Transformation In Streptococcus Research Articles

ComCDE (Competence) Operon Is Regulated by CcpA in Streptococcus pneumoniae D39.

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.

Mechanism of high-frequent horizontal gene transfer in Gram positive bacterial pathogens

Horizontal gene transfer through transconjugation and natural transformation plays a major role in the spread of antimicrobial resistance. Although the phenomenon of genetic element transmission has long been known, the rapid increase in the number of antimicrobial resistant bacteria in recent years and the accompanying accumulation of genomic information have revealed that horizontal gene transfer contributes to genome plasticity in various ways. The author reported the molecular mechanism of the antimicrobial activity of the accessory factor bacteriocin encoded by the junctional transfer plasmid of Enterococcus faecalis, a representative Gram-positive opportunistic pathogen that is concerned as highly antimicrobial resistant, and found diversity in the selfimmune system based on epidemiological studies. In addition, the author established a technique to visualize and quantify genomic recombination by natural transformation in Streptococcus pneumoniae which is also one of the most concerns for antimicrobial resistance and vaccine escape, at single cells level resolution in real time. Focuses on outcome from these research, this paper introduces the molecular mechanisms that promote horizontal gene transmission and the prospects for their technological application.

Coevolution of the bacterial pheromone ComS and sensor ComR fine-tunes natural transformation in streptococci

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic–aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

Antibiotics alter the window of competence for natural transformation in streptococci.

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 investigate the effect of antibiotic subinhibitory concentrations on sigX expression using a luciferase reporter, and correlate 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 nonsignificant effect on transformation, whereas streptomycin and the β-lactam ampicillin, inhibited both sigX expression and transformation. 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 do 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.

A programmed cell division delay preserves genome integrity during natural genetic transformation in Streptococcus pneumoniae

Competence for genetic transformation is a differentiation program during which exogenous DNA is imported into the cell and integrated into the chromosome. In Streptococcus pneumoniae, competence develops transiently and synchronously in all cells during exponential phase, and is accompanied by a pause in growth. Here, we reveal that this pause is linked to the cell cycle. At least two parallel pathways impair peptidoglycan synthesis in competent cells. Single-cell analyses demonstrate that ComM, a membrane protein induced during competence, inhibits both initiation of cell division and final constriction of the cytokinetic ring. Competence also interferes with the activity of the serine/threonine kinase StkP, the central regulator of pneumococcal cell division. We further present evidence that the ComM-mediated delay in division preserves genomic integrity during transformation. We propose that cell division arrest is programmed in competent pneumococcal cells to ensure that transformation is complete before resumption of cell division, to provide this pathogen with the maximum potential for genetic diversity and adaptation.

Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS.

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence.

Natural genetic transformation of Streptococcus pneumoniae, an important human pathogen, mediates horizontal gene transfer for the development of drug resistance, modulation of carriage and virulence traits, and evasion of host immunity. Transformation frequency differs greatly among pneumococcal clinical isolates, but the molecular basis and biological importance of this interstrain variability remain unclear. In this study, we characterized the transformation frequency and other associated phenotypes of 208 S. pneumoniae clinical isolates representing at least 30 serotypes. While the vast majority of these isolates (94.7%) were transformable, the transformation frequency differed by up to 5 orders of magnitude between the least and most transformable isolates. The strain-to-strain differences in transformation frequency were observed among many isolates producing the same capsule types, indicating no general association between transformation frequency and serotype. However, a statistically significant association was observed between the levels of transformation and colonization fitness/virulence in the hypertransformable isolates. Although nontransformable mutants of all the selected hypertransformable isolates were significantly attenuated in colonization fitness and virulence in mouse infection models, such mutants of the strains with relatively low transformability had no or marginal fitness phenotypes under the same experimental settings. This finding strongly suggests that the pneumococci with high transformation capability are "addicted" to a "hypertransformable" state for optimal fitness in the human host. This work has thus provided an intriguing hint for further investigation into how the competence system impacts the fitness, virulence, and other transformation-associated traits of this important human pathogen.

