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Competence‐induced fratricide in streptococci
Abstract
Competence for natural genetic transformation in Streptococcus pneumoniae is controlled by the extracellular concentration of the competence-stimulating peptide (CSP), an exported peptide pheromone. Upon entering the competent state, pneumococci start transcribing a number of CSP-responsive genes, termed the early and late competence (com) genes. Some of the proteins encoded by these com genes are absolutely required for DNA uptake and transformation, but most of them are dispensable. This finding indicates that the majority of CSP-regulated proteins in S. pneumoniae is involved in processes unrelated to natural genetic transformation. Recently, however, it became clear that the biological role of a few of the dispensable proteins might be linked to the transformation process. Although these proteins are not needed for transformation per se, they constitute a killing mechanism that could be used by competent cells to acquire DNA from non-competent pneumococci. This mechanism, termed fratricide, has so far only been described for pneumococci. In this manuscript, we review evidence that suggests the conservation of fratricide as well as the independent evolution of its genetic control and of its effectors in several species of the genus Streptococcus, and discuss its possible biological significance in relation to natural transformation.
... The BrpR/S system in S. aureus shares extensive homology to the ComC/D/E system in S. pneumoniae [28][29][30][31][32] as well as the ComC/D/E 2-component system and BrsR/M LRS in S. mutans [15][16][17][18]22,23]. Overall, the BrpR/S system shares the highest homology with the BrsR/M system in S. mutans [22]. ...
... Both S. mutans proteins comprise a LRS responsible for sensing a competence-stimulating peptide (CSP), regulating the production of a bacteriocin and inducing late-stage competence [22]. ComC/D/E is also tied to the generation of cell death in S. mutans through the secretion of a lethal factor protein [29,32]. ...
... The (E)-3-(2-(benzo[b]thiophen-2yl)vinyl)-(5)-methoxyphenol SK-03-92 lead drug is an analog of a stilbene structure that could have developed by convergent evolution to protect the sweet fern plant from bacterial colonization to force colonizing Gram-positive bacteria to enter late-stage competence through the production of CSP-like pheromones [33,34]. Part of this event would be induced cell lysis of the bacteria or competence-induced fratricide [29,32,34]. ...
Background: Staphylococcus aureus is a leading cause of skin and bloodstream infections in humans. Antibiotic resistant strains of S. aureus continue to be a problem in treating patients that are infected, so treatment options are needed. A drug discovery project identified SK-03-92 as a novel anti-staphylococcal drug, but the SK-03-92 mechanism of action is unknown. We hypothesized that a lethal factor was being released by the bacteria that killed siblings. Methods: In this study, filtration through molecular weight cut-off filters as well as boiling, trypsin treatment, and proteinase K treatment were used to ascertain what the lethal factor was released by SK-03-92 treated S. aureus cells. Results: Filtration through molecular weight cut-off filters demonstrated the lethal factor released by SK-03-92 treated S. aureus cells had a molecular cut-off between 10,000 Da and 30,000 Da that killed fresh S. aureus cells but was not released by untreated cells. Through proteinase K digestion, trypsin digestion, and boiling experiments, the lethal factor was shown to be a protein. Further experiments are needed to identify what proteins released following SK-03-92 treatment cause the death of S. aureus cells. Conclusions: The data show that SK-03-92 treatment causes S. aureus to release a lethal factor protein that kills S. aureus cells, suggesting a new mechanism of action for an antibacterial drug.
... The introduction of this special term was prompted by a necessity to differentiate this phenomenon from other types of cell lysis, such as heterolysis (or predation), which is directed against other unrelated species, or autolysis (which describes the self-destruction of cells). In the case of bacteria, the terms "fratricide" and "cannibalism" are often respectively associated with Streptococcus pneumoniae and other members of the Streptococcus genus and when speaking of "kin killing" in Bacillus subtilis populations [4,[11][12][13]. The term "siblicide" is also used occasionally [14,15], as well as "sobrinicide", to specify the type of killing observed between closely related species from the same phylotype [12]. ...
... In the case of bacteria, the terms "fratricide" and "cannibalism" are often respectively associated with Streptococcus pneumoniae and other members of the Streptococcus genus and when speaking of "kin killing" in Bacillus subtilis populations [4,[11][12][13]. The term "siblicide" is also used occasionally [14,15], as well as "sobrinicide", to specify the type of killing observed between closely related species from the same phylotype [12]. ...
... In bacterial allolysis, the most characterized and studied model of cell death is the streptococcal fratricide phenomenon. As there are earlier excellent reviews on this topic which provide the basic principles of fratricide [4,6,12], we will try to bring into focus new findings obtained in recent years and extend the context of such findings. Generally, fratricide in S. pneumoniae is associated with a cascade of events linked to entering a state of competence closely linked to the genetic transformation process. ...
In the context of a post-antibiotic era, the phenomenon of microbial allolysis, which is defined as the partial killing of bacterial population induced by other cells of the same species, may take on greater significance. This phenomenon was revealed in some bacterial species such as Streptococcus pneumoniae and Bacillus subtilis, and has been suspected to occur in some other species or genera, such as enterococci. The mechanisms of this phenomenon, as well as its role in the life of microbial populations still form part of ongoing research. Herein, we describe recent developments in allolysis in the context of its practical benefits as a form of cell death that may give rise to developing new strategies for manipulating the life and death of bacterial communities. We highlight how such findings may be viewed with importance and potential within the fields of medicine, biotechnology, and pharmacology.
... Alternatively, they could be degraded and, therefore, undetectable beyond mid-exponential phase. Interestingly, in S. thermophilus, this growth phase is associated with production of competence stimulating peptides along with possible fratricidal molecules, which kill non-competent sibling cells 22,[31][32][33][34] . These features provided some clues toward biocyclostreptin's biological activity, which we explored next. ...
... By contrast, S. thermophilus growth was insensitive to even high concentrations of the linear 8mer peptide. S. agalactiae was not affected by bicyclostreptin A. Given that bicyclostreptin A targets self or near-kin strains, it could be involved in an allolytic process, as has previously been described for extracellular protein toxins in Streptococcus pneumoniae [32][33][34][35] . Alternatively, bicyclostreptin A could represent a growth-curbing signal, used by streptococci to regulate growth as population density increases. ...
Microbial natural products comprise diverse architectures that are generated by equally diverse biosynthetic strategies. In peptide natural products, amino acid sidechains are frequently used as sites of modification to generate macrocyclic motifs. Backbone amide groups, among the most stable of biological moieties, are rarely used for this purpose. Here we report the discovery and biosynthesis of bicyclostreptins—peptide natural products from Streptococcus spp. with an unprecedented structural motif consisting of a macrocyclic β-ether and a heterocyclic sp3–sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon. Both reactions are installed, in that order, by two radical S-adenosylmethionine (RaS) metalloenzymes. Bicyclostreptins are produced at nM concentrations and are potent growth regulation agents in Streptococcus thermophilus. Our results add a distinct and unusual chemotype to the growing family of ribosomal peptide natural products, expand the already impressive catalytic scope of RaS enzymes, and provide avenues for further biological studies in human-associated streptococci. Bicyclostreptins are peptide natural products in which a macrocyclic β-ether and a heterocyclic sp3–sp3 linkage between a backbone amide nitrogen and an adjacent α-carbon are installed by two radical S-adenosylmethionine metalloenzymes.
... 12: 220143 has been a potent promoter of genetic variability and subsequent evolution. Competence is accomplished via horizontal gene transfer (HGT) in both Gram-positive and Gramnegative bacteria and has been well conserved in streptococci [18]. Competence for genetic transformation is an induced, temporary state [19]. ...
... Here, competent cells can trigger the lysis of non-competent cells, leading to the release of virulence factors, like the cytolytic toxin pneumolysin (Ply) from non-competent cells [18,54,55]. A slew of other factors are also involved in the killing of the non-competent cells, such as LytA, LytC and CbpD, along with a two-peptide bacteriocin (CibAB) [54]. ...
Understanding bacterial communication mechanisms is imperative to improve our current understanding of bacterial infectivity and find alternatives to current modes of antibacterial therapeutics. Both Gram-positive and Gram-negative bacteria use quorum sensing (QS) to regulate group behaviours and associated phenotypes in a cell-density-dependent manner. Group behaviours, phenotypic expression and resultant infection and disease can largely be attributed to efficient bacterial communication. Of particular interest are the communication mechanisms of Gram-positive bacteria known as streptococci. This group has demonstrated marked resistance to traditional antibiotic treatment, resulting in increased morbidity and mortality of infected hosts and an ever-increasing burden on the healthcare system. Modulating circuits and mechanisms involved in streptococcal communication has proven to be a promising anti-virulence therapeutic approach that allows managing bacterial phenotypic response but does not affect bacterial viability. Targeting the chemical signals bacteria use for communication is a promising starting point, as manipulation of these signals can dramatically affect resultant bacterial phenotypes, minimizing associated morbidity and mortality. This review will focus on the use of modified peptide signals in modulating the development of proliferative phenotypes in different streptococcal species, specifically regarding how such modification can attenuate bacterial infectivity and aid in developing future alternative therapeutic agents.
... The blp locus encodes a family of bacteriocins (pneumocins), which have been shown to promote competitive interactions between different strains of Spn (6). Additionally, a separate quorum-sensing system controlling the competence regulon causes lineage-independent killing of Spn (referred to as "fratricide") that have not achieved the necessary population size to turn on immunity factors also regulated by this system (7). The competitive fitness of clinical isolates and isogenic "capsule switch" strains, which differ only in the serotype-determining cps locus, have been demonstrated in mouse models of Spn colonization. ...
... Effects of Capsule Type on Pneumococcal Colonization and Transmission mBio mechanism. The strain that first reaches a threshold density senses its population size by the concentration of the competence-stimulating pheromone CSP, which then signals the upregulated expression of immunity factors ComM and CibC, among other competence-regulated genes (7). These immunity factors enable survival when the effectors of fratricide, the lysin CbpD and bacteriocins CibAB, are expressed in a later phase of the competence program. ...
Capsule is the major virulence factor and surface antigen of the opportunistic respiratory pathogen Streptococcus pneumoniae (Spn). Strains of Spn express at least 100 structurally and immunologically distinct types (serotypes) of capsule, but for unknown reasons only a few are common.
... Gram-positive bacteria use peptides as QS signal molecules, which generally have a narrow spectrum of activity. Among those peptides, the competence stimulating peptide (CSP) is an essential signal in a variety of Streptococcus species (S. mutans, S. gordonii, and S. intermedius), whose function is to control the levels of proteins involved in biofilm formation, competence development, bacteriocin synthesis, fraticide, and autolysis [51][52][53][54][55]. Autoinducer-2 (AI-2) is synthesized by the luxS gene in several genera of oral Gram-positive and Gram-negative bacteria and is considered a "universal language" for inter-species communication. ...
... Within the NITVK domain, the N1182 and V1185 residues are essential for recognition by Mfa1 [50]; however, it is the physical properties of the amino acids occupying the 1182 and 1185 positions which dictate activity. Substitution of basic amino acids for N1182 and substitution of hydrophobic residues for V1185 enhance binding to Mfa1, indicating that both electrostatic and hydrophobic interactions contribute to the BAR-Mfa1 interaction [51]. The VQDLL motif of BAR constitutes an α-helix that resembles the eukaryotic nuclear receptor box domain which is involved in protein-protein interactions through a hydrophobic or amphipathic α-helical motif [52]. ...
It is now well established that the oral microbiome plays a critical role in the health status of not only the oral cavity but other body sites away from the mouth. A transition from a commensal to a pathogenic oral microbiome causes an imbalance of oral homeostasis, a phenomenon called dysbiosis, which is a crucial concept to understand most oral diseases. In the present chapter, we briefly summarize the current knowledge on the function that oral microbes play in health and disease. We have divided the chapter into three major sections. In the first section, we cover the oral microbiome in health, where we describe the ecological forces that shape the microbial community as well as the composition and function of the commensal oral microbiome. In the second section, we focus on the transition from a commensal to a dysbiotic microbiome in the major oral diseases: caries and periodontal diseases. Finally, in the third section, we describe the role of members of the oral microbiome in systemic diseases, including cardiovascular disease, cancer, and adverse pregnancy outcomes.
... Comparison between lines a. and b. shows the higher expression found in samples treated with the pheromone. Stars followed by gene annotation represent non-annotated sequences in S. mitis genomes of killing factors [41,42]. Indeed, we identified a strongly upregulated bacteriocin locus in a single transcriptional unit located upstream of the comAB operon, with a conserved link to the late competence response by carrying two (in the type strain) or even three (in SK321) copies of the SigX box in their promoter regions (Fig. 5b). ...
