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1.  The response regulator ComE in Streptococcus mutans functions both as a transcription activator of mutacin production and repressor of CSP biosynthesis 
Microbiology (Reading, England)  2007;153(Pt 6):1799-1807.
In Streptococcus pneumoniae, competence and bacteriocin genes are controlled by two two-component systems, ComED and BlpRH, respectively. In Streptococcus mutans, both functions are controlled by the ComED system. Recent studies in S. mutans revealed a potential ComE binding site characterized by two 11 bp direct repeats shared by each of the bacteriocin genes responsive to the competence-stimulating peptide (CSP). Interestingly, this sequence was not found in the upstream region of the CSP structural gene comC. Since comC is suggested to be part of a CSP-responsive and ComE-dependent autoregulatory loop, it was of interest to determine how it was possible that the ComED system could simultaneously regulate bacteriocin expression and natural competence. Using the intergenic region IGS1499, shared by the CSP-responsive bacteriocin nlmC and comC, it was demonstrated that both genes are likely to be regulated by a bifunctional ComE. In a comE null mutant, comC gene expression was increased similarly to a fully induced wild-type. In contrast, nlmC gene expression was nearly abolished. Deletion of ComD exerted a similar effect on both genes to that observed with the comE null mutation. Electrophoretic mobility shift assays (EMSAs) with purified ComE revealed specific shift patterns dependent on the presence of one or both direct repeats in the nlmC–comC promoter region. The two direct repeats were also required for the promoter activity of both nlmC and comC. These results suggest that gene regulation of comC in S. mutans is fundamentally different from that reported for S. pneumoniae, which implicates a unique regulatory mechanism that allows the coordination of bacteriocin production with competence development.
doi:10.1099/mic.0.2007/005975-0
PMCID: PMC2062498  PMID: 17526837
2.  Characterization of DNA Binding Sites of the ComE Response Regulator from Streptococcus mutans▿† 
Journal of Bacteriology  2011;193(14):3642-3652.
In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.
doi:10.1128/JB.00155-11
PMCID: PMC3133340  PMID: 21602345
3.  Structural Basis of Response Regulator Inhibition by a Bacterial Anti-Activator Protein 
PLoS Biology  2011;9(12):e1001226.
Structure-function studies reveal that Rap proteins have distinct, nonoverlapping surfaces that interact with different cellular targets, and that for antiactivator RapF, one surface mimics DNA to bind a response regulator DNA binding domain, thereby sterically preventing the activity of this transcription transactivator.
The complex interplay between the response regulator ComA, the anti-activator RapF, and the signaling peptide PhrF controls competence development in Bacillus subtilis. More specifically, ComA drives the expression of genetic competence genes, while RapF inhibits the interaction of ComA with its target promoters. The signaling peptide PhrF accumulates at high cell density and upregulates genetic competence by antagonizing the interaction of RapF and ComA. How RapF functions mechanistically to inhibit ComA activity and how PhrF in turn antagonizes the RapF-ComA interaction were unknown. Here we present the X-ray crystal structure of RapF in complex with the ComA DNA binding domain. Along with biochemical and genetic studies, the X-ray crystal structure reveals how RapF mechanistically regulates ComA function. Interestingly, we found that a RapF surface mimics DNA to block ComA binding to its target promoters. Furthermore, RapF is a monomer either alone or in complex with PhrF, and it undergoes a conformational change upon binding to PhrF, which likely causes the dissociation of ComA from the RapF-ComA complex. Finally, we compare the structure of RapF complexed with the ComA DNA binding domain and the structure of RapH complexed with Spo0F. This comparison reveals that RapF and RapH have strikingly similar overall structures, and that they have evolved different, non-overlapping surfaces to interact with diverse cellular targets. To our knowledge, the data presented here reveal the first atomic level insight into the inhibition of response regulator DNA binding by an anti-activator. Compounds that affect the interaction of Rap and Rap-like proteins with their target domains could serve to regulate medically and commercially important phenotypes in numerous Bacillus species, such as sporulation in B. anthracis and sporulation and the production of Cry protein endotoxin in B. thuringiensis.
