Competence Regulon Research Articles

Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis.

Competence development in Streptococcus pneumoniae (pneumococcus) is tightly intertwined with virulence. In addition to genes encoding genetic transformation machinery, the competence regulon also regulates the expression of allolytic factors, bacteriocins, and cytotoxins. Pneumococcal competence system has been extensively interrogated in vitro where the short transient competent state upregulates the expression of three distinct phases of "early," "late," and "delayed" genes. Recently, we have demonstrated that the pneumococcal competent state develops naturally in mouse models of pneumonia-derived sepsis. To unravel the underlying adaptive mechanisms driving the development of the competent state, we conducted a time-resolved transcriptomic analysis guided by the spatiotemporal live in vivo imaging system of competence induction during pneumonia-derived sepsis. Mouse lungs infected by the serotype 2 strain D39 expressing a competent state-specific reporter gene (D39-ssbB-luc) were subjected to RNA sequencing guided by monitoring the competence development at 0, 12, 24, and, at the moribund state, >40 hours post-infection (hpi). Transcriptomic analysis revealed that the competence-specific gene expression patterns in vivo were distinct from those under in vitro conditions. There was significant upregulation of early, late, and some delayed phase competence-specific genes as early as 12 hpi, suggesting that the pneumococcal competence regulon is important for adaptation to the lung environment. Additionally, members of the histidine triad (pht) gene family were sharply upregulated at 12 hpi followed by a steep decline throughout the rest of the infection cycle, suggesting that Pht proteins participate in the early adaptation to the lung environment. Further analysis revealed that Pht proteins execute a metal ion-dependent regulatory role in competence induction.IMPORTANCEThe induction of pneumococcal competence for genetic transformation has been extensively studied in vitro but poorly understood during lung infection. We utilized a combination of live imaging and RNA sequencing to monitor the development of a competent state during acute pneumonia. Upregulation of competence-specific genes was observed as early as 12 hour post-infection, suggesting that the pneumococcal competence regulon plays an important role in adapting pneumococcus to the stressful lung environment. Among others, we report novel finding that the pneumococcal histidine triad (pht) family of genes participates in the adaptation to the lung environment and regulates pneumococcal competence induction.

Developing multispecies quorum-sensing modulators based on the Streptococcus mitis competence-stimulating peptide

Bacteria utilize quorum sensing (QS) to coordinate many group behaviors. As such, QS has attracted significant attention as a potential mean to attenuate bacterial infectivity without introducing selective pressure for resistance development. Streptococcus mitis, a human commensal, acts as a genetic diversity reservoir for Streptococcus pneumoniae, a prevalent human pathogen. S.mitis possesses a typical comABCDE competence regulon QS circuitry; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon QS circuitry in S.mitis are yet to be explored. We set out to delineate the competence regulon QS circuitry in S.mitis, including confirming the identity of the native CSP signal, evaluating the molecular mechanism that governs CSP interactions with histidine kinase receptor ComD leading to ComD activation, and defining the regulatory roles of the competence regulon QS circuitry in initiating various S.mitis phenotypes. Our analysis revealed important structure-activity relationship insights of the CSP signal and facilitated the development of novel CSP-based QS modulators. Our analysis also revealed the involvement of the competence regulon in modulating competence development and biofilm formation. Furthermore, our analysis revealed that the native S.mitis CSP signal can modulate QS response in S.pneumoniae. Capitalizing on this crosstalk, we developed a multispecies QS modulator that activates both the pneumococcus ComD receptors and the S.mitis ComD-2 receptor with high potencies. The novel scaffolds identified herein can be utilized to evaluate the effects temporal QS modulation has on S.mitis as it inhabits its natural niche.

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that other competence induction stimulus, like norfloxacin, is not conveyed by the HtrA-dependent pathway. This finding strongly suggests that competence can be induced by at least two independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus.

Investigating the Streptococcus sinensis competence regulon through a combination of transcriptome analysis and phenotypic evaluation.