... Bases divergent from the consensus are represented by lower case letters. Only the first genes within each induced transcriptionally active region are shown blpU preceded by a Blp-box, while in S. pneumoniae D39 there is also a transposase gene transcribed in the opposite direction [41]. This suggests that a shuffling between Blp and competence-induced bacteriocins might have occurred during the parallel evolution of the two species. ...
Background:
In streptococci of the mitis group, competence for natural transformation is a transient physiological state triggered by competence stimulating peptides (CSPs). Although low transformation yields and the absence of a widespread functional competence system have been reported for Streptococcus mitis, recent studies revealed that, at least for some strains, high efficiencies can be achieved following optimization protocols. To gain a deeper insight into competence in this species, we used RNA-seq, to map the global CSP response of two transformable strains: the type strain NCTC12261T and SK321.
Results:
All known genes induced by ComE in Streptococcus pneumoniae, including sigX, were upregulated in the two strains. Likewise, all sets of streptococcal SigX core genes involved in extracellular DNA uptake, recombination, and fratricide were upregulated. No significant differences in the set of induced genes were observed when the type strain was grown in rich or semi-defined media. Five upregulated operons unique to S. mitis with a SigX-box in the promoter region were identified, including two specific to SK321, and one specific to NCTC12261T. Two of the strain-specific operons coded for different bacteriocins. Deletion of the unique S. mitis sigX regulated genes had no effect on transformation.
Conclusions:
Overall, comparison of the global transcriptome in response to CSP shows the conservation of the ComE and SigX-core regulons in competent S. mitis isolates, as well as species and strain-specific genes. Although some S. mitis exhibit truncations in key competence genes, this study shows that in transformable strains, competence seems to depend on the same core genes previously identified in S. pneumoniae.
... Even more interesting is that this pathway has been implicated in allolysis in Streptococcus pneumoniae, where lysis is induced by the non-competent bacterial population by a group of competent and epigenetically different cells of the same species. This is thought to provide the DNA required for natural transformation (Claverys et al., 2007). ...
... PCD can also be used as a way to gain an evolutionary edge. The human pathogen Streptococcus pneumoniae has been shown to practice heterolysis/allolysis (lysis of a bacterial cell by another cell), instead of autolysis (lysis caused by the cell itself) (Claverys et al., 2007). During competence development, the population of competent cells kill the population of non-competent S. pneumonaie cells, releasing chromosomal DNA that may be used for natural transformation (Steinmoen et al., 2003). ...
For a long a time programmed cell death was thought to be a unique characteristic of higher eukaryotes, but evidence has accumulated showing that programmed cell death is a universal phenomenon in all life forms. Many different types of bacterial programmed cell death systems have been identified, rivalling the eukaryotic systems in diversity. Bacteria are singular, seemingly independently living organisms, however they are part of complex communities. Being part of a community seems indispensable for survival in different environments. This review is focussed on the mechanism of and reasons for bacterial programmed cell death in the context of bacterial communities.
... Griffith was the first scientist who discovered natural genetic transformation in S. pneumoniae in the year 1928, and later on, Avery and coworkers discovered that DNA transfers in transformation (Avery, MacLeod, & McCarty, 1944;Griffith, 1928). The transformation process transfer result in horizontal gene transfers is critical for evolution that facilities emergence of unique and novel traits resulting in antibiotic resistance (Claverys, Martin, & Håvarstein, 2007;Hakenbeck et al., 2001;Majewski, Zawadzki, Pickerill, Cohan, & Dowson, 2000). The genetic transformation and acquisition of novel genes facilitate bacteria to survive in stress and adverse environmental condition ( Johnsborg, Eldholm, & Håvarstein, 2007;Stewart & Carlson, 1986). ...
... The blp locus encodes a family of bacteriocins (pneumocins), which have been shown to promote competitive interactions between different strains [77]. In addition, a separate quorum-sensing system causes lineage-independent killing of pneumococci (known as fratricide) that have not achieved the required population size for immune activation also regulated by this system [78]. ...
Modern molecular technologies have revolutionized our understanding of bacterial epidemiology, but reported data across different settings remain under-integrated in common theoretical frameworks. Pneumococcus serotype co-colonization, caused by the polymorphic bacteria Streptococcus pneumoniae, has been increasingly investigated in recent years. While the global genomic diversity and serotype distribution of S. pneumoniae are well-characterized, there is limited information on how co-colonization patterns vary globally, critical for understanding bacterial evolution and dynamics. Gathering a rich dataset of cross-sectional pneumococcal colonization studies in the literature, we quantified patterns of transmission intensity and co-colonization prevalence in children populations across 17 geographic locations. Fitting these data to an SIS model with co-colonization under the assumption of similarity among interacting strains, our analysis reveals strong patterns of negative co-variation between transmission intensity ($R_0$) and susceptibility to co-colonization ($k$). In support of the stress-gradient hypothesis in ecology (SGH), pneumococcus serotypes appear to compete more in high-transmission settings and less in low-transmission settings, a trade-off which ultimately leads to a conserved ratio of single to co-colonization $\mu=1/(R_0-1)k$. Within our mathematical model, such conservation suggests preservation of 'stability-diversity-complexity' regimes in multi-strain coexistence. We find no major study differences in serotype composition, pointing to underlying adaptation of the same set of serotypes across environments. Our work highlights that understanding pneumococcus transmission patterns from global epidemiological data can benefit from simple analytical approaches that account for quasi-neutrality among strains, co-colonization, as well as variable environmental adaptation.
... In Streptococcus pneumoniae, fratricidal agents are produced in this growth phase, a process by which competent cells kill noncompetent sibling cells possibly as a means of increasing genetic diversity. 99,100 The timing of production of streptosactin, its potent self-killing activity, and other phenotypes observed have led to the proposal that streptosactin may act as a fratricidal agent in S. thermophilus. ...
Radical S-adenosylmethionine (RaS) enzymes have quickly advanced to one of the most abundant and versatile enzyme superfamilies known. Their chemistry is predicated upon reductive homolytic cleavage of a carbon-sulfur bond in cofactor S-adenosylmethionine forming an oxidizing carbon-based radical, which can initiate myriad radical transformations. An emerging role for RaS enzymes is their involvement in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a natural product family that has become known as RaS-RiPPs. These metabolites are especially prevalent in human and mammalian microbiomes because the complex chemistry of RaS enzymes gives rise to correspondingly complex natural products with minimal cellular energy and genomic fingerprint, a feature that is advantageous in microbes with small, host-adapted genomes in competitive environments. Herein, we review the discovery and characterization of RaS-RiPPs from the human microbiome with a focus on streptococcal bacteria. We discuss the varied chemical modifications that RaS enzymes introduce onto their peptide substrates and the diverse natural products that they give rise to. The majority of RaS-RiPPs remain to be discovered, providing an intriguing avenue for future investigations at the intersection of metalloenzymology, chemical ecology, and the human microbiome.
... Of all the topics discussed here, the mechanisms that eubacteria use to release these critical biofilm components is likely to vary the most amongst genera and species, and release of DNA will be no different. For example, in streptococci competent for transformation, there is a quorum sensing induced phenomenon termed fratricide, where bacteria induce autolysis, thus releasing DNA and the rest of the bacterial contents as common goods for the surviving bacteria [31]. To date, the best characterized release mechanism was determined for NTHI. ...
Bacterial biofilms contribute significantly to the antibiotic resistance, pathogenesis, chronicity and recurrence of bacterial infections. Critical to the stability and survival of extant biofilms is the extracellular DNA (eDNA)-dependent matrix which shields the resident bacteria from hostile environments, allows a sessile metabolic state, but also encourages productive interactions with biofilm-inclusive bacteria. Given the importance of the eDNA, approaches to this area of research have been to target not just the eDNA, but also the additional constituent structural components which appear to be widespread. Chief among these is a ubiquitous two-member family of bacterial nucleoid associated proteins (the DNABII proteins) responsible for providing structural integrity to the eDNA and thereby the biofilm. Moreover, this resultant novel eDNA-rich secondary structure can also be targeted for disruption. Here, we provide an overview of both what is known about the eDNA-dependent matrix, as well as the resultant means that have resulted in biofilm resolution. Results obtained to date have been highly supportive of continued development of DNABII-targeted approaches, which is encouraging given the great global need for improved methods to medically manage, or ideally prevent biofilm-dependent infections, which remains a highly prevalent burden worldwide.
... Sporulation is an energy-consuming and irreversible process which involves a commitment before its commencement among sub-populations of organisms in an environment. The cells either initiate the process of sporulation or resort to other alternatives such as cannibalism of isogenic sub-populations that have lost the competitive edge or form biofilms to avoid committing to the process (Claverys et al. 2007;González-Pastor, 2011). As nutrients from the cannibalised cells further deplete, the surviving sub-population then initiate sporulation. ...
Thermophilic Bacillus and their spores are important in dairy processing due to their ubiquity, resistance to high temperature, chemical inactivation and their biofilm forming potential. The presence of such microbial contaminants results in shelf-life reduction of processed foods serving as a potential hotspot of outbreaks and spoilage of post-processed milk. Therefore, this study seeks to characterise Bacillus subtilis and Bacillus velezensis from raw, pasteurised and packaged extended shelf-life (ESL) milk samples, determine their biofilm forming and spoilage potential as well as the effect of ultraviolet C (UVC) in the inactivation of their spores with the intent of mitigating their deleterious presence in the dairy processing plant.
The isolates were identified B. subtilis and B. velezensis with the potential of forming weak, moderate and strong biofilms with B. velezensis strain LPL-K103 (B44) with the most robust biofilm. All the isolates are novel sequence types (STs) using their multi-locus sequence type profile with the closest STs are 96 for B. velezensis and 128 for B. subtilis mostly isolated from the soil. The heat resistance profile indicated all 12 isolates are psychrotolerant as well as thermophilic with temperature ranges of 6 °C to 55 °C, 6 °C to 60 °C and 15 °C to 60 °C. All isolates can produce both lipolytic and proteolytic enzymes both in their planktonic and biofilm states.
The maximum lethality rate after UVC exposure is 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9 % from the sample B. velezensis strain LPL-K103 (B44). Flow cytometry analysis of UVC treated spore suspensions showed a divergence into subpopulations unaccounted for by plate counting on growth media which are: inactivated, live spores, dormant, sub-lethally injured and an unknown subpopulation. The Raman spectroscopy identified B. subtilis CECT 4002 (B4002) spores as the isolate possessing the highest concentration of Ca-DPA.
The spoilage potential of the isolates was determined by quantifying the concentrations of proteolytic and lipolytic enzymes produced by the biofilm and planktonic cells by using azocasein and p-nitrophenol palmitate (p-NPP) assays. In the planktonic cells, B48 has the highest proteolysis with 1033.6 ρL/CFU while B50 has highest lipolysis of 34.5 ρL/CFU. For the biofilms, B168 has the highest proteolysis and lipolysis per cell with a mean 3706 ρL and 179.9 ρL. The result of this study indicated that the spoilage potential (proteolysis and lipolysis) both of biofilms and planktonic culture are strain-dependent and that there seems to be a relationship between the strength or complexity of the biofilms and spoilage potential of the isolates. The study presents the significance of thermophilic B. subtilis B. velezensis and possible reason for their perpetuation in the dairy processing plant. The result linked the isolates to the raw milk used in the production of ESL milk-fed into the downstream processing line suggesting the survivability of the isolates by adaptation to the processing condition either as spores or as a community as in biofilms. The quality of the raw milk is thereby compromised which in turn affect the shelf-life of the final product. The result highlighted the effect of UVC in the inactivation of the spores and spore surface hydrophobicity are heterogenous with some strain-to-strain variations at molecular level among the organisms used.
... ComX activates the rest of the genes including the structural genes required for DNA transport. These transcription factors also activate fratricide effectors and bacteriocins, along with their respective immunity proteins [42,66,78,[84][85][86]. As in V. cholerae, co-regulation of these systems results in the activation of competence in an environment where there is likely to be DNA available for uptake. ...