Author Summary
Upon phosphorylation, bacterial proteins called response regulators bind to DNA promoters and activate or repress transcription. These response regulators are themselves regulated by anti-activator proteins, which can control response regulator activity without altering their phosphorylation state. We have determined the X-ray crystal structure of the anti-activator RapF complexed with the DNA-binding domain of the response regulator ComA. Our structure-function studies show that RapF disrupts the binding of ComA to DNA using a two-pronged mechanism. First, a RapF surface mimics DNA, and this DNA-like surface binds to nearly all of the ComA DNA-binding residues, thus blocking ComA's interaction with DNA. Second, RapF inhibits ComA dimerization. RapF is also regulated by the PhrF peptide; we find that the RapF-ComA interaction surface is distant from the proposed PhrF binding site. Furthermore, we found that RapF undergoes a conformational change upon binding to PhrF, which likely causes its dissociation from ComA. From these observations, we conclude that PhrF binding to RapF allosterically triggers its dissociation from ComA. Finally, we compared the RapF/ComA DNA-binding domain complex structure with the structure of another response regulator, Spo0F, complexed with the phosphatase RapH. This reveals that while RapF and RapH are structurally similar, they have evolved distinct, non-overlapping surfaces to interact with their different cellular targets.
doi:10.1371/journal.pbio.1001226
PMCID: PMC3246441  PMID: 22215984
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.
doi:10.1128/JB.184.10.2699-2708.2002
PMCID: PMC135014  PMID: 11976299
5.  Exit from Competence for Genetic Transformation in Streptococcus pneumoniae Is Regulated at Multiple Levels 
PLoS ONE  2013;8(5):e64197.
Development of natural competence in S. pneumoniae entails coordinated expression of two sets of genes. Early gene expression depends on ComE, a response regulator activated by the pheromone CSP (Competence-Stimulating-Peptide). Subsequently, an early gene product (the alternative sigma factor ComX) activates expression of late genes, establishing the competent state. Expression of both sets of genes is transient, rapidly shut off by a mechanism that depends on the late gene, dprA. It has been thought that the rapid shutoff of late gene expression is the combined result of auto-inhibition of ComE and the instability of ComX. However, this explanation seems incomplete, because of evidence for ComX-dependent repressor(s) that might also be important for shutting off the response to CSP and identifying dprA as such a gene. We screened individual late gene mutants to investigate further the roles of ComX-dependent genes in competence termination. A ΔdprA mutant displayed a prolonged late gene expression pattern, whereas mutants lacking cbpD, cibABC, cglEFG, coiA, ssbB, celAB, cclA, cglABCD, cflAB, or radA, exhibited a wild-type temporal expression pattern. Thus, no other gene than dprA was found to be involved in shutoff. DprA limits the amounts of ComX and another early gene product, ComW, by restriction of early gene expression rather than by promoting proteolysis. To ask if DprA also affects late gene expression, we decoupled late gene expression from early gene regulation. Because DprA did not limit ComX activity under these conditions, we also conclude that ComX activity is limited by another mechanism not involving DprA.
doi:10.1371/journal.pone.0064197
PMCID: PMC3661451  PMID: 23717566
6.  Identification of a New Regulator in Streptococcus pneumoniae Linking Quorum Sensing to Competence for Genetic Transformation 
Journal of Bacteriology  1999;181(16):5004-5016.
Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at −10 and −25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.
PMCID: PMC93990  PMID: 10438773
7.  Structural insights into the dimerization of the response regulator ComE from Streptococcus pneumoniae 
Nucleic Acids Research  2014;42(8):5302-5313.
Natural transformation contributes to the maintenance and to the evolution of the bacterial genomes. In Streptococcus pneumoniae, this function is reached by achieving the competence state, which is under the control of the ComD−ComE two-component system. We present the crystal and solution structures of ComE. We mimicked the active and non-active states by using the phosphorylated mimetic ComED58E and the unphosphorylatable ComED58A mutants. In the crystal, full-length ComED58A dimerizes through its canonical REC receiver domain but with an atypical mode, which is also adopted by the isolated RECD58A and RECD58E. The LytTR domain adopts a tandem arrangement consistent with the two direct repeats of its promoters. However ComED58A is monomeric in solution, as seen by SAXS, by contrast to ComED58E that dimerizes. For both, a relative mobility between the two domains is assumed. Based on these results we propose two possible ways for activation of ComE by phosphorylation.
doi:10.1093/nar/gku110
PMCID: PMC4005675  PMID: 24500202
8.  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.
doi:10.1128/JB.183.3.897-908.2001
PMCID: PMC94956  PMID: 11208787
9.  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
10.  Multilevel Control of Competence Development and Stress Tolerance in Streptococcus mutans UA159  
Infection and Immunity  2006;74(3):1631-1642.
