of the CiaR Regulon in Streptococcus mutans▿  
Journal of Bacteriology  2010;192(18):4669-4679.
The ciaRH operon in Streptococcus mutans contains 3 contiguous genes, ciaXRH. Unlike the CiaRH system in other streptococci, only the ciaH-null mutant displays defective phenotypes, while the ciaR-null mutant behaves like the wild type. The objective of this study was to determine the mechanism of this unusual property. We demonstrate that the ciaH mutation caused a >20-fold increase in ciaR transcript synthesis. A ciaRH double deletion reversed the ciaH phenotype, suggesting that overexpressed ciaR might be responsible for the observed ciaH phenotypes. When ciaR was forced to be overexpressed by a transcriptional fusion to the ldh promoter in the wild-type background, the same ciaH phenotypes were restored, confirming the involvement of overexpressed ciaR in the ciaH phenotypes. The ciaH mutation and ciaR overexpression also caused transcriptional alterations in 100 genes, with 15 genes upregulated >5-fold. Bioinformatics analysis identified a putative CiaR regulon consisting of 8 genes/operons, including the ciaXRH operon itself, all of which were upregulated. In vitro footprinting on 4 of the 8 promoters revealed a protected region of 26 to 28 bp encompassing two direct repeats, NTTAAG-n5-WTTAAG, 10 bp upstream of the −10 region, indicating direct binding of the CiaR protein to these promoters. Taken together, we conclude that overexpressed CiaR, as a result of either ciaH deletion or forced expression from a constitutive promoter, is a mediator in the CiaH-regulated phenotypes.
PMCID: PMC2937423  PMID: 20639331
17.  Competence for Genetic Transformation in Streptococcus pneumoniae: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW ▿  
Journal of Bacteriology  2009;191(10):3359-3366.
Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other “early” genes. ComX, a transient alternative sigma factor, drives transcription of “late” genes, many of which are essential for transformation. In vivo, ComW both stabilizes ComX against proteolysis by the ClpE-ClpP protease and stimulates its activity. Interestingly, stabilization of ComX by deletion of the gene encoding the ClpP protease did not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW and thus of its ComX stimulating activity, so that ComX might persist but lose its transcriptional activity. Western analysis revealed that ComW is indeed a transient protein, which is also stabilized by deletion of the gene encoding the ClpP protease. However, stabilizing both ComX and ComW did not prolong either ComX activity or the period of transformation, indicating that termination of the transcriptional activity of ComX is not dependent on proteolysis of ComW.
PMCID: PMC2687157  PMID: 19286798
18.  Pherotype Influences Biofilm Growth and Recombination in Streptococcus pneumoniae 
PLoS ONE  2014;9(3):e92138.
In Streptococcus pneumoniae the competence-stimulating peptide (CSP), encoded by the comC gene, controls competence development and influences biofilm growth. We explored the influence of pherotype, defined by the two major comC allelic variants (comC1 and comC2), on biofilm development and recombination efficiency. Among isolates recovered from human infections those presenting comC1 show a higher capacity to form in vitro biofilms. The influence of pherotype on biofilm growth was confirmed by experiments with isogenic strains differing in their comC alleles. Biofilm architecture evaluated by confocal laser scanning microscopy showed that strains carrying comC1 form biofilms that are denser and thicker than those carrying the comC2 allele. Isogenic strains carrying the comC1 allele yielded more transformants than those carrying the comC2 allele in both planktonic and biofilm growth. Transformation assays with comC knockout strains show that ComD1 needs lower doses of the signaling peptide to reach the same biological outcomes. In contrast to mixed planktonic growth, within mixed biofilms inter-pherotype genetic exchange is less frequent than that occurring between bacteria of the same pherotype. Since biofilms are a major bacterial lifestyle, these observations may explain the genetic differentiation between populations with different pherotypes reported previously. Considering that biofilms have been associated with colonization our results suggest that strains carrying the comC1 allele may be more transmissible and more efficient at persisting in carriage. Both effects may help explain the higher prevalence of the comC1 allele in the pneumococcal population.