The human pathogen Campylobacter jejuni is naturally competent for transformation with its own DNA. In this thesis work, we investigated key gene products of the transformation apparatus as well as the mechanism by which C. jejuni selects DNA for uptake and recombination. We demonstrate that two NTPases, CtsP and CtsE, are required for transformation. Their localization to the membrane is through a mechanism not generally observed with other competence ATPases. Further, CtsP interacts with another competence protein, CtsX, a single-pass transmembrane protein lacking significant homology to other proteins. Also investigated was the mechanism of DNA selection in C. jejuni. This bacterium is very selective in the DNA it uses during transformation, only using self or DNA derived from closely related strains. We demonstrate that this selection is based on adenine-methylation within a specific sequence motif, RAATTY. This site is broadly conserved in Campylobacter species and is over-represented in the C. jejuni genome. Methylation at RAATTY is conferred by CtsM, an orphan DNA methyltransferase also highly conserved in Campylobacter. CtsM is dispensable for transformation, but genomic DNA from a ctsM mutant serves as a transformation substrate with efficiency several orders of magnitude below DNA from a ctsM+ strain. A single methylated site is sufficient to confer wild type transformability to otherwise untransformable DNA. We also demonstrate that DNA lacking RAATTY methylation is transported inside the cell, implying that discrimination of methylated versus unmethylated DNA does not occur on the outer membrane. Finally, this work provides a potentially powerful tool to carry out rapid genome editing of C. jejuni, requiring only that an incoming homologous DNA fragment be RATTY-methylated for uptake and recombination. This is the first example of a methylation dependent mechanism of DNA selection during transformation.
... In pneumococcus and Bacillus, ComEA is not soluble, but localizes to the outer surface of the cell membrane, and is required for DNA uptake; it is not known whether it also acts as a ratchet to facilitate or drive DNA movement through the cell wall into the periplasm similarly to DNA uptake in competent Gram-negative species. However, in pneumo- With the discovery that expression of CbpD, LytA, and competence bacteriocin-like peptides is subject to direct competence regulation, and with demonstration that fratricide mediated by these products increases the efficiency of transformation in mixed-cell cultures dramatically (1000-fold) (Claverys et al., 2007;Johnsborg et al., 2008), it became reasonable to appreciate genetic transformation as reflecting a complex coordinated system of gene transfer from one living (noncompetent) cell to another (competent) living one. Specificity in the choice of victim cell is enhanced by speciesspecificity of CbpD, the principal competence lysin. ...
The competence pili of transformable Gram‐positive species are phylogenetically related to the diverse and widespread class of extracellular filamentous organelles known as type IV pili. In Gram‐negative bacteria, type IV pili act through dynamic cycles of extension and retraction to carry out diverse activities including attachment, motility, protein secretion, and DNA uptake. It remains unclear whether competence pili in Gram‐positive species exhibit similar dynamic activity, and their mechanism of action for DNA uptake remains unclear. They are hypothesized to either (1) leave transient cavities in the cell wall that facilitate DNA passage, (2) form static adhesins to enrich DNA near the cell surface for subsequent uptake by membrane‐embedded transporters, or (3) play an active role in translocating bound DNA via dynamic activity. Here, we use a recently described pilus labeling approach to demonstrate that competence pili in Streptococcus pneumoniae are highly dynamic structures that rapidly extend and retract from the cell surface. By labeling the principal pilus monomer, ComGC, with bulky adducts, we further demonstrate that pilus retraction is essential for natural transformation. Together, our results suggest that Gram positive competence pili in other species may also be dynamic and retractile structures that play an active role in DNA uptake. Competent pneumococci kill non‐competent cells on contact. Retractable DNA‐binding fibers in the type IV filament superfamily provide a key tool for retrieving DNA segments from cell wreckage for internalization and recombination.
... These differences, along with heterogeneity in cells' CSP-sensing potential will lead to slight timing differences of competence activation on a single-cell level, thereby leading to the formation of initial subpopulations of competent cells that then activate the rest of the population. Also, competent cells produce cell wall hydrolases and might reduce growth and kill non-competent siblings (Claverys et al., 2007). Interestingly, several factors, such as pH or antibiotics, can modify the rates at which single cells produce and/or sense CSP (Moreno-Gámez et al., 2017;Prudhomme et al., 2016). ...
Streptococcus pneumoniae can acquire antibiotic resistance by activation of competence and subsequent DNA uptake. Several antibiotics induce competence by disrupting protein-quality control or perturbing DNA replication. Here, we demonstrate that aztreonam (AZT) and clavulanic acid (CLA) also promote competence. We show that both compounds induce cell chain formation by targeting the D,D-carboxypeptidase PBP3. In support of the hypothesis that chain formation promotes competence, we demonstrate that an autolysin mutant ( lytB ) is hypercompetent. As competence is initiated by the binding of a small extracellular peptide (CSP) to a membrane-anchored receptor (ComD), we wondered if chain formation alters CSP diffusion and thereby sensing by ComD. Indeed, the presence of AZT or CLA affects competence synchronization by switching CSP-based quorum sensing to autocrine-like signaling, as CSP is retained to chained cells and no longer shared in a common pool. Together, these insights demonstrate the versatility of quorum sensing in integrating different stresses and highlight that certain antibiotics should be prescribed with care not to drive the spread of antibiotic resistance.
... In some cases, particularly among the Streptococci and Vibrio spp. competence is also an auto-parasitic process, as the first bacteria in a population to become competent kill their neighbors to ensure a source of DNA for transformation [49][50][51][52]. Finally, viral transduction results when a lysogenic (temperate) bacterial virus, or bacteriophage, excises itself from the host genome and inadvertently takes some of the host's genes along with its own genes and then reinserts these genes into the recipient's genome when it establishes a lysogenic state [1,7,15]. ...
The principle of monoclonality with regard to bacterial infections was considered immut-able prior to 30 years ago. This view, espoused by Koch for acute infections, has proven inadequate regarding chronic infections as persistence requires multiple forms of hetero-geneity among the bacterial population. This understanding of bacterial plurality emerged from a synthesis of what-were-then novel technologies in molecular biology and imaging science. These technologies demonstrated that bacteria have complex life cycles, poly-microbial ecologies, and evolve in situ via the horizontal exchange of genic characters. Thus, there is an ongoing generation of diversity during infection that results in far more highly complex microbial communities than previously envisioned. This perspective is based on the fundamental tenet that the bacteria within an infecting population display genotypic diversity, including gene possession differences, which result from horizontal gene transfer mechanisms including transformation, conjugation, and transduction. This understanding is embodied in the concepts of the supragenome/pan-genome and the distributed genome hypothesis (DGH). These paradigms have fostered multiple researches in diverse areas of bacterial ecology including host-bacterial interactions covering the gamut of symbiotic relationships including mutualism, commensalism, and parasitism. With regard to the human host, within each of these symbiotic relationships all bacterial species possess attributes that contribute to colonization and persistence; those species/strains that are pathogenic also encode traits for invasion and metastases. Herein we provide an update on our understanding of bacterial plurality and discuss potential applications in diagnostics, therapeutics, and vaccinology based on perspectives provided by the DGH with regard to the evolution of pathogenicity.
... Many microbial developmental programs utilize stochastic differentiation and programmed cell death as vital components of population fitness 4 . Selection for programmed cell death has been proposed to drive complex behaviors that delay commitment to costly cell fate decisions 5 , enable adaptation to environmental fluctuations 6 , eliminate competitor species 7 , reinforce biofilm structure 8 and promote colonization of hostile environments 9 . These behaviors represent divisions of labor between subpopulations of progenitor cells that propagate the species and sacrificial cells that provide a public good, analogous to germline and somatic cells from multicellular organisms. ...
Cooperation through division of labor underpins biological complexity for organisms and communities. In microbes, stochastic differentiation coupled to programmed cell death drives diverse altruistic behaviors that promote cooperation. Utilizing cell death for developmental multicellular programs requires control over differentiation rate to balance cell proliferation against the utility of sacrifice. However, these behaviors are often controlled by complex regulatory networks and have yet to be demonstrated from first principles. Here we engineered a synthetic developmental gene network that couples stochastic differentiation with programmed cell death to implement a two-member division of labor. Progenitor consumer cells were engineered to grow on cellobiose and differentiate at a controlled rate into self-destructive altruists that release an otherwise sequestered cellulase payload through autolysis. This circuit produces a developmental Escherichia coli consortium that utilizes cellulose for growth. We used an experimentally parameterized model of task switching, payload delivery and nutrient release to set key parameters to achieve overall population growth, liberating 14-23% of the available carbon. An inevitable consequence of engineering self-destructive altruism is the emergence of cheaters that undermine cooperation. We observed cheater phenotypes for consumers and altruists, identified mutational hotspots and developed a predictive model of circuit longeivity. This work introduces the altruistic developmental program as a tool for synthetic biology, demonstrates the utility of population dynamics models to engineer multicellular behaviors and provides a testbed for probing the evolutionary biology of self-destructive altruism.
... It is clear that at least two quorum sensing (QS) signal transduction pathways are critical for biofilm development: competence and Lux [7], [12][13][14][15]. The competence pathway has been the subject of intense investigation for decades [16][17][18][19][20][21][22][23][24][25][26][27][28]. Competence is activated by a classic two-component system (TCS) where the extracellular competence stimulating peptide (CSP, encoded by comC) binds to the surface exposed ComD histidine kinase receptor, inducing its autophosphorylation and the subsequent transfer of the phosphate group to its cognate regulator, ComE [23], [29]. ...
Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence , ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization. PLOS Pathogens | https://doi.org/10.1371/journal.ppat. Pneumococcal biofilms occur in chronic otitis media, chronic rhinosinusitis, and naso-pharyngeal colonization. These biofilms are an important component of pneumococcal epidemiology, particularly in influencing transmission, maintenance of asymptomatic col-onization, and development of disease. The transcriptional program initiated via signaling of the competence pathway is critical for productive biofilm formation and is a strong contributor of pneumococcal infection and adaptation. In this study, we have identified BriC, a previously uncharacterized peptide that serves as a bridge between the competence pathway and biofilm development. We show that briC is induced by ComE, the master regulator of competence, and promotes biofilm development. Moreover, our studies in the murine model demonstrate that BriC is a novel colonization enhancer. Our studies of briC regulation capture an instance of genomic plasticity, where natural variation in the briC promoter sequence reveals the existence of an additional competence-independent regulatory unit. This natural variation may be able to modify the extent to which competence contributes to biofilm development and to nasopharyngeal colonization across different pneumococcal lineages. In summary, this study introduces a colonization factor and reveals a molecular link between competence and biofilm development.
... The activation of the response regulator ComE drives the expression of early competence genes and the alternative sigma factor, ComX, which controls late gene expression (110). The lysis mechanisms autolysis and fratricide are induced during the competent state to release DNA from a subpopulation that is available for acquisition by the surviving cells (111)(112)(113)(114)(115)(116)(117). The regulation of competence induction is tightly controlled based on the inputs of secreted protein pheromone, growth phase, and stress stimuli (118). ...
Bacteria respond to changes in environmental conditions through adaptation to external cues. Frequently, bacteria employ nucleotide signaling molecules to mediate a specific, rapid response. Cyclic di-adenosine monophosphate (c-di-AMP) was recently discovered as a bacterial second messenger that is essential for viability in many species. In this review, we highlight recent work which has described the role of c-di-AMP in bacterial responses to various stress conditions. These studies show that depending on the lifestyle and environmental niche of the bacterial species, the c-di-AMP signaling network results in diverse outcomes, such as regulating osmolyte transport, controlling plant attachment, or providing a checkpoint for spore formation. c-di-AMP achieves this signaling specificity through expression of different classes of synthesis and catabolic enzymes as well as receptor proteins and RNAs, which will be summarized.
... Death' of other bacterial species within the biofilm to increase the inflammatory response and promotes its survival [60,216] Fratricide is the pathogenic process by which P. gingivalis kills and obtain DNAs fragments from noncompeting host cells and microbes by inducing their death [216,[218][219][220][221] During the process of programmed cell death, P. gingivalis promotes a fraction of the microbial population to perform a self-sacrificing 'suicide' and discharge its nutrients and extracellular DNA fragments into the environment. The discharged nutrients and extracellular DNA fragments are utilized by P. gingivalis and other members of the microbial community for their growth. ...
Porphyromonas. gingivalis (P. gingivalis)
is an obligate, asaccharolytic, gram-negative bacteria commonly associated with increased periodontal and systemic inflammation. P. gingivalis is known to survive and persist within the host tissues as it modulates the entire ecosystem by either engineering its environment or modifying the host’s immune response. It interacts with various host receptors and alters signaling pathways of inflammation, complement system, cell cycle, and apoptosis. P. gingivalis is even known to induce suicidal cell death of the host and other microbes in its vicinity with the emergence of pathobiont species. Recently, new molecular and immunological mechanisms and virulence factors of P. gingivalis that increase its chance of survival and immune evasion within the host have been discovered. Thus, the present paper aims to provide a consolidated update on the new intricate and unique molecular mechanisms and virulence factors of P. gingivalis associated with its survival, persistence, and immune evasion within the host.
