Natural competence in the genus Streptococcus: evidence that streptococci can change pherotype by interspecies recombinational exchanges.
ABSTRACT: 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.
Project description: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.
Project description:Natural transformation in the Gram-positive pathogen Streptococcus pneumoniae occurs when cells become "competent," a state that is induced in response to high extracellular concentrations of a secreted peptide signal called competence stimulating peptide (CSP) encoded by the comC locus. Two main CSP signal types (pherotypes) are known to dominate the pherotype diversity across strains. Using 4,089 fully sequenced pneumococcal genomes, we confirm that pneumococcal populations are highly genetically structured and that there is significant variation among diverged populations in pherotype frequencies; most carry only a single pherotype. Moreover, we find that the relative frequencies of the two dominant pherotypes significantly vary within a small range across geographical sites. It has been variously proposed that pherotypes either promote genetic exchange among cells expressing the same pherotype, or conversely that they promote recombination between strains bearing different pherotypes. We attempt to distinguish these hypotheses using a bioinformatics approach by estimating recombination frequencies within and between pherotypes across 4,089 full genomes. Despite underlying population structure, we observe extensive recombination between populations; additionally, we found significantly higher (although marginal) rates of genetic exchange between strains expressing different pherotypes than among isolates carrying the same pherotype. Our results indicate that pherotypes do not restrict, and may even slightly facilitate, recombination between strains; however, these marginal effects suggest the more likely possibility that the cause of CSP polymorphism lies outside of its effects on transformation. Our results suggest that the CSP balanced polymorphism does not causally underlie population differentiation. Therefore, when strains carrying different pherotypes encounter one another during cocolonization, genetic exchange can occur without restriction.
Project description: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.
Project description:The recent identification of Streptococcus pseudopneumoniae (pseudopneumococcus) has complicated classification schemes within members of the "mitis" streptococcal group. Accurate differentiation of this species is necessary for understanding its disease potential and identification in clinical settings. This work described the use of the competence-stimulatory peptide ComC sequence for identification of S. pseudopneumoniae. ComC sequences from clinical sources were determined for 17 strains of S. pseudopneumoniae, Streptococcus pneumoniae, and Streptococcus oralis. An additional 58 ComC sequences from a range of sources were included to understand the diversity and suitability of this protein as a diagnostic marker for species identification. We identified three pherotypes for this species, delineated CSP6.1 (10/14, 79%), CSP6.3 (3/14, 21%), and SK674 (1/14, 7%). Pseudopneumococcal ComC sequences formed a discrete cluster within those of other oral streptococci. This suggests that the comC sequence could be used to identify S. pseudopneumoniae, thus simplifying the study of the pathogenic potential of this organism. To avoid confusion between pneumococcal and pseudopneumococcal pherotypes, we have renamed the competence pherotype CSP6.1, formerly reported as an "atypical" pneumococcus, CSPps1 to reflect its occurrence in S. pseudopneumoniae.
Project description:Competence-stimulating peptide (CSP) and ComD of the streptococcal species are a pheromone and its receptor, respectively, involved in the regulation of competence for natural genetic transformation. We show here that these molecules have undergone positive selection. This study is the first report of positive selection due to competition among bacterial populations.
Project description:Bacillus subtilis strains communicate through the comQXPA quorum sensing (QS) system, which regulates genes expressed during early stationary phase. A high polymorphism of comQXP' loci was found in closely related strains isolated from desert soil samples separated by distances ranging from meters to kilometers. The observed polymorphism comprised four communication groups (pherotypes), such that strains belonging to the same pherotype exchanged information efficiently but strains from different pherotypes failed to communicate. To determine whether the same level of polymorphism in the comQXP' QS system could be detected at microscale, B. subtilis isolates were obtained from two separate 1-cm(3) soil samples, which were progressively divided into smaller sections. Cross-activation studies using pherotype-responsive reporter strains indicated the same number of communication pherotypes at microscale as previously determined at macroscale. Sequencing of the housekeeping gene gyrA and the QS comQ gene confirmed different evolutionary rates of these genes. Furthermore, an asymmetric communication response was detected inside the two pherotype clusters, suggesting continuous evolution of the QS system and possible development of new languages. To our knowledge, this is the first microscale study demonstrating the presence of different QS languages among isolates of one species, and the implications of this microscale diversity for microbial interactions are discussed.
Project description:<h4>Background</h4>The boundaries of bacterial species and the mechanisms underlying bacterial speciation are matters of intense debate. Theoretical studies have shown that recombination acts as a strong cohesive force preventing divergence in bacterial populations. Streptococcus pneumoniae populations have the telltale signs of high recombination with competence implicated as the major driving force behind gene exchange. Competence in S. pneumoniae is triggered by a quorum-sensing mechanism controlled by the competence-stimulating peptide pheromone.<h4>Results</h4>We studied the distribution of the two major pherotypes in the pneumococcal population and their association with serotype, antimicrobial resistance and genetic lineage. Using multilocus sequence data we evaluated pherotype influence on the dynamics of horizontal gene transfer. We show that pherotype is a clonal property of pneumococci. Standard population genetic analysis and multilocus infinite allele model simulations support the hypothesis that two genetically differentiated populations are defined by the major pherotypes.<h4>Conclusion</h4>Severe limitations to gene flow can therefore occur in bacterial species in the absence of geographical barriers and within highly recombinogenic populations. This departure from panmixia can have important consequences for our understanding of the response of pneumococci to human imposed selective pressures such as vaccination and antibiotic use.
Project description:Streptococcus mutans ( S. mutans) is a Gram-positive human pathogen that is one of the major contributors to dental caries, a condition with an economic cost of over $100 billion per year in the United States. S. mutans secretes a 21-amino-acid peptide termed the competence stimulating peptide (21-CSP) to assess its population density in a process termed quorum sensing (QS) and to initiate a variety of phenotypes such as biofilm formation and bacteriocin production. 21-CSP is processed by a membrane bound protease SepM into active 18-CSP, which then binds to the ComD receptor. This study seeks to determine the molecular mechanism that ties 21-CSP:SepM recognition and 18-CSP:ComD receptor binding and to identify QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in both 21-CSP and 18-CSP and assessed the ability of the mutated analogs to modulate QS. We identified residues that are important to SepM recognition and ComD receptor binding. Our results shed light on the S. mutans competence QS pathway at the molecular level. Moreover, our structural insights of the CSP signal can be used to design QS-based anti-infective therapeutics against S. mutans.
Project description:The competence stimulating peptide (CSP) plays a key role in the regulation of pneumococcal quorum sensing (QS), a communication system that is critical to the infectivity of pneumococci. CSP functions through binding and activating a transmembrane receptor, ComD. Molecules that can modulate pneumococcal QS through intercepting CSP:ComD interaction may serve as new generation of antibacterial agents to treat pneumococcal infections. In this work, we systematically modified the N-terminus of CSP1, a region that is essential to ComD activation, to identify detailed structural features of the N-terminus that are responsible for its function. Our results revealed structural features that are optimal to achieve receptor activation and structure-activity trends that improve our understanding of CSP:ComD interaction, all of which will contribute to the design of novel pneumococcal QS modulators with higher potency and improved pharmacological properties.
Project description:Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.