Genetic competence appears to be important in establishment of biofilms and tolerance of environmental insults. We report here that the development of competence is controlled at multiple levels in a complex network that includes two signal-transducing two-component systems (TCS). Using Streptococcus mutans strain UA159, we demonstrate that the histidine kinase CiaH, but not the response regulator CiaR, causes a dramatic decrease in biofilm formation and in transformation efficiency. Inactivation of comE or comD had no effect on stress tolerance, but transformability of the mutants was poor and was not restored by addition of competence-stimulating peptide (CSP). Horse serum (HS) or bovine serum albumin (BSA) had no impact on transformability of any strains. Interestingly, though, the presence of HS or BSA in combination with CSP was required for efficient induction of comD, comX, and comYA, and induction was dependent on ComDE and CiaH, but not CiaR. Inactivation of comC, encoding CSP, had no impact on transformation, and CiaH was shown to be required for optimal comC expression. This study reveals that S. mutans integrates multiple environmental signals through CiaHR and ComDE to coordinate induction of com genes and that CiaH can exert its influence through CiaR and as-yet-unidentified regulators. The results highlight critical differences in the role and regulation of CiaRH and com genes in different S. mutans isolates and between S. mutans and Streptococcus pneumoniae, indicating that substantial divergence in the role and regulation of TCS and competence genes has occurred in streptococci.
doi:10.1128/IAI.74.3.1631-1642.2006
PMCID: PMC1418624  PMID: 16495534
11.  ComE, a Competence Protein from Neisseria gonorrhoeae with DNA-Binding Activity 
Journal of Bacteriology  2001;183(10):3160-3168.
Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 × 104-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.
doi:10.1128/JB.183.10.3160-3168.2001
PMCID: PMC95217  PMID: 11325945
12.  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.
doi:10.1128/JB.187.13.4392-4400.2005
PMCID: PMC1151753  PMID: 15968048
13.  Regulation of the Competence Pathway as a Novel Role Associated with a Streptococcal Bacteriocin▿† 
Journal of Bacteriology  2011;193(23):6552-6559.
The oral biofilm organism Streptococcus mutans must face numerous environmental stresses to survive in its natural habitat. Under specific stresses, S. mutans expresses the competence-stimulating peptide (CSP) pheromone known to induce autolysis and facilitate the uptake and incorporation of exogenous DNA, a process called DNA transformation. We have previously demonstrated that the CSP-induced CipB bacteriocin (mutacin V) is a major factor involved in both cellular processes. Our objective in this work was to characterize the role of CipB bacteriocin during DNA transformation. Although other bacteriocin mutants were impaired in their ability to acquire DNA under CSP-induced conditions, the ΔcipB mutant was the only mutant showing a sharp decrease in transformation efficiency. The autolysis function of CipB bacteriocin does not participate in the DNA transformation process, as factors released via lysis of a subpopulation of cells did not contribute to the development of genetic competence in the surviving population. Moreover, CipB does not seem to participate in membrane depolarization to assist passage of DNA. Microarray-based expression profiling showed that under CSP-induced conditions, CipB regulated ∼130 genes, among which are the comDE locus and comR and comX genes, encoding critical factors that influence competency development in S. mutans. We also discovered that the CipI protein conferring immunity to CipB-induced autolysis also prevented the transcriptional regulatory activity of CipB. Our data suggest that besides its role in cell lysis, the S. mutans CipB bacteriocin also functions as a peptide regulator for the transcriptional control of the competence regulon.
doi:10.1128/JB.05968-11
PMCID: PMC3232909  PMID: 21984782
14.  Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans 
Journal of Bacteriology  2012;194(6):1307-1316.
The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N5-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.
doi:10.1128/JB.06071-11
PMCID: PMC3294852  PMID: 22228735
15.  Identification and Characterization of ComE and ComF, Two Novel Pilin-Like Competence Factors Involved in Natural Transformation of Acinetobacter sp. Strain BD413 
Applied and Environmental Microbiology  1999;65(10):4568-4574.