... Le contact physique de la donatrice et de la réceptrice est nécessaire dans le processus de conjugaison. Dans le cas de la transformation, la condition sine qua non reste la persistance dans l'environnement de l'ADN sous forme libre (qui peut également provenir de la lyse des cellules soeurs non compétentes par celles compétentes (Claverys et al., 2007)) et dela concordance entre cette persistance et l'état physiologique de compétence de la bactérie (Nielsen et al., 1998;Cohan, 2002b). ...
Les Streptomyces sont des bactéries de la rhizosphère qui contribuent à la fertilité des sols (recyclage de la matière organique), et à la croissance et la santé des plantes. Elles possèdent parmi les plus grands génomes bactériens (12 Mb) et présentent une variabilité génétique importante. Cette variabilité connue au niveau interspécifique n’a jamais été abordée à l’échelle de la population, c’est-à-dire entre individus sympatriques appartenant à la même espèce (souches sœurs) au sein de la même niche écologique. L’objectif de ce travail est de rechercher cette diversité dans les populations de l’écosystème sol forestier, d’approcher sa dynamique et son rôle fonctionnel. Après séquençage et comparaison des génomes complets, nous avons observé une grande diversité génomique en termes de taille, de présence/absence d’éléments extrachromosomiques, mais également en terme de présence/absence de gènes le long du chromosome. Un grand nombre d’événements d’insertions et délétions (indels) comprenant de 1 à 241 gènes différencient les individus de la population. Au vu des liens phylogénétiques étroits entre les individus, l’ancêtre commun de la population est récent, aussi la diversité génomique résulterait d’un flux massif et rapide de gènes. La forte prévalence d’éléments conjugatifs intégrés dans la population suggère que la conjugaison est le moteur prépondérant de cette diversité génomique. La production différentielle de métabolites spécialisés (antibiotiques) a également été utilisée pour estimer l’impact de la diversité génétique sur le fonctionnement de la population. Nous avons pu montrer que cette production était liée à des gènes spécifiques de souches et qu’elle pouvait constituer un bien commun pour la population. Nous proposons que l’évolution rapide du génome participe au maintien des mécanismes de cohésion sociale chez ces bactéries du sol.
... They may also select DNA with specific uptake sequences to avoid integrating exogenous DNA (20). In few cases, competent bacteria kill their own kin cells, enriching their surrounding with clonal DNA (38). Considering all the barriers to the acquisition of foreign DNA, transformation would mainly act as a conservative mechanism instead of a means of genetic diversification and mixing (36). ...
Natural transformation is the acquisition, controlled by bacteria, of extracellular DNA and is one of the most common mechanisms of horizontal gene transfer, promoting the spread of resistance genes. However, its evolutionary function remains elusive, and two main roles have been proposed: (i) the new gene acquisition and genetic mixing within bacterial populations and (ii) the removal of infectious parasitic mobile genetic elements (MGEs). While the first one promotes genetic diversification, the other one promotes the removal of foreign DNA and thus genome stability, making these two functions apparently antagonistic. Using a computational model, we show that intermediate transformation rates, commonly observed in bacteria, allow the acquisition then removal of MGEs. The transient acquisition of costly MGEs with resistance genes maximizes bacterial fitness in environments with stochastic stress exposure. Thus, transformation would ensure both a strong dynamic of the bacterial genome in the short term and its long-term stabilization.
... Previous studies by our lab have shown that the expression of competence-dependent allolytic factors, including LytA, CbpD, and CibAB, benefits both acute pneumonia and bacteremia infections through the release of pneumolysin (26,28). In addition, in vitro studies have shown that these allolytic factors contribute to fratricide, which increases DNA availability by killing a subpopulation of noncompetent pneumococcus (54)(55)(56)(57)(58)(59). Fratricide was shown to be important for genetic transformation inside pneumococcal biofilms (60), which has been proposed to resemble nasopharyngeal colonization (20). ...
The competence regulon of pneumococcus regulates both genetic transformation and virulence. However, competence induction during host infection has not been examined. By using the serotype 2 strain D39, we transcriptionally-fused the firefly luciferase ( luc ) to competence-specific genes, and spatiotemporally monitored the competence development in a mouse model of pneumonia-derived sepsis. Contrary to the universally-reported short transient burst of competent state in vitro , naturally-developed competent state was prolonged and persistent during pneumonia-derived sepsis. Competent state began at approximately 20 hours post infection (hpi) and facilitated systemic invasion and sepsis development, and progressed in different manners. In some mice, acute pneumonia quickly led to sepsis and death, accompanied by increasing intensity of the competence signal. In the remaining mice, pneumonia lasted longer with the competence signal decreasing at first, but increasing as the infection became septic. The concentration of pneumococcal inoculum (1x10 ⁶ – 1x10 ⁸ CFU/mouse) and post-infection lung bacterial burden did not appreciably impact the kinetics of competence induction. Exogenously-provided competence stimulating peptide 1 (CSP1) failed to modulate the onset kinetics of competence development in vivo . The competence shut off regulator DprA was highly expressed during pneumonia-derived sepsis, but failed to turn off the competent state in mice. Competent D39 bacteria propagated the competence signal through cell-to-cell contact rather than the classically described quorum sensing mechanism. Finally, clinical pneumococcal strains of different serotypes were also able to develop natural competence during pneumonia-derived sepsis.
... They may also select DNA with specific uptake sequences to avoid integrating exogenous DNA 33 . In some cases, competent bacteria kill their own kin cells, enriching their surrounding with clonal DNA 34 . Considering all the barriers to the acquisition of foreign DNA, transformation would mainly act as a conservative mechanism instead of a mean of genetic diversification and mixing 31 . ...
Horizontal gene transfer (HGT) is known to promote the spread of genes in bacterial communities, which is of primary importance to human health when these genes provide resistance to antibiotics. Among the main HGT mechanisms, natural transformation stands out as being widespread and encoded by the bacterial core genome. From an evolutionary perspective, transformation is often viewed as a mean to generate genetic diversity and mixing within bacterial populations. However, another recent paradigm proposes that its main evolutionary function would be to cure bacterial genomes from their parasitic mobile genetic elements (MGEs). Here, we propose to combine these two seemingly opposing points of view because MGEs, although costly for bacterial cells, can carry functions that are point-in-time beneficial to bacteria under stressful conditions (e.g. antibiotic resistance genes under antibiotic exposure). Using computational modeling, we show that, in stochastic environments (unpredictable stress exposure), an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes but costly for the replication of host cells. By ensuring such reversible genetic diversification (acquisition then removal of MGEs), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, which would explain its striking conservation.
... Ce mécanisme est illustré par la figure 2.9 page 44 et expliqué ci-après : L'induction de la compétence pourrait être due en partie à des signaux environnementaux lors de la formation de biofilms mais ce mécanisme reste encore mal compris. L'état de compétence implique l'expression transitoire des gènes précoces et tardifs de la compétence (Claverys, Martin et Håvarstein, 2007 ;Johnsborg et al., 2008). Deux opérons sont d'abord activés, comAB qui code pour un ABC (ATP-Binding-Cassette) transporteur (Chandler et Morrison, 1987) et comCDE qui code pour le peptide de stimulation de la compétence (CSP) sous forme inactive (Håvarstein, Coomaraswamy et Morrison, 1995), ComC/ComD le récepteur de CSP et ComE le régulateur de réponse du système à deux composantes ComDE (Håvarstein et al., 1996). ...
La paroi des bactéries à Gram positif se compose du peptidoglycane (PG) et des acides téichoïques (TA). Leur étude a révélé de nouveaux mécanismes de régulation chez le pathogène humain Streptococcus pneumoniae. Nous avons montré que la O-acétylation intervient précocement dans la biosynthèse du PG, participe à sa maturation et à la division cellulaire. Nous avons développé une approche innovante basée sur la chimie click pour le marquage in vivo des TAs, et révélé que leur synthèse est septale et corrélée à celle du PG. Le PG et les TAs contribuent aussi à réguler l'activité enzymatique de l'autolysine majeur du pneumocoque LytA: la O-acétylation du PG protège les cellules en division de l'autolyse par LytA et les TAs, sur lesquels elle se fixe, régulent sa localisation de surface. Pour conclure, ce travail souligne le rôle coopératif du PG et des TAs dans la synthèse de la paroi, la division cellulaire et la régulation de composants de la surface bactérienne.
... The pneumococcal Com regulon has been studied primarily as a mechanism for DNA acquisition and genetic transformation. There is heterogeneity in the population during QS such that not all cells activate the Com regulon in response to CSP at the same time, and pneumococcal fratricide, a process whereby competent cells target non-competent cells for lysis and DNA acquisition, occurs 33 . The release of DNA and virulence factors, such as pneumolysin, are thought to be advantageous for the remaining population. ...
Competition among microorganisms is a key determinant of successful host colonization and persistence. For Streptococcus pneumoniae, lower than predicted rates of co-colonizing strains suggest a competitive advantage for resident bacteria over newcomers. In light of evolutionary theory, we hypothesized that S. pneumoniae use owner–intruder asymmetries to settle contests, leading to the disproportionate success of the initial resident ‘owner’, regardless of the genetic identity of the ‘intruder’. We investigated the determinants of within-host competitive success utilizing S. pneumoniae colonization of the upper respiratory tract of infant mice. Within 6 h, colonization by the resident inhibited colonization by an isogenic challenger. The competitive advantage of the resident was dependent on quorum sensing via the competence (Com) regulon and downstream choline binding protein D (CbpD) and on the competence-induced bacteriocins A and B (CibAB) implicated in fratricide. CbpD and CibAB are highly conserved across pneumococcal lineages, indicating evolutionary advantages for asymmetric competitive strategies within the species. Mathematical modelling supported a significant role for quorum sensing via the Com regulon in competition, even for strains with different competitive advantages. Our study suggests that asymmetric owner–intruder competitive strategies do not require complex cognition and are used by a major human pathogen to determine ‘ownership’ of human hosts. © 2018, The Author(s), under exclusive licence to Springer Nature Limited.
... The highlighted area shows the temporal window in which cells were able to take up DNA. (Claverys et al., 2007). Interestingly, several factors, such as pH or antibiotics, can modify the rates at which single cells produce and/or sense CSP (Moreno-Gá mez et al., 2017;Prudhomme et al., 2016). ...
Streptococcus pneumoniae can acquire antibiotic resistance by activation of competence and subsequent DNA uptake. Here, we demonstrate that aztreonam (ATM) and clavulanic acid (CLA) promote competence. We show that both compounds induce cell chain formation by targeting the D,D-carboxypeptidase PBP3. In support of the hypothesis that chain formation promotes competence, we demonstrate that an autolysin mutant (ΔlytB) is hypercompetent. Since competence is initiated by the binding of a small extracellular peptide (CSP) to a membrane-anchored receptor (ComD), we wondered whether chain formation alters CSP diffusion kinetics. Indeed, ATM or CLA presence affects competence synchronization by shifting from global to local quorum sensing, as CSP is primarily retained to chained cells, rather than shared in a common pool. Importantly, autocrine-like signaling prolongs the time window in which the population is able to take up DNA. Together, these insights demonstrate the versatility of quorum sensing and highlight the importance of an accurate antibiotic prescription. Streptococcus pneumoniae can take up exogenous DNA by activating competence. Aztreonam and clavulanic acid can induce competence by targeting PBP3, leading to cell chaining. Cell chaining reshapes quorum sensing to autocrine-like signaling and increases the time window in which cells can take up DNA, potentially accelerating the spread of antibiotic resistance.
... It is clear that at least two quorum sensing (QS) signal transduction pathways are critical for biofilm development: competence and Lux [7], [12][13][14][15]. The competence pathway has been the subject of intense investigation for decades [16][17][18][19][20][21][22][23][24][25][26][27][28]. Competence is activated by a classic two-component system (TCS) where the extracellular competence stimulating peptide (CSP, encoded by comC) binds to the surface exposed ComD histidine kinase receptor, inducing its autophosphorylation and the subsequent transfer of the phosphate group to its cognate regulator, ComE [23], [29]. ...
Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC, and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE-deletion induced biofilm defect in vitro, and partially in vivo. These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.
... Competence-stimulating peptide has also been shown to induce fratricide, killing, and lysis of noncompetent cells in the environment. The biological role of fratricide may be to provide DNA for genetic exchange (62). ...
Bacteria within the oral cavity live primarily as complex, polymicrobial biofilms. Dental biofilms are necessary etiological factors for dental caries and periodontal diseases but have also been implicated in diseases outside the oral cavity. Biofilm is the preferred lifestyle for bacteria, and biofilms are found on almost any surface in nature. Bacteria growing within a biofilm exhibit an altered phenotype. Substantial changes in gene expression occur when bacteria are in close proximity or physical contact with one another or with the host. This may facilitate nutritional co‐operation, cell–cell signaling, and gene transfer, including transfer of antibiotic‐resistance genes, thus rendering biofilm bacteria with properties other than those found in free‐floating, planktonic bacteria. We will discuss biofilm properties and possible consequences for future prophylaxis.
