Dynamic Modeling of Streptococcus pneumoniae Competence Provides Regulatory Mechanistic Insights Into Its Tight Temporal Regulation.
ABSTRACT: 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.
Project description:Natural bacterial transformation is a genetically programmed process allowing genotype alterations that involves the internalization of DNA and its chromosomal integration catalyzed by the universal recombinase RecA, assisted by its transformation-dedicated loader, DNA processing protein A (DprA). In Streptococcus pneumoniae, the ability to internalize DNA, known as competence, is transient, developing suddenly and stopping as quickly. Competence is induced by the comC-encoded peptide, competence stimulating peptide (CSP), via a classic two-component regulatory system ComDE. Upon CSP binding, ComD phosphorylates the ComE response-regulator, which then activates transcription of comCDE and the competence-specific ?(X), leading to a sudden rise in CSP levels and rendering all cells in a culture competent. However, how competence stops has remained unknown. We report that DprA, under ?(X) control, interacts with ComE?P to block ComE-driven transcription, chiefly impacting ?(X) production. Mutations of dprA specifically disrupting interaction with ComE were isolated and shown to map mainly to the N-terminal domain of DprA. Wild-type DprA but not ComE interaction mutants affected in vitro binding of ComE to its promoter targets. Once introduced at the dprA chromosomal locus, mutations disrupting DprA interaction with ComE altered competence shut-off. The absence of DprA was found to negatively impact growth following competence induction, highlighting the importance of DprA for pneumococcal physiology. DprA has thus two key roles: ensuring production of transformants via interaction with RecA and competence shut-off via interaction with ComE, avoiding physiologically detrimental consequences of prolonged competence. Finally, phylogenetic analyses revealed that the acquisition of a new function by DprA impacted its evolution in streptococci relying on ComE to regulate comX expression.
Project description:Competence for genetic transformation in Streptococcus pneumoniae is coordinated by the competence-stimulating peptide (CSP), which induces a sudden and transient appearance of competence during exponential growth in vitro. Models of this quorum-sensing mechanism have proposed sequential expression of several regulatory genes followed by induction of target genes encoding DNA-processing-pathway proteins. Although many genes required for transformation are known to be expressed only in response to CSP, the relative timing of their expression has not been established. Overlapping expression patterns for the genes cinA and comD (G. Alloing, B. Martin, C. Granadel, and J. P. Claverys, Mol. Microbiol. 29:75-83, 1998) suggest that at least two distinct regulatory mechanisms may underlie the competence cycle. DNA microarrays were used to estimate mRNA levels for all known competence operons during induction of competence by CSP. The known competence regulatory operons, comAB, comCDE, and comX, exhibited a low or zero initial (uninduced) signal, strongly increased expression during the period between 5 and 12 min after CSP addition, and a decrease nearly to original values by 15 min after initiation of exposure to CSP. The remaining competence genes displayed a similar expression pattern, but with an additional delay of approximately 5 min. In a mutant defective in ComX, which may act as an alternate sigma factor to allow expression of the target competence genes, the same regulatory genes were induced, but the other competence genes were not. Finally, examination of the expression of 60 candidate sites not previously associated with competence identified eight additional loci that could be induced by CSP.
Project description:Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.
Project description:Competence is a widespread bacterial differentiation program driving antibiotic resistance and virulence in many pathogens. Here, we studied the spatiotemporal localization dynamics of the key regulators that master the two intertwined and transient transcription waves defining competence in Streptococcus pneumoniae. The first wave relies on the stress-inducible phosphorelay between ComD and ComE proteins, and the second on the alternative sigma factor ?X, which directs the expression of the DprA protein that turns off competence through interaction with phosphorylated ComE. We found that ComD, ?X and DprA stably co-localize at one pole in competent cells, with ?X physically conveying DprA next to ComD. Through this polar DprA targeting function, ?X mediates the timely shut-off of the pneumococcal competence cycle, preserving cell fitness. Altogether, this study unveils an unprecedented role for a transcription ? factor in spatially coordinating the negative feedback loop of its own genetic circuit.
Project description:Pneumococcal bacteriocins (pneumocins) are antibacterial toxins that mediate intra-species competition within the human host. However, the triggers of pneumocin expression are poorly understood. Using RNA-sequencing, we mapped the regulon of the pneumocin cluster (blp) of Streptococcus pneumoniae D39. Furthermore, by analogy with pneumococcal competence, we show that several antibiotics activate the blp-genes. Using real-time gene expression measurements we show that while the promoter driving expression of the two-component regulatory system blpR/H is constitutive, the remaining blp-promoters that control pneumocin expression, immunity and the inducer peptide BlpC, are pH-dependent and induced in the late exponential phase. Intriguingly, competence for genetic transformation, mediated by the paralogous ComD/E two-component quorum system, is induced by the same environmental cues. To test for interplay between these regulatory systems, we quantified the regulatory response to the addition of synthetic BlpC and competence-stimulating peptide (CSP). Supporting the idea of such interplay, we found that immediately upon addition of CSP, the blp-promoters were activated in a comD/E-dependent manner. After a delay, blp-expression was highly induced and was strictly dependent on blpRH and blpC. This raised the question of the mechanism of BlpC export, since bioinformatic analysis showed that the genes encoding the putative exporter for BlpC, blpAB, are not intact in strain D39 and most other strains. By contrast, all sequenced pneumococcal strains contain intact comAB genes, encoding the transport system for CSP. Consistent with the idea that comAB mediate BlpC export, we finally show that high-level expression of the blp-genes requires comAB. Together, our results demonstrate that regulation of pneumocin expression is intertwined with competence, explaining why certain antibiotics induce blp-expression. Antibiotic-induced pneumocin expression might therefore have unpredictable consequences on pneumococcal colonization dynamics by activating genes that mediate intra-specific interference competition.