Although the high level of competence for natural transformation of Acinetobacter sp. strain BD413 has been the subject of numerous studies, only two competence genes, comC and comP, have been identified to date. By chromosomal walking analysis we found two overlapping open reading frames, designated comE and comF, starting 61 bp downstream of comC. comE and comF are expressed as stable proteins in Escherichia coli, thus proving that they are indeed coding regions, but expression was successful only with 5′-deleted genes. ComE and ComF are similar to pilins and pilin-like components. Both genes were mutated, and the phenotypes of the mutants were analyzed. Natural transformation in comF mutants is 1,000-fold reduced, whereas comE mutants exhibit 10-fold-reduced transformation frequencies. This is clear evidence that comE and comF are involved in natural transformation. However, ComE and ComF are specific for DNA translocation, since comE and comF defects affected neither piliation nor lipase secretion. These results suggest that the type IV pili, the general protein secretion pathway, and the DNA translocation machinery in Acinetobacter sp. strain BD413 are evolutionary related but functionally distinct systems.
PMCID: PMC91608  PMID: 10508090
16.  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.
doi:10.1371/journal.ppat.1002241
PMCID: PMC3164649  PMID: 21909280
17.  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.
doi:10.1128/JB.01363-10
PMCID: PMC3133041  PMID: 21317319
18.  The secretion ATPase ComGA is required for the binding and transport of transforming DNA 
Molecular microbiology  2011;81(3):818-830.
Summary
Transformation requires specialized proteins to facilitate the binding and uptake of DNA. The genes of the B. subtilis comG operon (comGA–G) are required for transformation and to assemble a structure, the pseudopilus, in the cell envelope. No role for the pseudopilus has been established and the functions of the individual comG genes are unknown. We show that among the comG genes, only comGA is absolutely required for DNA binding to the cell surface. ComEA, an integral membrane DNA binding protein plays a minor role in the initial binding step, while an unidentified protein which communicates with ComGA must be directly responsible for binding to the cell. We show that the use of resistance to DNAse to measure “DNA uptake” reflects the movement of transforming DNA to a protected state in which it is not irreversibly associated with the protoplast, and presumably resides outside the cell membrane, in the periplasm or associated with the cell wall. We suggest that ComGA is needed for the acquisition of DNAse-resistance as well as for the binding of DNA to the cell surface. Finally, we show that the pseudopilus is required for DNA uptake and we offer a revised model for the transformation process.
doi:10.1111/j.1365-2958.2011.07730.x
PMCID: PMC3781931  PMID: 21707789
Transformation; Bacillus subtilis; secretion ATPase; DNA uptake; ComGA
19.  Characterization of comQ and comX, Two Genes Required for Production of ComX Pheromone in Bacillus subtilis 
Journal of Bacteriology  2002;184(2):410-419.
Many microbes use secreted peptide-signaling molecules to stimulate changes in gene expression in response to high population density, a process called quorum sensing. ComX pheromone is a modified 10-amino-acid peptide used by Bacillus subtilis to modulate changes in gene expression in response to crowding. comQ and comX are required for production of ComX pheromone. We found that accumulation of ComX pheromone in culture supernatant paralleled cell growth, indicating that there was no autoinduction of production of ComX pheromone. We overexpressed comQ and comX separately and together and found that overexpression of comX alone was sufficient to cause an increase in production of ComX pheromone and early induction of a quorum-responsive promoter. These results indicate that the extracellular concentration of ComX pheromone plays a major role in determining the timing of the quorum response and that expression of comX is limiting for production of ComX pheromone. We made alanine substitutions in the residues that comprise the peptide backbone of ComX pheromone. Analysis of these mutants highlighted the importance of the modification for ComX pheromone function and identified three residues (T50, G54, and D55) that are unlikely to interact with proteins involved in production of or response to ComX pheromone. We have also identified and mutated a putative isoprenoid binding domain of ComQ. Mutations in this domain eliminated production of ComX pheromone, consistent with the hypothesis that ComQ is involved in modifying ComX pheromone and that the modification is likely to be an isoprenoid.
doi:10.1128/JB.184.2.410-419.2002
PMCID: PMC139578  PMID: 11751817
20.  Characterization of irvR, a Novel Regulator of the irvA-Dependent Pathway Required for Genetic Competence and Dextran-Dependent Aggregation in Streptococcus mutans▿  
Journal of Bacteriology  2008;190(21):7268-7274.