PMCID: PMC3960169  PMID: 24646937
19.  Inactivation of the ciaH Gene in Streptococcus mutans Diminishes Mutacin Production and Competence Development, Alters Sucrose-Dependent Biofilm Formation, and Reduces Stress Tolerance  
Infection and Immunity  2004;72(8):4895-4899.
Many clinical isolates of Streptococcus mutans produce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of S. mutans in dental plaque. In this study, inactivation of a histidine kinase gene, ciaH, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.
PMCID: PMC470703  PMID: 15271957
20.  Microfluidic study of competence regulation in Streptococcus mutans: environmental inputs modulate bimodal and unimodal expression of comX 
Molecular microbiology  2012;86(2):258-272.
Streptococcus mutans regulates genetic competence through a complex network that receives inputs from a number of environmental stimuli, including two signaling peptides designated as CSP and XIP. The response of the downstream competence genes to these inputs shows evidence of stochasticity and bistability and has been difficult to interpret. We have used microfluidic, single-cell methods to study how combinations of extracellular signals shape the response of comX, an alternative sigma factor governing expression of the late competence genes. We find that the composition of the medium determines which extracellular signal (XIP or CSP) can elicit a response from comX and whether that response is unimodal or bimodal across a population of cells. In a chemically defined medium, exogenous CSP does not induce comX, whereas exogenous XIP elicits a comX response from all cells. In complex medium, exogenous XIP does not induce comX, whereas CSP elicits a bimodal comX response from the population. Interestingly, bimodal behavior required an intact copy of comS, which encodes the precursor of XIP. The comS-dependent capability for both unimodal and bimodal response suggests that a constituent – most likely peptides – of complex medium interacts with a positive feedback loop in the competence regulatory network.
PMCID: PMC3468698  PMID: 22845615
single-cell; bistability; quorum sensing; gene regulation; feedback; transformation
21.  Two Distinct Functions of ComW in Stabilization and Activation of the Alternative Sigma Factor ComX in Streptococcus pneumoniae 
Journal of Bacteriology  2005;187(9):3052-3061.
Natural genetic transformation in Streptococcus pneumoniae is controlled by a quorum-sensing system, which acts through the competence-stimulating peptide (CSP) for transient activation of genes required for competence. More than 100 genes have been identified as CSP regulated by use of DNA microarray analysis. One of the CSP-induced genes required for genetic competence is comW. As the expression of this gene depended on the regulator ComE, but not on the competence sigma factor ComX (σX), and as expression of several genes required for DNA processing was affected in a comW mutant, comW appears to be a new regulatory gene. Immunoblotting analysis showed that the amount of the σX protein is dependent on ComW, suggesting that ComW may be directly or indirectly involved in the accumulation of σX. As σX is stabilized in clpP mutants, a comW mutation was introduced into the clpP background to ask whether the synthesis of σX depends on ComW. The clpP comW double mutant accumulated an amount of σX higher (threefold) than that seen in the wild type but was not transformable, suggesting that while comW is not needed for σX synthesis, it acts both in stabilization of σX and in its activation. Modification of ComW with a histidine tag at its C or N terminus revealed that both amino and carboxyl termini are important for increasing the stability of σX, but only the N terminus is important for stimulating its activity.
PMCID: PMC1082825  PMID: 15838032
22.  Inhibition of Competence Development, Horizontal Gene Transfer and Virulence in Streptococcus pneumoniae by a Modified Competence Stimulating Peptide 
PLoS Pathogens  2011;7(9):e1002241.
Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.