Project description:In Streptococcus mutans, the alternative sigma factor ComX controls entry into genetic competence. Competence stimulating peptide (CSP) induces bimodal expression of comX, with only a fraction of the population becoming transformable. Curiously, the bimodality of comX is affected by peptides in the growth medium and by carbohydrate source. CSP elicits bimodal expression of comX in media rich in small peptides, but CSP elicits no response in defined media lacking small peptides. In addition, growth on certain sugars increases the proportion of the population that activates comX in response to CSP. By investigating the connection between media and comX bimodality, we find evidence for two mechanisms that modulate transcriptional positive feedback in the ComRS system, where comX bimodality originates. We find that the endopeptidase PepO suppresses the ComRS feedback loop, most likely by degrading the XIP/ComS feedback signal. Deletion of pepO eliminates comX bimodality, leading to a unimodal comX response to CSP in both defined and complex media. We also find that CSP stimulates the ComRS feedback system by upregulating comR in a carbohydrate source-dependent fashion. Our data provide mechanistic insight into how S. mutans regulates bimodality and explain the puzzle of growth medium effects on competence induction by CSP.
Project description:Natural transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration. The period of competence induced by CSP lasts only 15 min (quarter-height peak width). The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions. To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an Escherichia coli overexpression strain and core RNA polymerase was purified from a comX-deficient S. pneumoniae strain. The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes ssbB, cinA, cglA, celA, and dalA and was inhibited by anti-ComX antibody, but not by anti-sigma(70) antibody. Western blotting using antibodies specific for ComX, sigma(70), and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and sigma(A) remained between 60 and 125% of its normal level. ComX reached a molar ratio to RNA polymerase of at least 1.5. We conclude that ComX is unstable and acts as a competence-specific sigma factor.
Project description:The com operon of naturally transformable streptococcal species contains three genes, comC, comD, and comE, involved in the regulation of competence. The comC gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The comD and comE genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the comD-encoded protein being a receptor for CSP. Here we report on the genetic variability of comC and comD within Streptococcus pneumoniae isolates. Comparative analysis of sequence variations of comC and comD shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many S. pneumoniae strains in the laboratory, there is a clear correlation between the presence of a particular comC allele and the cognate comD allele. These findings effectively rule out the possibility that the presence of noncognate comC and comD alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional com pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of S. mitis and S. oralis. The CSPs from these isolates are much more closely related to those from S. pneumoniae than to most CSPs previously reported from S. mitis and S. oralis, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of S. pneumoniae that are under strong selective pressure.
Project description:Streptococcus mutans regulates genetic competence through a complex network that receives inputs from a number of environmental stimuli, including two signalling peptides designated as CSP and XIP. The response of the downstream competence genes to these inputs shows evidence of stochasticity and bistability and has been difficult to interpret. We have used microfluidic, single-cell methods to study how combinations of extracellular signals shape the response of comX, an alternative sigma factor governing expression of the late competence genes. We find that the composition of the medium determines which extracellular signal (XIP or CSP) can elicit a response from comX and whether that response is unimodal or bimodal across a population of cells. In a chemically defined medium, exogenous CSP does not induce comX, whereas exogenous XIP elicits a comX response from all cells. In complex medium, exogenous XIP does not induce comX, whereas CSP elicits a bimodal comX response from the population. Interestingly, bimodal behaviour required an intact copy of comS, which encodes the precursor of XIP. The comS-dependent capability for both unimodal and bimodal response suggests that a constituent - most likely peptides - of complex medium interacts with a positive feedback loop in the competence regulatory network.
Project description:Streptococcus sanguinis is an important component of dental plaque and a leading cause of infective endocarditis. Genetic competence in S. sanguinis requires a quorum sensing system encoded by the early comCDE genes, as well as late genes controlled by the alternative sigma factor, ComX. Previous studies of Streptococcus pneumoniae and Streptococcus mutans have identified functions for the >100-gene com regulon in addition to DNA uptake, including virulence. We investigated this possibility in S. sanguinis. Strains deleted for the comCDE or comX master regulatory genes were created. Using a rabbit endocarditis model in conjunction with a variety of virulence assays, we determined that both mutants possessed infectivity equivalent to that of a virulent control strain, and that measures of disease were similar in rabbits infected with each strain. These results suggest that the com regulon is not required for S. sanguinis infective endocarditis virulence in this model. We propose that the different roles of the S. sanguinis, S. pneumoniae, and S. mutans com regulons in virulence can be understood in relation to the pathogenic mechanisms employed by each species.