Previous studies identified irvA as a normally repressed but highly inducible transcription regulator capable of repressing mutacin I gene expression in Streptococcus mutans. In this study, we aimed to identify and characterize the regulator(s) responsible for repressing the expression of irvA. An uncharacterized open reading frame (SMU.1398) located immediately adjacent to irvA and annotated as a putative transcription repressor was identified as a likely candidate. The results of mutation studies confirmed that the expression of irvA was greatly increased in the SMU.1398 background. Mutation of SMU.1398 (“irvR”) abolished genetic competence and reduced the expression of the late competence genes/operons comEA, comY, and dprA without affecting the expression of the known competence regulators comC, comED, or comX. In addition, irvR was found to be a potent negative regulator of dextran-dependent aggregation (DDAG) and gbpC expression. Each of these irvR mutant phenotypes could be rescued with a double mutation of irvA or complemented by introducing a wild-type copy of irvR on a shuttle vector. These data indicate that the repression of irvA is critically dependent upon irvR and that irvA repression is essential for the development of genetic competence and the proper control of DDAG in S. mutans.
doi:10.1128/JB.00967-08
PMCID: PMC2580701  PMID: 18757533
21.  Activation of the SMU.1882 Transcription by CovR in Streptococcus mutans 
PLoS ONE  2010;5(11):e15528.
In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor.
doi:10.1371/journal.pone.0015528
PMCID: PMC2989922  PMID: 21124877
22.  ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. 
Journal of Bacteriology  1993;175(10):3182-3187.
ComA is a response regulator protein of Bacillus subtilis which is required for the transcription of several genes which are involved in late-growth expression and in responses to environmental stress. Among these genes are degQ, gsiA, and srfA. The last is an operon needed for the development of genetic competence, surfactin production, and normal sporulation. We show here that partially purified ComA protein, isolated from an overproducing Escherichia coli strain, is phosphorylated in vitro by incubation with acetyl phosphate and that ComA could bind specifically to a DNA fragment containing the promoter of srfA and associated sequences. The binding affinity is enhanced when ComA is phosphorylated. DNase I protection analysis identified two protected sites located upstream from the srfA promoter. The presence of DNase I-hypersensitive bonds induced by ComA binding which are located between the protected sequences is consistent with a model for ComA action involving the bending of DNA.
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PMCID: PMC204641  PMID: 8387999
23.  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.
doi:10.1128/IAI.01696-07
PMCID: PMC2446691  PMID: 18443092
24.  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.
doi:10.1128/JB.187.9.3052-3061.2005
PMCID: PMC1082825  PMID: 15838032
25.  Competence without a Competence Pheromone in a Natural Isolate of Streptococcus infantis 
Journal of Bacteriology  2002;184(13):3426-3432.
Many streptococcal species belonging to the mitis and anginosus phylogenetic groups are known to be naturally competent for genetic transformation. Induction of the competent state in these bacteria is regulated by a quorum-sensing mechanism consisting of a secreted peptide pheromone encoded by comC and a two-component regulatory system encoded by comDE. Here we report that a natural isolate of a mitis group streptococcus (Atu-4) is competent for genetic transformation even though it has lost the gene encoding the competence pheromone. In contrast to other strains, induction of competence in Atu-4 is not regulated by cell density, since highly diluted cultures of this strain are still competent. Interestingly, competence in the Atu-4 strain is lost if the gene encoding the response regulator ComE is disrupted, demonstrating that this component of the quorum-sensing apparatus is still needed for competence development. These results indicate that mutations in ComD or ComE have resulted in a gain-of-function phenotype that allows competence without a competence pheromone. A highly similar strain lacking comC was isolated independently from another individual, suggesting that strains with this phenotype are able to survive in nature in competition with wild-type strains.
doi:10.1128/JB.184.13.3426-3432.2002
PMCID: PMC135153  PMID: 12057935

Results 1-25 (1133299)