Streptococcus sinensis is a recently identified member of the Mitis group of streptococci. This species has been associated with infective endocarditis; however its mechanisms of pathogenesis and virulence are not fully understood. This study aimed to investigate the influence of the competence-stimulating peptide (CSP) and the competence regulon quorum-sensing circuitry (ComABCDE) on subsequent gene transcription and expression, as well as resultant phenotypes. In this study we confirmed the native CSP identity, ascertained when endogenous CSP was produced and completed a transcriptome-wide analysis of all genes following CSP exposure. RNA sequencing analysis revealed the upregulation of genes known to be associated with competence, biofilm formation and virulence. As such, a variety of phenotypic assays were utilized to assess the correlation between increased mRNA expression and potential phenotype response, ultimately gaining insight into the effects of CSP on both gene expression and developed phenotypes. The results indicated that the addition of exogenous CSP aided in competence development and successful transformation, yielding an average transformation efficiency comparable to that of other Mitis group streptococci. Additional studies are needed to further delineate the effects of CSP exposure on biofilm formation and virulence. Overall, this study provides novel information regarding S. sinensis and provides a substantial foundation on which this species and its role in disease pathogenesis can be further investigated.

Attenuating the Streptococcus pneumoniae Competence Regulon Using Urea-Bridged Cyclic Dominant-Negative Competence-Stimulating Peptide Analogs.

Streptococcus pneumoniae (pneumococcus) is a prevalent human pathogen that utilizes the competence regulon quorum sensing circuitry to acquire antibiotic resistance and initiate its attack on the human host. Therefore, targeting the competence regulon can be applied as an anti-infective approach with minimal pressure for resistance development. Herein, we report the construction of a library of urea-bridged cyclic dominant-negative competence-stimulating peptide (dnCSP) derivatives and their evaluation as competitive inhibitors of the competence regulon. Our results reveal the first pneumococcus dual-action CSPs that inhibit the group 1 pneumococcus competence regulon while activating the group 2 pneumococcus competence regulon. Structural analysis indicates that the urea-bridge cyclization stabilizes the bioactive α-helix conformation, while in vivo studies using a mouse model of infection exhibit that the lead dual-action dnCSP, CSP1-E1A-cyc(Dab6Dab10), attenuates group 1-mediated mortality without significantly reducing the bacterial burden. Overall, our results pave the way for developing novel therapeutics against this notorious pathogen.

Pharmacological Evaluation of Synthetic Dominant-Negative Peptides Derived from the Competence-Stimulating Peptide of Streptococcus pneumoniae.

The competence regulon of Streptococcus pneumoniae (pneumococcus) is a quorum-sensing circuitry that regulates the ability of this pathogen to acquire antibiotic resistance or perform serotype switching, leading to vaccine-escape serotypes, via horizontal gene transfer, as well as initiate virulence. Induction of the competence regulon is centered on binding of the competence-stimulating peptide (CSP) to its cognate receptor, ComD. We have recently synthesized multiple dominant-negative peptide analogs capable of inhibiting competence induction and virulence in S. pneumoniae. However, the pharmacodynamics and safety profiles of these peptide drug leads have not been characterized. Therefore, in this study, we compared the biostability of cyanine-7.5-labeled wild-type CSPs versus dominant-negative peptide analogs (dnCSPs) spatiotemporally by using an IVIS Spectrum in vivo imaging system. Moreover, in vitro cytotoxicity and in vivo toxicity were evaluated. We conclude that our best peptide analog, CSP1-E1A-cyc(Dap6E10), is an attractive therapeutic agent against pneumococcal infection with superior safety and pharmacokinetics profiles.

Elucidating the Role and Structure-Activity Relationships of the Streptococcus oligofermentans Competence-Stimulating Peptide.