... In Streptococcus pneumoniae, competence is a transient physiological state induced by activation of a genetic program in response to specific conditions, such as environmental stress Fontaine et al., 2015). The competence state allows natural transformation, fratricide , and biofilm formation (Oggioni et al., 2006;Claverys et al., 2007;Trappetti et al., 2011;Vidal et al., 2013), and it contributes to virulence (Zhu et al., 2015;Lin et al., 2016). Upon addition of the synthetic auto-inducer competence-stimulating peptide (CSP) (Håvarstein et al., 1995) to laboratory exponential cultures, competence develops abruptly and nearly simultaneously in virtually all the cells, and then, after about 20 min, declines almost as quickly (Alloing et al., 1998). ...
In the human pathogen Streptococcus pneumoniae, the gene regulatory circuit leading to the transient state of competence for natural transformation is based on production of an auto-inducer that activates a positive feedback loop. About 100 genes are activated in two successive waves linked by a central alternative sigma factor ComX. This mechanism appears to be fundamental to the biological fitness of S. pneumoniae. We have developed a knowledge-based model of the competence cycle that describes average cell behavior. It reveals that the expression rates of the two competence operons, comAB and comCDE, involved in the positive feedback loop must be coordinated to elicit spontaneous competence. Simulations revealed the requirement for an unknown late com gene product that shuts of competence by impairing ComX activity. Further simulations led to the predictions that the membrane protein ComD bound to CSP reacts directly to pH change of the medium and that blindness to CSP during the post-competence phase is controlled by late DprA protein. Both predictions were confirmed experimentally.
... In a biofilm model, pneumocin production resulted in an increase in transformation efficiency (11). The competence regulon itself encodes so-called "fratricide effectors" which target and kill noncompetent pneumococci (24). The role of fratricide seems to be to increase access to extracellular DNA during competence (25,26). ...
Significance
The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) participates in horizontal gene transfer through genetic competence and produces antimicrobial peptides called “bacteriocins.” Here, we show that the competence and bacteriocin-related ABC transporters ComAB and BlpAB share the same substrate pool, resulting in bidirectional crosstalk between competence and bacteriocin regulation. We also clarify the role of each transporter in bacteriocin secretion and show that, based on their transporter content, pneumococcal strains can be separated into a majority opportunist group that uses bacteriocins only to support competence and a minority aggressor group that uses bacteriocins in broader contexts. Our findings will impact how bacteriocin regulation and production is modeled in the many other bacterial species that use ComAB/BlpAB-type transporters.
... The analysis of bacterial surface-associated proteins is challenging because of their physico-chemical properties and autolysis or fratricide committed during bacterial growth [12][13][14] . Hitherto, classical gel-based proteomic approaches have been applied for analysis of surface-exposed bacterial ligands and their receptors on the host cells [15][16][17] . ...
The mechanisms by which Streptococcus pneumoniae penetrates the blood-brain barrier (BBB), reach the CNS and causes meningitis are not fully understood. Adhesion of bacterial cells on the brain microvascular endothelial cells (BMECs), mediated through protein-protein interactions, is one of the crucial steps in translocation of bacteria across BBB. In this work, we proposed a systematic workflow for identification of cell wall associated ligands of pneumococcus that might adhere to the human BMECs. The proteome of S. pneumoniae was biotinylated and incubated with BMECs. Interacting proteins were recovered by affinity purification and identified by data independent acquisition (DIA). A total of 44 proteins were identified from which 22 were found to be surface-exposed. Based on the subcellular location, ontology, protein interactive analysis and literature review, five ligands (adhesion lipoprotein, endo-β-N-acetylglucosaminidase, PhtA and two hypothetical proteins, Spr0777 and Spr1730) were selected to validate experimentally (ELISA and immunocytochemistry) the ligand-BMECs interaction. In this study, we proposed a high-throughput approach to generate a dataset of plausible bacterial ligands followed by systematic bioinformatics pipeline to categorize the protein candidates for experimental validation. The approach proposed here could contribute in the fast and reliable screening of ligands that interact with host cells.
... Pneumococci encode for a diverse number of bacteriocins like BlpC, BlpN, BlpM, CibAB (Dawid et al., 2007;Guiral et al., 2005;Kjos et al., 2016;Lux et al., 2007;Wholey et al., 2016). Most of them function during the competence state to ensure the elimination of non-competent cells and the release of their DNA, nutrients, and virulence factors (Claverys et al., 2007;Guiral et al., 2005;Wholey et al., 2016). TCS13 or BlpRH modulates the expression of pneumococcal antimicrobial peptides (AMPs) bacteriocins like BlpC (de Saizieu et al., 2000;Kjos et al., 2016;Lux et al., 2007;Reichmann and Hakenbeck, 2000;Wholey et al., 2016). ...
Streptococcus pneumoniae is a human pathobiont possessing a diverse array of multifunctional proteins essential for bacterial fitness and virulence. Gene expression is tightly controlled by regulatory components and among the pneumococcal sensorial tools, two‑component regulatory systems (TCS) are the most widespread and conserved. This review aims to provide a comprehensive analysis of original studies on pneumococcal TCS on a functional level. Despite a rather chaotic nomenclature, the current available information on pneumococcal regulation by these systems can be conveniently addressed, according to the regulation of pathophysiological cell processes and the responses to detectable environmental signals. Pneumococcal pathophysiological processes driven by TCS can be further categorized into competence and fratricide, bacteriocin production, and virulence factors expression. Conversely, detectable environmental signals by pneumococci can be grouped into antibiotics and cell wall perturbations, environmental stress, and nutrients acquisition. This review summarizes the state of the art on pneumococcal TCS based on an integral approach and thus providing insights into the regulatory network(s) of S. pneumoniae.
Bacterial biofilms are intricate ecosystems of microbial communities that adhere to various surfaces and are enveloped by an extracellular matrix composed of polymeric substances. Within the context of bacterial biofilms, extracellular DNA (eDNA) originates from cell lysis or is actively secreted, where it exerts a significant influence on the formation, stability, and resistance of biofilms to environmental stressors. The exploration of eDNA within bacterial biofilms holds paramount importance in research, with far-reaching implications for both human health and the environment. An enhanced understanding of the functions of eDNA in biofilm formation and antibiotic resistance could inspire the development of strategies to combat biofilm-related infections and improve the management of antibiotic resistance. This comprehensive review encapsulates the latest discoveries concerning eDNA, encompassing its origins, functions within bacterial biofilms, and significance in bacterial pathogenesis.
Natural transformation plays an important role in the formation of drug-resistant bacteria. Exploring the regulatory mechanism of natural transformation can aid the discovery of new antibacterial targets and reduce the emergence of drug-resistant bacteria. Competence is a prerequisite of natural transformation in Streptococcus pneumoniae, in which comCDE operon is the core regulator of competence. To date, only ComE has been shown to directly regulate comCDE transcription. In this study, a transcriptional regulator, the catabolite control protein A (CcpA), was identified that directly regulated comCDE transcription. We confirmed that CcpA binds to the cis-acting catabolite response elements (cre) in the comCDE promoter region to regulate comCDE transcription and transformation. Moreover, CcpA can coregulate comCDE transcription with phosphorylated and dephosphorylated ComE. Regulation of comCDE transcription and transformation by CcpA was also affected by carbon source signals. Together, these insights demonstrate the versatility of CcpA and provide a theoretical basis for reducing the emergence of drug-resistant bacteria.
IMPORTANCE Streptococcus pneumoniae is a major cause of bacterial infections in humans, such as pneumonia, bacteremia, meningitis, otitis media, and sinusitis. Like most streptococci, S. pneumoniae is naturally competent and employs this ability to augment its adaptive evolution. The current study illustrates CcpA, a carbon catabolite regulator, can participate in the competence process by regulating comCDE transcription, and this process is regulated by different carbon source signals. These hidden abilities are likely critical for adaptation and colonization in the environment.
The combination of next-generation DNA sequencing technologies and bioinformatics has revitalized natural product discovery. Using a bioinformatic search strategy, we recently identified ∼600 gene clusters in otherwise overlooked streptococci that code for ribosomal peptide natural products synthesized by radical S-adenosylmethionine enzymes. These grouped into 16 subfamilies and pointed to an unexplored microbiome biosynthetic landscape. Here we report the structure, biosynthesis and function of one of these natural product groups, which we term enteropeptins, from the gut microbe Enterococcus cecorum. We show three reactions in the biosynthesis of enteropeptins that are each catalysed by a different family of metalloenzymes. Among these, we characterize the founding member of a widespread superfamily of Fe–S-containing methyltransferases, which, together with an Mn²⁺-dependent arginase, installs N-methylornithine in the peptide sequence. Biological assays with the mature product revealed bacteriostatic activity only against the producing strain, extending an emerging theme of fratricidal or self-inhibitory metabolites in microbiome firmicutes.
Humans live in large and extensive societies and spend much of their time interacting socially. Likewise, most other animals also interact socially. Social behaviour is of constant fascination to biologists and psychologists of many disciplines, from behavioural ecology to comparative biology and sociobiology. The two major approaches used to study social behaviour involve either the mechanism of behaviour - where it has come from and how it has evolved, or the function of the behaviour studied. With guest articles from leaders in the field, theoretical foundations along with recent advances are presented to give a truly multidisciplinary overview of social behaviour, for advanced undergraduate and graduate students. Topics include aggression, communication, group living, sexual behaviour and co-operative breeding. With examples ranging from bacteria to social mammals and humans, a variety of research tools are used, including candidate gene approaches, quantitative genetics, neuro-endocrine studies, cost-benefit and phylogenetic analyses and evolutionary game theory.
Competence refers to the specialized physiological state in which bacteria undergo transformation through the internalization of exogenous DNA in a controlled and genetically encoded process that leads to genotypic and, in many cases, phenotypic changes. Natural transformation was first described in Streptococcus pneumoniae and has since been demonstrated in numerous species, including Bacillus subtilis and Neisseria gonorrhoeae. Homologs of the genes encoding the DNA uptake machinery for natural transformation have been reported to be present in several lactic acid bacteria, including Lactobacillus spp., Streptococcus thermophilus, and Lactococcus spp. In this review, we collate current knowledge of the phenomenon of natural transformation in Gram-positive bacteria. Furthermore, we describe the mechanism of competence development and its regulation in model bacterial species. We highlight the importance and opportunities for the application of these findings in the context of bacterial starter cultures associated with food fermentations as well as current limitations in this area of research.
Streptococcus pneumoniae is an opportunistic respiratory human pathogen that poses a continuing threat to human health. Natural competence for genetic transformation in S. pneumoniae plays an important role in aiding pathogenicity and it is the best‐characterized feature to acquire antimicrobial resistance genes by a frequent process of recombination. In S. pneumoniae, competence, along with virulence factor production, is controlled by a cell‐density communication mechanism termed the competence regulon. In this review, we present the recent advances in the development of alternative methods to attenuate the pathogenicity of S. pneumoniae by targeting the various stages of the non‐essential competence regulon communication system. We mainly focus on new developments related to competitively intercepting the competence regulon signaling through the introduction of promising dominant‐negative Competence Stimulating Peptide (CSP) scaffolds. We also discuss recent reports on antibiotics that can block CSP export by disturbing the proton motive force across the membrane and various ways to control the pneumococcal pathogenicity by activating the counter signaling circuit and targeting the pneumococcal proteome.
Abstract
Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that causes otitis media, sinusitis, pneumonia, meningitis and sepsis. The progression to this pathogenic lifestyle is preceded by asymptomatic colonization of the nasopharynx. This colonization is associated with biofilm formation; the competence pathway influences the structure and stability of biofilms. However, the molecules that link the competence pathway to biofilm formation are unknown. Here, we describe a new competence-induced gene, called briC , and demonstrate that its product promotes biofilm development and stimulates colonization in a murine model. We show that expression of briC is induced by the master regulator of competence, ComE. Whereas briC does not substantially influence early biofilm development on abiotic surfaces, it significantly impacts later stages of biofilm development. Specifically, briC expression leads to increases in biofilm biomass and thickness at 72h. Consistent with the role of biofilms in colonization, briC promotes nasopharyngeal colonization in the murine model. The function of BriC appears to be conserved across pneumococci, as comparative genomics reveal that briC is widespread across isolates. Surprisingly, many isolates, including strains from clinically important PMEN1 and PMEN14 lineages, which are widely associated with colonization, encode a long briC promoter. This long form captures an instance of genomic plasticity and functions as a competence-independent expression enhancer that may serve as a precocious point of entry into this otherwise competence-regulated pathway. Moreover, overexpression of briC by the long promoter fully rescues the comE -deletion induced biofilm defect in vitro , and partially in vivo . These findings indicate that BriC may bypass the influence of competence in biofilm development and that such a pathway may be active in a subset of pneumococcal lineages. In conclusion, BriC is a part of the complex molecular network that connects signaling of the competence pathway to biofilm development and colonization.