Regulation of competence for natural transformation in streptococci

Natural DNA transformation is a lateral gene transfer mechanism during which bacteria take up naked DNA from their environment and stably integrate it in their genome. The proteins required for this process are conserved between species and are produced during a specific physiological state known as competence. Although natural transformation drives genome plasticity and adaptability, it is also likely to cause deleterious effects in the chromosome of the recipient bacteria and negatively impact cell growth. The competence window is thus generally tightly regulated in response to species-specific environmental conditions and limited to a proportion of the cell population. In streptococci species, the entry into competence is dictated by the amount of the competence sigma factor σX, the master regulator of natural transformation in those species. The Streptococcus genus includes 7 phylogenetic groups that have evolved different regulatory circuits to govern natural transformation. Here, we review the current knowledge on transcriptional and post-transcriptional mechanisms that control the activity of σX at the whole population and the single-cell level, with an emphasis on growth conditions that modulate their activation. Recent findings regarding competence regulation by the ComCDE and ComRS cell–cell signalling pathways and the Clp proteolytic system are specifically highlighted.

Competence for genetic transformation in Streptococcus pneumoniae: mutations in σA bypass the comW requirement.

Competence for genetic transformation in the genus Streptococcus depends on an alternative sigma factor, σ(X), for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of σ(X) is coordinated between cells by transcription factors linked to peptide pheromone signals. In Streptococcus pneumoniae, elevated σ(X) is insufficient for development of full competence without coexpression of a second competence-specific protein, ComW. ComW, shared by eight species in the Streptococcus mitis and Streptococcus anginosus groups, is regulated by the same pheromone circuit that controls σ(X), but its role in expression of the σ(X) regulon is unknown. Using the strong, but not absolute, dependence of transformation on comW as a selective tool, we collected 27 independent comW bypass mutations and mapped them to 10 single-base transitions, all within rpoD, encoding the primary sigma factor subunit of RNA polymerase, σ(A). Eight mapped to sites in rpoD region 4 that are implicated in interaction with the core β subunit, indicating that ComW may act to facilitate competition of the alternative sigma factor σ(X) for access to core polymerase.

Conservative Sex and the Benefits of Transformation in Streptococcus pneumoniae

Natural transformation has significant effects on bacterial genome evolution, but the evolutionary factors maintaining this mode of bacterial sex remain uncertain. Transformation is hypothesized to have both positive and negative evolutionary effects on bacteria. It can facilitate adaptation by combining beneficial mutations into a single individual, or reduce the mutational load by exposing deleterious alleles to natural selection. Alternatively, it may expose transformed cells to damaged or otherwise mutated environmental DNA and is energetically expensive. Here, we examine the long-term effects of transformation in the naturally competent species Streptococcus pneumoniae by evolving populations of wild-type and competence-deficient strains in chemostats for 1000 generations. Half of these populations were exposed to periodic mild stress to examine context-dependent benefits of transformation. We find that competence reduces fitness gain under benign conditions; however, these costs are reduced in the presence of periodic stress. Using whole genome re-sequencing, we show that competent populations fix fewer new mutations and that competence prevents the emergence of mutators. Our results show that during evolution in benign conditions competence helps maintain genome stability but is evolutionary costly; however, during periods of stress this same conservativism enables cells to retain fitness in the face of new mutations, showing for the first time that the benefits of transformation are context dependent.

Competence in Streptococcus pneumoniae Is a Response to an Increasing Mutational Burden

Competence for genetic transformation in Streptococcus pneumoniae has previously been described as a quorum-sensing trait regulated by a secreted peptide pheromone. Recently we demonstrated that competence is also activated by reduction in the accuracy of protein biosynthesis. We have now investigated whether errors upstream of translation in the form of random genomic mutations can provide a similar stimulus. Here we show that generation of a mutator phenotype in S. pneumoniae through deletions of mutX, hexA or hexB enhanced the expression of competence. Similarly, chemical mutagenesis with the nucleotide analog dPTP promoted development of competence. To investigate the relationship between mutational load and the activation of competence, replicate lineages of the mutX strain were serially passaged under conditions of relaxed selection allowing random accumulation of secondary mutations. Competence increased with propagation in these lineages but not in control lineages having wild-type mutX. Resequencing of these derived strains revealed between 1 and 9 single nucleotide polymorphisms (SNPs) per lineage, which were broadly distributed across the genome and did not involve known regulators of competence. Notably, the frequency of competence development among the sequenced strains correlated significantly with the number of nonsynonymous mutations that had been acquired. Together, these observations provide support for the hypothesis that competence in S. pneumoniae is regulated in response to the accumulated burden of coding mutations in the bacterial genome. In contrast to previously described DNA damage response systems that are activated by physical lesions in the chromosome, this pneumococcal pathway may represent a unique stress response system that monitors the coding integrity of the genome.