Streptococcus oligofermentans is an early colonizer of the oral microbiome with documented bactericidal activity against the oral pathogen Streptococcus mutans. S. oligofermentans has been observed to possess the typical comABCDE competence regulon found within most oral streptococci; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon is yet to be explored. Herein, we have both confirmed the identity of the S. oligofermentans CSP and utilized a wide range of phenotypic assays to characterize its regulatory role in competence, biofilm formation, and hydrogen peroxide formation. To determine the importance of each amino acid residue in CSP/ComD binding, we performed systematic replacement of amino acid residues within the S. oligofermentans CSP and developed a luciferase-based reporter system to assess the ability of these mutated analogues to modulate the competence regulon. Additionally, we performed CD analysis on mutated CSP analogues to determine the correlation between the peptide secondary structure and QS activation. To further explore S. oligofermentans' potential as a biotherapeutic against S. mutans infection, lead QS activators and inhibitors were used in interspecies competition assays to assess the effect of QS modulation on interactions between these two species. Lastly, we have documented a lack of S. oligofermentans-induced cytotoxicity, highlighting the potential of this native flora as a biotherapeutic with minimal health risks.

Tight Interplay between Replication Stress and Competence Induction in Streptococcus pneumoniae.

Cells respond to genome damage by inducing restorative programs, typified by the SOS response of Escherichia coli. Streptococcus pneumoniae (the pneumococcus), with no equivalent to the SOS system, induces the genetic program of competence in response to many types of stress, including genotoxic drugs. The pneumococcal competence regulon is controlled by the origin-proximal, auto-inducible comCDE operon. It was previously proposed that replication stress induces competence through continued initiation of replication in cells with arrested forks, thereby increasing the relative comCDE gene dosage and expression and accelerating the onset of competence. We have further investigated competence induction by genome stress. We find that absence of RecA recombinase stimulates competence induction, in contrast to SOS response, and that double-strand break repair (RexB) and gap repair (RecO, RecR) initiation effectors confer a similar effect, implying that recombinational repair removes competence induction signals. Failure of replication forks provoked by titrating PolC polymerase with the base analogue HPUra, over-supplying DnaA initiator, or under-supplying DnaE polymerase or DnaC helicase stimulated competence induction. This induction was not correlated with concurrent changes in origin-proximal gene dosage. Our results point to arrested and unrepaired replication forks, rather than increased comCDE dosage, as a basic trigger of pneumococcal competence.

Strategies to Attenuate the Competence Regulon in Streptococcus pneumoniae.

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 (dnCSP) scaffolds. We also discuss recent reports on antibiotics that can block CSP export by disturbing the proton motive force (PMF) across the membrane and various ways to control the pneumococcal pathogenicity by activating the counter signaling circuit and targeting the pneumococcal proteome.

Spatial Correlations and Distribution of Competence Gene Expression in Biofilms of Streptococcus mutans.

Streptococcus mutans is an important pathogen in the human oral biofilm. It expresses virulent behaviors that are linked to its genetic competence regulon, which is controlled by comX. Expression of comX is modulated by two diffusible signaling peptides, denoted CSP and XIP, and by other environmental cues such as pH and oxidative stress. The sensitivity of S. mutans competence to environmental inputs that may vary on microscopic length scales raises the question of whether the biofilm environment creates microniches where competence and related phenotypes are concentrated, leading to spatial clustering of S. mutans virulence behaviors. We have used two-photon microscopy to characterize the spatial distribution of comX expression among individual S. mutans cells in biofilms. By analyzing correlations in comX activity, we test for spatial clustering that may suggest localized competence microenvironments. Our data indicate that both competence-signaling peptides diffuse efficiently through the biofilm. XIP elicits a population-wide response. CSP triggers a Poisson-like, spatially random comX response from a subpopulation of cells that is homogeneously dispersed. Our data indicate that competence microenvironments if they exist are small enough that the phenotypes of individual cells are not clustered or correlated to any greater extent than occurs in planktonic cultures.

Structure Activity Relationship Study of the XIP Quorum Sensing Pheromone in Streptococcus mutans Reveal Inhibitors of the Competence Regulon.

