A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation

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.     

Identification of the streptococcal competence-pheromone receptor

Competence for genetic transformation in certain species of streptococci has been known for many years to be induced by a secreted protease-sensitive pheromone, referred to as the competence factor or activator, which acts as a quorum-sensing signal to co-ordinate expression of late competence genes. We recently reported identification of the pheromone of Streptococcus pneumoniae strain Rx as a small unmodified peptide, which was termed competence-stimulating peptide (CSP). By identifying the gene ( comC ) encoding the Rx CSP we were able to show that it is synthesized as a precursor peptide containing an N-terminal double-glycine type leader. In the present work, we describe two alleles of the corresponding gene from Streptococcus gordonii strains Challis and NCTC 7865, which are strains with distinct competence pheromones and corresponding specific pheromone reactivities. In addition, the nucleic acid sequences of two genes located downstream of comC were determined; interestingly, these genes encode a two-component signal transduction system. We therefore speculated that their products, a histidine kinase (ComD) and its cognate response regulator (ComE), act downstream of the CSP in competence regulation. By tracing the CSP specificity of the competence response in these strains to strain-specific alleles of comD , we obtained evidence demonstrating that the histidine kinase ComD is the competence-pheromone receptor.     

Genetic variation in comC, the gene encoding competence-stimulating peptide (CSP) in Streptococcus mutans

The genetic variability in comC , the gene encoding the quorum-sensing molecule, competence-stimulating peptide (CSP) in Streptococcus mutans is reported. Seven comC alleles encoding three distinct mature CSPs were identified among 36 geographically diverse strains, although, compared with Streptococcus pneumoniae , the amount of predicted amino acid sequence variation is low. In agreement with other studies, significant variation was found in the natural competence for DNA uptake in these strains. However, there was no correlation between the CSP genotype and the ability to transform these strains. Representative strains encoding each of the CSP variants became competent in response to synthetic CSPs of each type. Therefore, in contrast to S. pneumoniae , comC alleles in S. mutans are functionally equivalent and there is no evidence of pherotype specificity.     

Natural competence in the genus Streptococcus: evidence that streptococci can change pherotype by interspecies recombinational exchanges.

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.     

Natural genetic transformation of Streptococcus mutans growing in biofilms

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.     

Competence for genetic transformation in encapsulated strains of Streptococcus pneumoniae: two allelic variants of the peptide pheromone.

The nucleotide sequence of comC, the gene encoding the 17-residue competence-stimulating peptide (CSP) of Streptococcus pneumoniae (L. S. Havarstein, G. Coomaraswamy, and D. A. Morrison, Proc. Natl. Acad. Sci. USA 92:11140-11144, 1995) was determined with 42 encapsulated strains of different serotypes. A new allele, comC2, was found in 13 strains, including the type 3 Avery strain, A66, while all others carried a gene (now termed comC1) identical to that originally described for strain Rx1. The predicted mature product of comC2 is also a heptadecapeptide but differs from that of comC1 at eight residues. Both CSP-1 and CSP-2 synthetic peptides were used to induce competence in the 42 strains; 48% of the strains became competent after the addition of the synthetic peptide, whereas none were transformable without the added peptides.     

Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae

Regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, genes involved in DNA uptake and recombination and a link between these two gene sets. The alternative sigma factor ComX provides this link. ComE, the response regulator of the quorum-sensing system, is required for expression of ComX and other early genes. However, an unknown ComE-dependent regulator is also required for competence when comX is expressed under control of the raffinose-responsive promoter of the aga operon. The gene comW (SP0018) is required for a high level of competence and is regulated by the quorum-sensing system, but its function is unknown. To explore its role further, comW was cloned into the multicopy plasmid pMSP3535, under the control of a nisin-inducible promoter (P(N)), and transformed into pneumococcal strains containing a raffinose-inducible comX gene (P(R)::comX). Further introduction of a comE deletion blocked the endogenous CSP signal transduction pathway. In the resulting strain, competence was independent of CSP but depended on treatment with both nisin and raffinose, showing that coexpression of comW and comX complemented the comE deficiency. ComX protein accumulation and expression of a late competence gene in the above strain support the conclusion that ComW is a new positive factor involved in competence regulation.     

Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide

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.     

Evolution of Streptococcus pneumoniae and its close commensal relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.     