In the human pathogen Streptococcus pneumoniae , the gene regulatory circuit leading to the transient state of competence for natural transformation is based on production of an auto-inducer that activates a positive feedback loop. About one hundred genes are activated in two successive waves linked by a central alternative sigma factor ComX. This mechanism appears to be fundamental to the biological fitness of S. pneumoniae. We have developed a knowledge-based model of the competence cycle that describes average cell behavior. It reveals that the expression rates of the two competence operon, comAB and comCDE , involved in the positive feedback loop must be coordinated to elicit spontaneous competence. Simulations revealed the requirement for an unknown late com gene product that shuts of competence by impairing ComX activity. Further simulations led to the predictions that the membrane protein ComD bound to CSP reacts directly to pH change of the medium and that blindness to CSP during the post-competence phase is controlled by late DprA protein. Both predictions were confirmed experimentally.
Mammalian microbiomes encode thousands of biosynthetic gene clusters (BGCs) and represent a new frontier in natural product research. We recently found an abundance of quorum sensing-regulated BGCs in mammalian microbiome streptococci that code for ribosomally synthesized and post-translationally modified peptides (RiPPs) and contain one or more radical S-adenosylmethionine (RaS) enzymes, a versatile superfamily known for catalyzing some of the most unusual reactions in biology. In the current work, we target a wide-spread group of streptococcal RiPP BGCs and elucidate both the reaction carried out by its encoded RaS enzyme and identify its peptide natural product, which we name streptosactin. Streptosactin is the first sactipeptide identified from Streptococcus spp.; it contains two sequential four amino acid sactionine macrocycles, an unusual topology for this compound family. Bioactivity assays reveal potent but narrow-spectrum activity against the producing strain and its closest relatives that carry the same BGC, suggesting streptosactin may be a long-suspected fratricidal agent of Streptococcus thermophilus. Our results highlight mammalian streptococci as a rich source of unusual enzymatic chemistries and bioactive natural products.
While programmed cell death was once thought to be exclusive to eukaryotic cells, there are now abundant examples of well regulated cell death mechanisms in bacteria. The mechanisms by which bacteria undergo programmed cell death are diverse, and range from the use of toxin-antitoxin systems, to prophage-driven cell lysis. Moreover, some bacteria have learned how to coopt programmed cell death systems in competing bacteria. Interestingly, many of the potential reasons as to why bacteria undergo programmed cell death may parallel those observed in eukaryotic cells, and may be altruistic in nature. These include protection against infection, recycling of nutrients, to ensure correct morphological development, and in response to stressors. In the following chapter, we discuss the molecular and signaling mechanisms by which bacteria undergo programmed cell death. We conclude by discussing the current open questions in this expanding field.
Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.
The mitis group of streptococci comprises species that are common colonizers of the naso-oral-pharyngeal tract of humans. Streptococcus pneumoniae and Streptococcus mitis are close relatives and share ~60–80% of orthologous genes, but still present striking differences in pathogenic potential toward the human host. S. mitis has long been recognized as a reservoir of antibiotic resistance genes for S. pneumoniae, as well as a source for capsule polysaccharide variation, leading to resistance and vaccine escape. Both species share the ability to become naturally competent, and in this context, competence-associated killing mechanisms such as fratricide are thought to play an important role in interspecies gene exchange. Here, we explore the general mechanism of natural genetic transformation in the two species and touch upon the fundamental clinical and evolutionary implications of sharing similar competence, fratricide mechanisms, and a large fraction of their genomic DNA.
Streptococcus pneumoniae transformation occurs within a short competence window, during which the alternative sigma factor X (SigX) is activated to orchestrate the expression of genes allowing extracellular DNA uptake and recombination. Importantly, antibiotic stress promotes transcriptional changes that may affect more than 20% of the S. pneumoniae genome, including competence genes. These can be activated or repressed, depending on the antibiotic agent. For most antibiotics, however, it remains unknown whether transcriptional effects on competence translate into altered transformability. Here we investigated the effect of antibiotic subinhibitory concentrations on sigX expression using a luciferase reporter, and correlated for the first time with transformation kinetics. Induction of sigX expression by ciprofloxacin and novobiocin correlated with increased and prolonged transformability in S. pneumoniae. The prolonged effect of ciprofloxacin on competence and transformation was also observed in the streptococcal relatives Streptococcus mitis and Streptococcus mutans. In contrast, tetracycline and erythromycin, which induced S. pneumoniae sigX expression, had either an inhibitory or a non‐significant effect on transformation, whereas streptomycin and the β‐lactam ampicillin, inhibited both sigX expression and transformation. This is particularly relevant, as β‐lactams are important antibiotics in the arsenal against pneumococcal infections. Thus, the results show that antibiotics may vary in their effects on competence, ranging from inhibitory to stimulatory effects, and that responses affecting transcription of sigX not always correlate with the transformation outcomes. Antibiotics that increase or decrease transformation are of particular clinical relevance, as they may alter the ability of S. pneumoniae to escape vaccines and antibiotics.
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Afin de faire face à différents types de stress et s'adapter à de nouveaux environnements, les bactéries ont développé de nombreux mécanismes génétiquement régulés. La compétence pour la transformation naturelle est un processus qui favorise le transfert horizontal de gènes. Si les espèces phylogénétiquement éloignées partagent des mécanismes conservés d'intégration et de remaniement de l'ADN, les circuits de régulation de la compétence ne sont toutefois pas universels mais adaptés au mode de vie de chaque espèce. Chez les bactéries Gram-positives, les cascades de régulation de Streptococcus pneumoniae et Bacillus subtilis sont les mieux documentées. Si de nombreux modèles mathématiques ont été établis pour étudier différents aspects de la régulation des compétences chez B. subtilis, un seul modèle à échelle de population a été développé pour S. pneumoniae, il y a plus de dix ans, sur la base d'hypothèses contestées par de nouvelles données expérimentales. Nous avons développé, chez S. pneumoniae, un modèle fondé sur la connaissance de la régulation de la compétence qui intègre les éléments biologiques essentiels connus à ce jour. La cohérence structurelle de la topologie du réseau est confirmée par le formalisme des réseaux de Petri. Le réseau est ensuite transformé en un ensemble d'équations différentielles ordinaires pour étudier son comportement dynamique. La cinétique des protéines a été estimée en utilisant des données de luminescence et l'estimation des paramètres a été contrainte à partir des connaissances disponibles. Après avoir testé des modèles alternatifs, nous avons proposé l'existence d'un produit de gène tardif supplémentaire pouvant inhiber l'action de ComW, l'activateur du facteur sx. Nous apportons également un nouvel éclairage sur cette cascade de régulation en prédisant la cinétique de composantes du système qui pourraient être impliquées dans des comportements spécifiques. Ce modèle consolide les connaissances expérimentales acquises sur la régulation de la compétence chez S. pneumoniae. De plus, il peut être appliqué aux autres espèces de streptocoques appartenant aux groupes mitis et anginosus puisqu'ils partagent le même circuit régulateur. À l'échelle populationnelle, la transition vers l'état de compétence se produit d'abord dans une sous-population de cellules et se propage ensuite dans toute la population par contact physique cellule à cellule. En permettant la simulation du comportement d'une cellule individuelle, le modèle pourra servir de module dans la conception d'un modèle d'une population bactérienne composée de cellules hétérogènes.
Lantibiotics are polycyclic peptides containing unusual amino acids, which have binding specificity for bacterial cells, targeting the bacterial cell wall component lipid II to form pores and thereby lyse the cells. Yet several members of these lipid II–targeted lantibiotics are too short to be able to span the lipid bilayer and cannot form pores, but somehow they maintain their antibacterial efficacy. We describe an alternative mechanism by which members of the lantibiotic family kill Gram-positive bacteria by removing lipid II from the cell division site (or septum) and thus block cell wall synthesis.
Choline binding proteins are virulence determinants present in several Gram-positive bacteria. Because anchorage of these proteins to the cell wall through their choline binding domain is essential for bacterial virulence, their release from the cell surface is considered a powerful target for a weapon against these pathogens. The first crystal structure of a choline binding domain, from the toxin-releasing enzyme pneumococcal major autolysin (LytA), reveals a novel solenoid fold consisting exclusively of beta-hairpins that stack to form a left-handed superhelix. This unique structure is maintained by choline molecules at the hydrophobic interface of consecutive hairpins and may be present in other choline binding proteins that share high homology to the repeated motif of the domain.
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.
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.
Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.
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.
We have identified in the Streptococcus pneumoniaegenome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing
systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial
peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a
regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating
peptide ComC. The putative translation product of blpC, a small gene located downstream ofblpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters.
Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*)
induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since
insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions
of theblp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended −10 promoter elements.
We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response
regulator.
Strains of Streptococcus mutans produce at least three mutacins, I, II, and III. Mutacin II is a member of subgroup AII in the lantibiotic family of bacteriocins, and mutacins I and III belong to subgroup AI in the lantibiotic family. In this report, we characterize two mutacins produced by UA140, a group I strain of S. mutans. One is identical to the lantibiotic mutacin I produced by strain CH43 (F. Qi et al., Appl. Environ. Microbiol. 66:3221-3229, 2000); the other is a nonlantibiotic bacteriocin, which we named mutacin IV. Mutacin IV belongs to the two-peptide, nonlantibiotic family of bacteriocins produced by gram-positive bacteria. Peptide A, encoded by gene nlmA, is 44 amino acids (aa) in size and has a molecular mass of 4,169 Da; peptide B, encoded by nlmB, is 49 aa in size and has a molecular mass of 4,826 Da. Both peptides derive from prepeptides with glycines at positions -2 and -1 relative to the processing site. Production of mutacins I and IV by UA140 appears to be regulated by different mechanisms under different physiological conditions. The significance of producing two mutacins by one strain under different conditions and the implication of this property in terms of the ecology of S. mutans in the oral cavity are discussed.
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.
As part of a series of longitudinal studies on the development of the indigenous microflora of the upper respiratory tract, the establishment of streptococci in the oral cavity and nasopharynx and IgA1 protease production by the early streptococcal flora was examined in 50 healthy Caucasian infants at the ages of 2, 6, 12, 18 and 24 months. In the oral cavity, streptococci were found in all infants on every sampling occasion, Streptococcus mitis biovar 1 being the main finding in each age group. S. salivarius and S. mitis biovar 2 reached their highest prevalence during the first year of life, whereas the prevalence of S. oralis and S. sanguis showed no significant increase before 12 months of age. Salivary streptococci mainly consisted of the above-mentioned species during the follow-up period. In contrast to the oral cavity, no stable colonisation pattern was observed for viridans streptococci in the nasopharynx. S. mitis biovar 1 and S. pneumoniae, a traditional respiratory pathogen, were the principal streptococcal species among nasopharyngeal isolates. IgA1 protease production by early streptococci was common in infancy. Among the oral streptococcal microflora, S. mitis biovar 1 (especially during the first year of life) and S. oralis and S. sanguis constituted the main species responsible for this enzyme activity. In the nasopharynx, IgA1 protease was produced by S. mitis biovar 1, S. oralis and S. pneumoniae. In conclusion, streptococcal colonisation differs in these two close habitats in the upper respiratory tract.
Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins (N-acetylmuramoyl-L-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, gamma-D-glutamyl-L-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze gamma-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage lambda. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.
Several streptococcal species are able to take up naked DNA from the environment and integrate it into their genomes by homologous recombination. This process is called natural transformation. In Streptococcus pneumoniae and related streptococcal species, competence for natural transformation is induced by a peptide pheromone through a quorum-sensing mechanism. Recently we showed that induction of the competent state initiates lysis and release of DNA from a subfraction of the bacterial population and that the efficiency of this process is influenced by cell density. Here we have further investigated the nature of this cell density-dependent release mechanism. Interestingly, we found that competence-induced pneumococci lysed competence-deficient cells of the same strain during cocultivation and that the efficiency of this heterolysis increased as the ratio of competent to noncompetent cells increased. Furthermore, our results indicate that the lysins made by competent pneumococci are not released into the growth medium. More likely, they are anchored to the surface of the competent cells by choline-binding domains and cause lysis of noncompetent pneumococci through cell-to-cell contact.