A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

Exit from Competence for Genetic Transformation in Streptococcus pneumoniae Is Regulated at Multiple Levels

Development of natural competence in S. pneumoniae entails coordinated expression of two sets of genes. Early gene expression depends on ComE, a response regulator activated by the pheromone CSP (Competence-Stimulating-Peptide). Subsequently, an early gene product (the alternative sigma factor ComX) activates expression of late genes, establishing the competent state. Expression of both sets of genes is transient, rapidly shut off by a mechanism that depends on the late gene, dprA. It has been thought that the rapid shutoff of late gene expression is the combined result of auto-inhibition of ComE and the instability of ComX. However, this explanation seems incomplete, because of evidence for ComX-dependent repressor(s) that might also be important for shutting off the response to CSP and identifying dprA as such a gene. We screened individual late gene mutants to investigate further the roles of ComX-dependent genes in competence termination. A ΔdprA mutant displayed a prolonged late gene expression pattern, whereas mutants lacking cbpD, cibABC, cglEFG, coiA, ssbB, celAB, cclA, cglABCD, cflAB, or radA, exhibited a wild-type temporal expression pattern. Thus, no other gene than dprA was found to be involved in shutoff. DprA limits the amounts of ComX and another early gene product, ComW, by restriction of early gene expression rather than by promoting proteolysis. To ask if DprA also affects late gene expression, we decoupled late gene expression from early gene regulation. Because DprA did not limit ComX activity under these conditions, we also conclude that ComX activity is limited by another mechanism not involving DprA.

Low Species Barriers in Halophilic Archaea and the Formation of Recombinant Hybrids

Speciation of sexually reproducing organisms requires reproductive barriers. Prokaryotes reproduce asexually butoften exchange DNA by lateral gene transfer mechanisms and recombination [1], yet distinct lineages are still observed. Thus, barriers to gene flow such as geographic isolation, genetic incompatibility or a physiological inability to transfer DNA represent potential underlying mechanisms behind preferred exchange groups observed in prokaryotes [2-6]. In Bacteria, experimental evidence showed that sequence divergence impedes homologous recombination between bacterial species [7-11]. Here we study interspecies gene exchange in halophilic archaea that possess a parasexual mechanism of genetic exchange that is functional between species [12, 13]. In this process, cells fuse forming a diploid state containing the full genetic repertoire of both parental cells, which facilitates genetic exchange and recombination. Later, cells separate, occasionally resulting in hybrids of the parental strains [14]. We show high recombination frequencies between Haloferax volcanii and Haloferax mediterranei, two species that have an average nucleotide sequence identity of 86.6%. Whole genome sequencing of Haloferax interspecies hybrids revealed the exchange of chromosomal fragments ranging from 310Kb to 530Kb. These results show that recombination barriers may be more permissive in halophilic archaea than they are in bacteria.

Competence in Streptococcus pneumoniae Is Regulated by the Rate of Ribosomal Decoding Errors

ABSTRACTCompetence for genetic transformation in Streptococcus pneumoniae develops in response to accumulation of a secreted peptide pheromone and was one of the initial examples of bacterial quorum sensing. Activation of this signaling system induces not only expression of the proteins required for transformation but also the production of cellular chaperones and proteases. We have shown here that activity of this pathway is sensitively responsive to changes in the accuracy of protein synthesis that are triggered by either mutations in ribosomal proteins or exposure to antibiotics. Increasing the error rate during ribosomal decoding promoted competence, while reducing the error rate below the baseline level repressed the development of both spontaneous and antibiotic-induced competence. This pattern of regulation was promoted by the bacterial HtrA serine protease. Analysis of strains with the htrA (S234A) catalytic site mutation showed that the proteolytic activity of HtrA selectively repressed competence when translational fidelity was high but not when accuracy was low. These findings redefine the pneumococcal competence pathway as a response to errors during protein synthesis. This response has the capacity to address the immediate challenge of misfolded proteins through production of chaperones and proteases and may also be able to address, through genetic exchange, upstream coding errors that cause intrinsic protein folding defects. The competence pathway may thereby represent a strategy for dealing with lesions that impair proper protein coding and for maintaining the coding integrity of the genome.