Author Summary
Streptococcus pneumoniae is a major cause of pneumonia, ear infection and meningitis. Antibiotic resistance among S. pneumoniae isolates is increasingly a major clinical problem. The acquisition of antibiotic resistance genes in S. pneumoniae is controlled by a peptide pheromone called competence-stimulating peptide (CSP). CSP binds to a receptor called ComD, which in turn activates its cognate transcription factor ComE to initiate DNA uptake and integration into the S. pneumoniae genome. CSP-ComD/E also regulates the expression of virulence factors required for infection. In this study, multiple synthetic analogues of CSP pheromone were examined for their ability to inhibit acquisition of exogenous DNA, and to control infection by S. pneumoniae in mice. Two of these analogues, CSP1-E1A and CSP2-E1A, competitively inhibit the ability of S. pneumoniae to acquire the streptomycin resistance rpsL gene and the capsule gene cap3A during mouse models of acute pneumonia and bacteremia. CSP1-E1A also reduces mouse mortality during lung infection by S. pneumoniae. This is the first demonstration of the use of CSP analogues to attenuate virulence and to inhibit acquisition of an antibiotic resistance gene in S. pneumoniae. Because the CSP-ComD/E system is conserved among many pathogenic bacteria, CSP analogues may be applicable to reduce the spread of antibiotic resistance genes and to treat infections.
PMCID: PMC3164649  PMID: 21909280
23.  DNA Binding-Uptake System: a Link between Cell-to-Cell Communication and Biofilm Formation 
Journal of Bacteriology  2005;187(13):4392-4400.
DNA has recently been described as a major structural component of the extracellular matrix in biofilms. In streptococci, the competence-stimulating peptide (CSP) cell-to-cell signal is involved in competence for genetic transformation, biofilm formation, and autolysis. Among the genes regulated in response to the CSP are those involved in binding and uptake of extracellular DNA. We show in this study that a functional DNA binding-uptake system is involved in biofilm formation. A comGB mutant of Streptococcus mutans deficient in DNA binding and uptake, but unaffected in signaling, showed reduced biofilm formation. During growth in the presence of DNase I, biofilm was reduced in the wild type to levels similar to those found with the comGB mutant, suggesting that DNA plays an important role in the wild-type biofilm formation. We also showed that growth in the presence of synthetic CSP promoted significant release of DNA, with similar levels in the wild type and in the comGB mutant. The importance of the DNA binding-uptake system in biofilm formation points to possible novel targets to fight infections.
PMCID: PMC1151753  PMID: 15968048
24.  Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain wicky. 
Journal of Bacteriology  1996;178(19):5831-5835.
Streptococcus gordonii Wicky becomes competent only after stimulation with conditioned medium from strain Challis as a source of competence factor (CF). A 3.2-kbp genomic fragment from Challis was found to impart spontaneous competence on Wicky by a complementation assay. Wicky clones containing the fragment secreted a heat-sensitive activity that induced competence in Wicky and in a comA insertion mutant of Challis. Activity was localized to a putative open reading frame, comX, with the potential to encode a 52-amino-acid peptide. comX had no similarity to known sequences, and a comX::ermAM insertion mutant of Challis transformed normally and secreted CF. These data suggest that a CF-independent pathway for competence induction exists in S. gordonii.
PMCID: PMC178432  PMID: 8824638
25.  An Extracelluar Protease, SepM, Generates Functional Competence-Stimulating Peptide in Streptococcus mutans UA159 
Journal of Bacteriology  2012;194(21):5886-5896.
Cell-cell communication in Gram-positive bacteria often depends on the production of extracellular peptides. The cariogenic bacterium Streptococcus mutans employs so-called competence-stimulating peptide (CSP) to stimulate mutacin (bacteriocin) production and competence development through the activation of the ComDE two-component pathway. In S. mutans, CSP is secreted as a 21-residue peptide; however, mass spectrometric analysis of culture supernatant indicates the presence of an 18-residue proteolytically cleaved species. In this study, using a transposon mutagenesis screening, we identified a cell surface protease that is involved in the processing of 21-residue CSP to generate the 18-residue CSP. We named this protease SepM for streptococcal extracellular protease required for mutacin production. We showed that the truncated 18-residue peptide is the biologically active form and that the specific postexport cleavage is a prerequisite to activate the ComDE two-component signal transduction pathway. We also showed that the CSP and the mutacins are exported outside the cell by the same ABC transporter, NlmTE. Our study further confirmed that the ComDE two-component system is absolutely necessary for mutacin production in S. mutans.
PMCID: PMC3486099  PMID: 22923597

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