Members of the bacterial genus Streptococcus are responsible for causing a wide variety of infections in humans. Many Streptococci use quorum-sensing systems to regulate several physiological properties, including the ability to incorporate foreign DNA, tolerate acid, form biofilms, and become virulent. These quorum-sensing systems are primarily made of small soluble signal peptides that are detected by neighboring cells via a histidine kinase/response regulator pair.
Streptococcus pneumoniae secretes two different peptide pheromones used for intercellular communication. These peptides, which have completely unrelated
primary structures, activate two separate signal transduction pathways, ComABCDE and BlpABCSRH, which regulate natural genetic
transformation and bacteriocin production, respectively. Each signal transduction pathway contains a response regulator (ComE
and BlpR, respectively) that activates transcription of target genes by binding to similar, but not identical, imperfect direct
repeat motifs. In general the direct repeat binding sites are specific for one or the other of the two response regulators,
ensuring that competence development and bacteriocin production are regulated separately. However, in the present study we
show that the rate of transcription of an operon, encoding an ABC transporter of unknown function, can be stimulated by both
peptide pheromones. We also show that this cross-induction is due to a hybrid direct repeat motif that can respond to both
ComE and BlpR. To our knowledge this kind of convergent gene regulation by two separate two-component regulatory systems has
not been described before in bacteria.
Several studies have shown that nasopharyngeal sampling is more sensitive than oropharyngeal sampling for the detection of pneumococcal carriage in children. The data for adults are limited and conflicting. This study was part of a larger study of pneumococcal carriage on the Navajo and White Mountain Apache Reservation following a clinical trial of a seven-valent pneumococcal conjugate vaccine. Persons aged 18 years and older living in households with children enrolled in the vaccine trial were eligible. We collected both nasopharyngeal and oropharyngeal specimens by passing a flexible calcium alginate wire swab either nasally to the posterior nasopharynx or orally to the posterior oropharynx. Swabs were placed in skim milk-tryptone-glucose-glycerin medium and frozen at -70 degrees C. Pneumococcal isolation was performed by standard techniques. Analyses were based on specimens collected from 1,994 adults living in 1,054 households. Nasopharyngeal specimens (11.1%; 95% confidence interval [CI], 9.8 and 12.6%) were significantly more likely to grow pneumococci than were oropharyngeal specimens (5.8%; 95% CI, 4.8 to 6.9%) (P < 0.0001). Few persons had pneumococcal growth from both specimens (1.7%). Therefore, both tests together were more likely to identify pneumococcal carriage (15.2%; 95% CI, 13.7 to 16.9%) than either test alone. Although we found that nasopharyngeal sampling was more sensitive than oropharyngeal sampling, nasopharyngeal sampling alone would have underestimated the prevalence of pneumococcal carriage in this adult population. Sampling both sites may give more accurate results than sampling either site alone in studies of pneumococcal carriage in adults.
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.
Natural competence for genetic transformation is the best-characterized feature of the major human pathogen Streptococcus pneumoniae. Recent studies have shown the virulence of competence-deficient mutants to be attenuated, but the nature of the connection between competence and virulence remained unknown. Here we document the release, triggered by competent cells, of virulence factors (e.g., the cytolytic toxin pneumolysin) from noncompetent cells. This phenomenon, which we name allolysis, involves a previously undescribed bacteriocin system consisting of a two-peptide bacteriocin, CibAB, and its immunity factor, CibC; the major autolysin, LytA, and lysozyme, LytC; and a proposed new amidase, CbpD. We show that CibAB are absolutely required for allolysis, whereas LytA and LytC can be supplied either by the competent cells or by the targeted cells. We propose that allolysis constitutes a competence-programmed mechanism of predation of noncompetent cells, which benefits to the competent cells and contributes to virulence by coordinating the release of virulence factors.
• allolysis bacteriocin
• competence
• predation
• virulence
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.
Streptococcus pneumoniae is an important human pathogen that is able to take up naked DNA from the environment by a quorum-sensing-regulated process
called natural genetic transformation. This property enables members of this bacterial species to efficiently acquire new
properties that may increase their ability to survive and multiply in the human host. We have previously reported that induction
of the competent state in a liquid culture of Streptococcus pneumoniae triggers lysis of a subfraction of the bacterial population resulting in release of DNA. We have also proposed that such
competence-induced DNA release is an integral part of natural genetic transformation that has evolved to increase the efficiency
of gene transfer between pneumococci. In the present work, we have further elucidated the mechanism behind competence-induced
cell lysis by identifying a putative murein hydrolase, choline-binding protein D (CbpD), as a key component of this process.
By using real-time PCR to estimate the amount of extracellular DNA in competent relative to noncompetent cultures, we were
able to show that competence-induced cell lysis and DNA release are strongly attenuated in a cbpD mutant. Ectopic expression of CbpD in the presence or absence of other competence proteins revealed that CbpD is essentially
unable to cause cell lysis on its own but depends on at least one additional protein expressed during competence.
ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity.
The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The
1.75 Å crystal structure of the targeting domain shows an all-β fold similar to typical SH3s but with unique features. The
structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially
interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various
bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of
the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly
conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.
Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational
analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by
the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).
Sreptococcus mutans is the primary causative agent involved in dental caries in humans. Among important virulence factors of this pathogen, its ability to form and sustain a polysaccharide-encased biofilm (commonly called dental plaque) is vital not only to its survival and persistence in the oral cavity, but also for its pathogenicity as well. This chapter focuses on the S. mutans' biofilm phenotype and how this mode of growth is regulated by its density-dependent quorum sensing (QS) system primarily comprised of the Competence Stimulating Peptide (CSP) and the ComD/ComE two-component signal transduction system. In addition to biofilm formation, the CSP-mediated QS system in S. mutans also affects its acidogenicity, aciduricity, genetic transformation and bacteriocin production. Interestingly, it has also been discovered that these properties are optimally expressed in cells derived from a biofilm as opposed to a free-floating planktonic mode of growth. Hence, strategies targeting S. mutans' QS system to attenuate biofilm formation and/or virulence are currently being used to develop therapeutic or preventative measures against dental caries. Recently, it was discovered that the addition of CSP in large concentrations (relative to amounts used for normal competence development) resulted in growth arrest and eventual cell death, thus paving way for CSP-mediated targeted killing of S. mutans. In addition to the QS system, effects of other two-component signal transduction systems on the biofilm phenotype of S. mutans are also discussed.
Of the 13 two-component signal transduction systems (TCS) identified in Streptococcus pneumoniae, two, ComDE and CiaRH, are known to affect competence for natural genetic transformation. ComD and ComE act together with the comC-encoded competence-stimulating peptide (CSP) and with ComAB, the CSP-dedicated exporter, to co-ordinate activation of genes required for differentiation to competence. Several lines of evidence suggest that the CiaRH TCS and competence regulation are interconnected, including the observation that inactivation of the CiaR response regulator derepresses competence. However, the nature of the interconnection remains poorly understood. Interpretation of previous transcriptome analyses of ciaR mutants was complicated by competence derepression in the mutants. To circumvent this problem, we have used microarray analysis to investigate the transition from non-competence to competence in a comC-null wild-type strain and its ciaR derivative after the addition of CSP. This study increased the number of known CSP-induced genes from approximate to 47 to 105 and revealed approximate to 42 genes with reduced expression in competent cells. Induction of the CiaR regulon, as well as the entire HrcA and part of the CtsR stress response regulons, was observed in wild-type competent cells. Enhanced induction of stress response genes was detected in ciaR competent cells. In line with these observations, CSP was demonstrated to trigger growth arrest and stationary phase autolysis in ciaR cells. Taken together, these data strongly suggest that differentiation to competence imposes a temporary stress on cells, and that the CiaRH TCS is required for the cells to exit normally from the competent state.
Streptococcus pneumoniae has re-emerged as a major cause of morbidity and mortality throughout the world and its continuous increase in antimicrobial resistance is rapidly becoming a leading cause of concern for public health. This review is focussed on the analysis of recent insights on the study of capsular polysaccharide biosynthesis, and cell wall (murein) hydrolases, two fundamental pneumococcal virulence factors. Besides, we have also re-evaluated the molecular biology of the pneumococcal phage, their possible role in pathogenicity and in the shaping of natural populations of S. pneumoniae. Precise knowledge of the topics reviewed here should facilitate the rationale to move towards the design of alternative ways to combat pneumococcal disease.
In naturally-competent streptococci such as Streptococcus pneumoniae, expression of the late competence operons is regulated by ComX (σX), the competence-specific alternative σ factor. In this study, duplicate genes (comR1 and comR2) encoding the putative ComX homologue of the oral bacterium Streptococcus gordonii were identified. Like the identical twin comX loci of S. pneumoniae, both comR determinants are independently functional as well as responsive to the ComDE signal transduction system activated by competence-stimulating peptide. However, in contrast to the comX system, nucleotide sequence analyses in combination with in trans complementation studies with a comR null mutant demonstrate that the identical 83 bp tracts (Region I) located immediately upstream of the comR structural genes are insufficient to confer wild-type competence levels. Wild-type transformation levels required additional distal nonhomologous DNA segments (Region II). Our findings suggest that alternative regulatory elements, under overall control of the ComDE pathway, may influence expression of the comR loci.
Streptococcus pneumoniae has re-emerged as a major cause of morbidity and mortality throughout the world and its continuous increase in antimicrobial resistance is rapidly becoming a leading cause of concern for public health. This review is focussed on the analysis of recent insights on the study of capsular polysaccharide biosynthesis, and cell wall (murein) hydrolases, two fundamental pneumococcal virulence factors. Besides, we have also re-evaluated the molecular biology of the pneumococcal phage, their possible role in pathogenicity and in the shaping of natural populations of S. pneumoniae. Precise knowledge of the topics reviewed here should facilitate the rationale to move towards the design of alternative ways to combat pneumococcal disease.
Recently published taxonomic studies of viridans streptococci have resulted in several changes in the nomenclature and definition of oral streptococcal species. With this background, the ecology of streptococci in the oropharyngeal cavities was reinvestigated. The results based on the examination of 1426 streptococcal isolates confirmed and extended earlier findings. Apart from mature supragingival plaque, which contained a mixture of all orally encountered streptococci, each site showed a characteristic streptococcal flora. Initial dental plaque formation is primarily associated with Streptococcus sanguis, Streptococcus mitis biovar 1 and Streptococcus oralis. Our investigation showed that S. sanguis and S. mitis biovar 1 were the most prominent streptococci, also on buccal mucosa. In contrast, S. oralis was almost exclusively found in initial dental plaque. Streptococcus gordonii, formerly part of S. sanguis, was found in small numbers on the oropharyngeal mucosa and in mature supragingival plaque. The dorsum of the tongue was dominated by S. mitis biovar 2 and Streptococcus salivarius, the latter of which was predominant also on the pharyngeal mucosa. Streptococcus anginosus was by far the most predominant streptococcus in subgingival plaque. Immunoglobulin A1 (IgA1) protease-producing streptococci were primarily isolated from initial dental plaque and from the buccal mucosa. This lends further support to the concept of IgA1 proteases being important for the ability of streptococci to evade the local immune defence during their initial colonization of certain oral surfaces.
SH3 domains mediate many important protein-protein interactions. The molecular basis of the binding of these domains to their ligands has been revealed, making it possible to identify SH3-binding sites in new proteins.
Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides containing N-terminal extensions (leader peptides) which are cleaved off during maturation. Most non-lantibiotics and also some lantibiotics have leader peptides of the so-called double-glycine type. These leader peptides share consensus sequences and also a common processing site with two conserved glycine residues in positions -1 and -2. The double-glycine-type leader peptides are unrelated to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the sec pathway. Their processing sites are also different from typical signal peptidase cleavage sites, suggesting that a different processing enzyme is involved. Peptide bacteriocins are exported across the cytoplasmic membrane by a dedicated ATP-binding cassette (ABC) transporter. Here we show that the ABC transporter is the maturation protease and that its proteolytic domain resides in the N-terminal part of the protein. This result demonstrates that the ABC transporter has a dual function: (i) removal of the leader peptide from its substrate, and (ii) translocation of its substrate across the cytoplasmic membrane. This represents a novel strategy for secretion of bacterial proteins.