Peptide-Regulated Gene Depletion System Developed for Use in Streptococcus pneumoniae

To facilitate the study of pneumococcal genes that are essential for viability or normal cell growth, we sought to develop a tightly regulated, titratable gene depletion system that interferes minimally with normal cellular functions. A possible candidate for such a system is the recently discovered signal transduction pathway regulating competence for natural transformation in Streptococcus thermophilus. This pathway, which is unrelated to the ComCDE pathway used for competence regulation in Streptococcus pneumoniae, has not been fully elucidated, but it is known to include a short unmodified signaling peptide, ComS*, an oligopeptide transport system, Ami, and a transcriptional activator, ComR. The transcriptional activator is thought to bind to an inverted repeat sequence termed the ECom box. We introduced the ComR protein and the ECom box into the genome of S. pneumoniae R6 and demonstrated that addition of synthetic ComS* peptide induced the transcription of a luciferase gene inserted downstream of the ECom box. To determine whether the ComRS system could be used for gene depletion studies, the licD1 gene was inserted behind the chromosomally located ECom box promoter by using the Janus cassette. Then, the native versions of licD1 and licD2 were deleted, and the resulting mutant was recovered in the presence of ComS*. Cultivation of the licD1 licD2 double mutant in the absence of ComS* gradually affected its ability to grow and propagate, demonstrating that the ComRS system functions as intended. In the present study, the ComRS system was developed for use in S. pneumoniae. In principle, however, it should work equally well in many other Gram-positive species.

Competence for Genetic Transformation in Streptococcus pneumoniae : Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW

Competence for genetic transformation in Streptococcus pneumoniae is a transient physiological state whose development is coordinated by a peptide pheromone (CSP) and its receptor, which activates transcription of two downstream genes, comX and comW, and 15 other "early" genes. ComX, a transient alternative sigma factor, drives transcription of "late" genes, many of which are essential for transformation. In vivo, ComW both stabilizes ComX against proteolysis by the ClpE-ClpP protease and stimulates its activity. Interestingly, stabilization of ComX by deletion of the gene encoding the ClpP protease did not extend the period of competence. We considered the hypothesis that the rapid decay of competence arises from a rapid loss of ComW and thus of its ComX stimulating activity, so that ComX might persist but lose its transcriptional activity. Western analysis revealed that ComW is indeed a transient protein, which is also stabilized by deletion of the gene encoding the ClpP protease. However, stabilizing both ComX and ComW did not prolong either ComX activity or the period of transformation, indicating that termination of the transcriptional activity of ComX is not dependent on proteolysis of ComW.

RadC, a Misleading Name?

The pfam04002 annotation describes RadC as a bacterial DNA repair protein. Although the radC gene is expressed specifically during competence for genetic transformation in Streptococcus pneumoniae, we report that radC mutants exhibit normal uptake and processing of transforming DNA. They also display normal sensitivity to DNA-damaging agents, providing no support for the rad epithet.

Competence‐induced fratricide in streptococci

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.

Transformation in Streptococcus pneumoniae: formation of eclipse complex in a coiA mutant implicates CoiA in genetic recombination

CoiA is a transient protein expressed specifically during competence and required for genetic transformation in Streptococcus pneumoniae, but not for DNA uptake. It is widely conserved among Gram-positive bacteria but its function is unknown. Here we report that although the rate of DNA uptake was not affected in a coiA mutant, the internalized donor DNA did not recombine into the host chromosome to form a physical and genetic heteroduplex. Instead, DNA taken up by a coiA mutant accumulated in the form of a single-stranded (ss) DNA-protein complex indistinguishable from the eclipse complex formed as a recombination intermediate in wild-type competent cells. Internalized donor DNA in a dprA mutant did not accumulate either as ss DNA or as an eclipse complex. Together, these results establish that a coiA mutant exhibits a phenotype different from that of dprA or recA mutants, and that CoiA functions at a later step in promoting recombination during genetic transformation in Streptococcus pneumoniae.