框内缺失突变法构建变形链球菌comE基因突变株

变形链球菌的种内密度感应系统由com基因家族编码控制。膜受体ComD接受密度感应信号后激活菌体内的反应调节子ComE,ComE作为启动子可以调节一系列相关基因的表达。应用框内缺失突变法（in-frame deletion）,通过两次同源性重组,成功构建了变形链球菌comE基因突变株IFD140ΔcomE。由于框内缺失突变没有引入任何的遗传标记物,所以有效地避免了传统的基因失活方法——插入重复(insertion duplication) 和等位交换(allelic exchange),所导致的下游基因极性效应（polar effect）。经过PCR、测序分析及RT-PCR,证实IFD140ΔcomE仅在comE基因内部缺失了717bp的片段,未引入任何外源性DNA,且comE下游的comD基因可以正常转录,无极性效应产生。对IFD140Δcom表型特征的研究发现,在液体培养基中IFD140Δcom更易发生沉淀性生长和贴壁性生长。菌体呈长链状排列。IFD140ΔcomE的成功构建,为进一步研究变形链球菌种内密度感应系统奠定基础。     

Competence without a competence pheromone in a natural isolate of Streptococcus infantis

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.     

Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in "atypical" pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcal alleles. Conversely, the piaA gene was found to be specific to typical pneumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.     

Identification and characterization of ComE and ComF, two novel pilin-like competence factors involved in natural transformation of Acinetobacter sp. strain BD413.

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.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

Regulation of Streptococcus pneumoniae clp Genes and Their Role in Competence Development and Stress Survival

In vitro mariner transposon mutagenesis of Streptococcus pneumoniae chromosomal DNA was used to isolate regulatory mutants affecting expression of the comCDE operon, encoding the peptide quorum-sensing two-component signal transduction system controlling competence development. A transposon insertion leading to increased comC expression was found to lie directly upstream from the S. pneumoniae clpP gene, encoding the proteolytic subunit of the Clp ATP-dependent protease, whose expression in Bacillus subtilis is controlled by the CtsR repressor. In order to examine clp gene regulation in S. pneumoniae, a detailed analysis of the complete genome sequence was performed, indicating that there are five likely CtsR-binding sites located upstream from the clpE, clpP, and clpL genes and the ctsR-clpC and groESL operons. The S. pneumoniae ctsR gene was cloned under the control of an inducible promoter and used to demonstrate regulation of the S. pneumoniae clpP and clpE genes and the clpC and groESL operons by using B. subtilis as a heterologous host. The CtsR protein of S. pneumoniae was purified and shown to bind specifically to the clpP, clpC, clpE, and groESL regulatory regions. S. pneumoniae Delta ctsR, Delta clpP, Delta clpC, and Delta clpE mutants were constructed by gene deletion/replacement. ClpP was shown to act as a negative regulator, preventing competence gene expression under inappropriate conditions. Phenotypic analyses also indicated that ClpP and ClpE are both required for thermotolerance. Contrary to a previous report, we found that ClpC does not play a major role in competence development, autolysis, pneumolysin production, or growth at high temperature of S. pneumoniae.     

Gene expression analysis of the Streptococcus pneumoniae competence regulons by use of DNA microarrays

Competence for genetic transformation in Streptococcus pneumoniae is coordinated by the competence-stimulating peptide (CSP), which induces a sudden and transient appearance of competence during exponential growth in vitro. Models of this quorum-sensing mechanism have proposed sequential expression of several regulatory genes followed by induction of target genes encoding DNA-processing-pathway proteins. Although many genes required for transformation are known to be expressed only in response to CSP, the relative timing of their expression has not been established. Overlapping expression patterns for the genes cinA and comD (G. Alloing, B. Martin, C. Granadel, and J. P. Claverys, Mol. Microbiol. 29:75-83, 1998) suggest that at least two distinct regulatory mechanisms may underlie the competence cycle. DNA microarrays were used to estimate mRNA levels for all known competence operons during induction of competence by CSP. The known competence regulatory operons, comAB, comCDE, and comX, exhibited a low or zero initial (uninduced) signal, strongly increased expression during the period between 5 and 12 min after CSP addition, and a decrease nearly to original values by 15 min after initiation of exposure to CSP. The remaining competence genes displayed a similar expression pattern, but with an additional delay of approximately 5 min. In a mutant defective in ComX, which may act as an alternate sigma factor to allow expression of the target competence genes, the same regulatory genes were induced, but the other competence genes were not. Finally, examination of the expression of 60 candidate sites not previously associated with competence identified eight additional loci that could be induced by CSP.