Colicin V is a ribosomally synthesized antimicrobial peptide produced by Escherichia coli. Four recently characterized genes, arranged in two convergent operons on the plasmid pCoIV-K30, are required for colicin V synthesis, export and immunity. We report the purification and N-terminal amino acid sequencing of the colicin V protein. Our results demonstrate that the colicin V primary translation product, which consists of 103 amino acids, is proteolytically processed. A leader peptide, consisting of 15 amino acid residues, is removed from the N-terminus during maturation of colicin V. This leader peptide is not related to the N-terminal signal sequences which direct proteins across the cytoplasmic membrane via the Sec pathway. The molecular mass of colicin V, obtained by mass spectrometry analysis, showed that the peptide consists of only unmodified amino acids. The deduced amino acid sequence of the leader peptide was highly homologous to the N-terminal extensions found in non-lantibiotic, peptide bacteriocins produced by Gram-positive bacteria. These findings strongly indicate that colicin V belongs to a family of small peptide bacteriocins that have been found previously only among the Gram-positive lactic acid bacteria.
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Bacteriophage muralytic enzymes degrade the cell wall envelope of staphylococci to release phage particles from the bacterial
cytoplasm. Murein hydrolases of staphylococcal phages φ11, 80α, 187, Twort, and φPVL harbor a central domain that displays
sequence homology to knownN-acetylmuramyl-l-alanyl amidases; however, their precise cleavage sites on the staphylococcal peptidoglycan have thus far not been determined.
Here we examined the properties of the φ11 enzyme to hydrolyze either the staphylococcal cell wall or purified cell wall anchor
structures attached to surface protein. Our results show that the φ11 enzyme has d-alanyl-glycyl endopeptidase as well asN-acetylmuramyl-l-alanyl amidase activity. Analysis of a deletion mutant lacking the amidase-homologous sequence, φ11(Δ181–381), revealed that
the d-alanyl-glycyl endopeptidase activity is contained within the N-terminal 180 amino acid residues of the polypeptide chain.
Sequences similar to this N-terminal domain are found in the murein hydrolases of staphylococcal phages but not in those of
phages that infect other Gram-positive bacteria such as Listeria or Bacillus.
The kinetics of global changes in transcription patterns during competence development in Streptococcus pneumoniae was analysed with high-density arrays. Four thousand three hundred and one clones of a S. pneumoniae library, covering almost the entire genome, were amplified by PCR and gridded at high density onto nylon membranes. Competence was induced by the addition of CSP (competence stimulating peptide) to S. pneumoniae cultures grown to the early exponential phase. RNA was extracted from samples at 5 min intervals (for a period of 30 min) after the addition of CSP. Radiolabelled cDNA was generated from isolated total RNA by random priming and the probes were hybridized to identical high density arrays. Genes whose transcription was induced or repressed during competence were identified. Most of the genes previously known to be competence induced were detected together with several novel genes that all displayed the characteristic transient kinetics of competence-induced genes. Among the newly identified genes many have suggested functions compatible with roles in genetic transformation. Some of them may represent new members of the early or late competence regulons showing competence specific consensus sequences in their promoter regions. Northern experiments and mutational analysis were used to confirm some of the results.
The peptide SpiP of Streptococcus pneumoniae regulates the induction of a complex signal transduction system spiR1spiR2spiH. Distinct alleles of spiP and the receptor histidine protein kinase gene spiH were recognized in different pneumococcal clones. The spi system in strain KNR7/87 is adjacent to a bacteriocin gene cluster encoding putative double glycine-type bacteriocins, immunity proteins, and translocator proteins. A direct repeat element upstream of the spiR1 promoter and another three potential transcription start sites within the bacteriocin cluster indicate that SpiP functions as an inducing peptide for bacteriocin synthesis in S. pneumoniae.
Bacteria, which often are subjected to fluctuations in nutrients, temperature, radiation, pH, etc., adapt to the physico-chemical environment they live in by making the appropriate changes in their gene expression patterns. During the last decades it has become increasingly clear that bacteria, in addition, have a "social life", and that changes in gene expression can also be elicited by the presence of other bacteria. Traditionally bacteria have been viewed as solitary organisms that in general do not interact with other bacteria in a coordinated manner. Recent advances in the field of bacterial cell-to-cell communication has proved this to be a misconception, and mounting evidence now show that bacterial group behaviour is ubiquitous in nature. Competence for natural genetic transformation in Streptococcus pneumoniae, which has been studied for more than seventy years, has become a paradigm for intercellular communication and cell density dependent regulation of gene expression in Gram-positive bacteria. There has been rapid progress recently in elucidating the molecular mechanisms behind regulation of natural competence in S. pneumoniae. In this review, we describe the current status of our knowledge of natural competence in this bacterium, with particular emphasis on the early phase of competence induction.
Bacteriocins from lactic acid bacteria are ribosomally produced peptides (usually 30-60 amino acids) that display potent antimicrobial activity against certain other Gram-positive organisms. They function by disruption of the membrane of their targets, mediated in at least some cases by interaction of the peptide with a chiral receptor molecule (e.g., lipid II or sugar PTS proteins). Some bacteriocins are unmodified (except for disulfide bridges), whereas others (i.e. lantibiotics) possess extensive post-translational modifications which include multiple monosulfide (lanthionine) bridges and dehydro amino acids as well as possible keto amide residues at the N-terminus. Most known bacteriocins are biologically active as single peptides. However, there is a growing class of two peptide systems, both unmodified and lantibiotic, which are fully active only when both partners are present (usually 1:1). In some cases, neither peptide has activity by itself, whereas in others, the activity of one is enhanced by the other. This review discusses the classification, structure, production, regulation, biological activity, and potential applications of such two-peptide bacteriocins.
An exhaustive review published ten years ago reported natural genetic transformation, a potential mechanism for intra- and interspecies gene transfer, in approximately 40 species belonging to different taxonomic and trophic groups. Since then, considerable progress has been made in characterizing DNA-uptake machineries and regulatory circuits controlling their expression in cells competent for genetic transformation. In this article, in light of the recent description of a Group A streptococcal isolate capable of DNA transfer in mixed cultures, we discuss whether the detection in completely sequenced microbial genomes of intact homologues of key competence-regulatory and/or DNA-uptake proteins enables the prediction of new transformable species.
Cleavage of peptidoglycan plays an important role in bacterial cell division, cell growth and cell lysis. Here, we reveal that several known peptidoglycan amidases fall into a family, which includes many proteins of previously unknown function. The family includes two different peptidoglycan cleavage activities: L-muramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity. The family includes the amidase portion of the bifunctional glutathionylspermidine synthase/amidase enzyme from bacteria and pathogenic trypanosomes. The glutathionylspermidine synthase is thought to be a key component of the alternative pathway in trypanosomes for protection from oxygen-radical damage and has been proposed as a potential drug target. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain is often found in association with other domains that cleave peptidoglycan. The large number of multifunctional hydrolases suggests that they might act in a cooperative manner to cleave specialized substrates.
Of the 13 two-component signal transduction systems (TCS) identified in Streptococcus pneumoniae, two, ComDE and CiaRH, are known to affect competence for natural genetic transformation. ComD and ComE act together with the comC-encoded competence-stimulating peptide (CSP) and with ComAB, the CSP-dedicated exporter, to co-ordinate activation of genes required for differentiation to competence. Several lines of evidence suggest that the CiaRH TCS and competence regulation are interconnected, including the observation that inactivation of the CiaR response regulator derepresses competence. However, the nature of the interconnection remains poorly understood. Interpretation of previous transcriptome analyses of ciaR mutants was complicated by competence derepression in the mutants. To circumvent this problem, we have used microarray analysis to investigate the transition from non-competence to competence in a comC-null wild-type strain and its ciaR derivative after the addition of CSP. This study increased the number of known CSP-induced genes from approximately 47 to 105 and revealed approximately 42 genes with reduced expression in competent cells. Induction of the CiaR regulon, as well as the entire HrcA and part of the CtsR stress response regulons, was observed in wild-type competent cells. Enhanced induction of stress response genes was detected in ciaR competent cells. In line with these observations, CSP was demonstrated to trigger growth arrest and stationary phase autolysis in ciaR cells. Taken together, these data strongly suggest that differentiation to competence imposes a temporary stress on cells, and that the CiaRH TCS is required for the cells to exit normally from the competent state.
Natural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum-sensing system mediated by a peptide pheromone called competence-stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX, which encodes an alternative sigma factor. Late genes were dependent on ComX for CSP-induced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP-responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP-inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation.
The release of chromosomal DNA into culture media has been reported for several naturally transformable bacterial species, but a direct link between competence development and the liberation of DNA is generally lacking. Based on the analysis of strains with mutations in competence-regulatory genes and the use of conditions favouring or preventing competence, we provide evidence that DNA release is triggered by the induction of competence in Streptococcus pneumoniae. Kinetic analyses revealed that whereas competence was maximal 20 min after addition of competence-stimulating peptide, and then decreased, the amount of liberated DNA continued to increase and reached a maximum in stationary phase, when cells are no longer competent for DNA uptake. These data are not consistent with the proposal that release of DNA by a fraction of the population is coordinated with uptake by the remainder. Moreover, we observed that an unidentified DNase was specifically induced or released in competent cultures, and that together with the major pneumococcal endonuclease, EndA, it could degrade released DNA. Nearby complete abolition of release in a mutant lacking both the major autolysin, LytA, and the autolytic lysozyme, LytC, indicated that DNA liberation occurs by LytA-LytC-dependent cell lysis. These observations suggest that competence-dependent DNA release is one facet of a more general phenomenon of sensitization to autolysis that reaches its maximum in stationary phase.
It is important to ensure DNA availability when bacterial cells develop competence. Previous studies in Streptococcus pneumoniae demonstrated that the competence-stimulating peptide (CSP) induced autolysin production and cell lysis of its own non-competent cells, suggesting a possible active mechanism to secure a homologous DNA pool for uptake and recombination. In this study, we found that in Streptococcus mutans CSP induced co-ordinated expression of competence and mutacin production genes. This mutacin (mutacin IV) is a non-lantibiotic bacteriocin which kills closely related Streptococcal species such as S. gordonii. In mixed cultures of S. mutans and S. gordonii harbouring a shuttle plasmid, plasmid DNA transfer from S. gordonii to S. mutans was observed in a CSP and mutacin IV-dependent manner. Further analysis demonstrated an increased DNA release from S. gordonii upon addition of the partially purified mutacin IV extract. On the basis of these findings, we propose that Streptococcus mutans, which resides in a multispecies oral biofilm, may utilize the competence-induced bacteriocin production to acquire transforming DNA from other species living in the same ecological niche. This hypothesis is also consistent with a well-known phenomenon that a large genomic diversity exists among different S. mutans strains. This diversity may have resulted from extensive horizontal gene transfer.
Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon which offers food scientists the possibility of directing or preventing the development of specific bacterial species in food. This can be particularly useful in preservation or food safety applications, but also has implications for the development of desirable flora in fermented food. In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to foodstuffs, helping processors extend their control over the food flora long after manufacture.
In 1971, Tomasz and Zanati discovered that competent pneumococci have a tendency to form aggregates when pelleted by centrifugation and resuspended in 0.01 N HCl by brief vortexing. Interestingly, no clumping was observed with parallel cultures of non-competent cells treated in the same way. We set out to elucidate the mechanism behind this striking phenomenon, and were able to show that it depends on extracellular DNA that is presumably released by so-called competence-induced cell lysis. Competence-induced cell lysis, which was first described a few years ago, seems to rely on the concerted action of several murein hydrolases. Our results confirmed and extended previous findings by showing that competence-induced aggregation is abolished in a lytA-lytC double mutant, and absolutely requires CbpD and its N-terminal CHAP amidase domain. Furthermore, we discovered a novel competence stimulating peptide (CSP)-induced immunity protein, encoded by the early competence gene comM (spr1762), which protects competent pneumococci against their own lysins. Together, the murein hydrolases and the immunity protein constitutes a CSP-controlled mechanism that allows competent pneumococci to commit fratricide by killing non-competent pneumococci sharing the same ecological niche. Through such predatory behaviour, pneumococci can get access to transforming DNA and nutrients, promote the release of virulence factors, and at the same time get rid of competitors.
The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.
Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.
Bacterial transformation, a programmed mechanism for genetic exchange originally discovered in Streptococcus pneumoniae, is widespread in bacteria. It is based on the uptake and integration of exogenous DNA into the recipient genome. This review examines whether induction of competence for genetic transformation is a general response to stress in gram-positive bacteria. It compares data obtained with bacteria chosen for their different lifestyles, the soil-dweller Bacillus subtilis and the major human pathogen S. pneumoniae. The review focuses on the relationship between competence and other global responses in B. subtilis, as well as on recent evidence for competence induction in response to DNA damage or antibiotics and for the ability of S. pneumoniae to use competence as a substitute for SOS. This comparison reveals that the two species use different fitness-enhancing strategies in response to stress conditions. Whereas B. subtilis combines competence and SOS induction, S. pneumoniae relies only on competence to generate genetic diversity through transformation.