The dental cariogenic pathogen Streptococcus mutans coordinates competence for genetic transformation via two peptide pheromones, competence stimulating peptide (CSP) and comX-inducing peptide (XIP). CSP is sensed by the comCDE system and induces competence indirectly, whereas XIP is sensed by the comRS system and induces competence directly. In chemically defined media (CDM), after uptake by oligopeptide permease, XIP interacts with the cytosolic receptor ComR to form the XIP::ComR complex that activates the expression of comX, an alternative sigma factor that initiates the transcription of late-competence genes. In this study, we set out to determine the molecular mechanism of XIP::ComR interaction. To this end, we performed systematic replacement of the amino acid residues in the XIP pheromone and assessed the ability of the mutated analogs to modulate the competence regulon in CDM. We were able to identify structural features that are important to ComR binding and activation. Our structure-activity relationship insights led us to construct multiple XIP-based inhibitors of the comRS pathway. Furthermore, when comCDE and comRS were both stimulated with CSP and XIP, respectively, a lead XIP-based inhibitor was able to maintain the inhibitory activity. Last, phenotypic assays were used to highlight the potential of XIP-based inhibitors to attenuate pathogenicity in S. mutans and to validate the specificity of these compounds to the comRS pathway within the competence regulon. The XIP-based inhibitors developed in this study can be used as lead scaffolds for the design and development of potential therapeutics against S. mutans infections.

Streptococcus pneumoniae Elaborates Persistent and Prolonged Competent State during Pneumonia-Derived Sepsis.

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. In contrast to the universally reported short transient burst of competent state in vitro, the naturally developed competent state was prolonged and persistent during pneumonia-derived sepsis. The competent state began at approximately 20 h postinfection (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 (1 × 106 to 1 × 108 CFU/mouse) and postinfection 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 shutoff 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.

Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human pathogen that utilizes the competence regulon, a quorum-sensing circuitry, to acquire antibiotic resistance genes and initiate its attack on the human host. Interception of the competence regulon can therefore be utilized to study S. pneumoniae cell-cell communication and behavioral changes, as well as attenuate S. pneumoniae infectivity. Herein we report the design and synthesis of cyclic dominant negative competence-stimulating peptide (dnCSP) analogs capable of intercepting the competence regulon in both S. pneumoniae specificity groups with activities at the low nanomolar range. Structural analysis of lead analogs provided important insights as to the molecular mechanism that drives CSP receptor binding and revealed that the pan-group cyclic CSPs exhibit a chimeric hydrophobic patch conformation that resembles the hydrophobic patches required for both ComD1 and ComD2 binding. Moreover, the lead cyclic dnCSP, CSP1-E1A-cyc(Dap6E10), was found to possess superior pharmacological properties, including improved resistance to enzymatic degradation, while remaining nontoxic. Lastly, CSP1-E1A-cyc(Dap6E10) was capable of attenuating mouse mortality during acute pneumonia caused by both group 1 and group 2 S. pneumoniae strains. This cyclic pan-group dnCSP is therefore a promising drug lead scaffold against S. pneumoniae infections that could be administered individually or utilized in combination therapy to augment the effects of current antimicrobial agents.

Biological Evaluation of Native Streptococcal Competence Stimulating Peptides Reveal Potential Crosstalk Between Streptococcus mitis and Streptococcus pneumoniae and a New Scaffold for the Development of S. pneumoniae Quorum Sensing Modulators.

Streptococcus pneumoniae, an opportunistic human pathogen, acquires genes from its neighboring species of the mitis group of streptococci, which confer antibiotic resistances and allow it to produce diverse virulence factors. Most species of the mitis group are naturally competent, and they utilize the competence stimulating peptide (CSP) and the CSP-dependent competence regulon, a conserved quorum sensing (QS) circuit, to regulate their competence behavior. The dependence of the mitis group on this communication pathway makes QS a potential target to control their behavior. In this work, we sought to evaluate the impact of the native pheromones of the adjacent species of S. pneumoniae to modulate the activity of the S. pneumoniae competence regulon. Our results revealed the potential role of S. mitis as a modulator of QS in S. pneumoniae. Most importantly, our analysis also revealed that by using the native pheromone of S. mitis as a template, highly potent pan-group agonists and antagonists of the pneumococcal competence regulon could be developed. The newly developed QS modulators may have therapeutic utility in treating pneumococcal infections.

Development of Streptococcus pneumoniae Pan-Group Quorum-Sensing Modulators.

The rapid increase in multidrug-resistant pathogens is a major health concern that could bring mankind back to the pre-antibiotic era. Streptococcus pneumoniae is a highly recombinogenic opportunistic pathogen that causes a variety of deadly diseases and rapidly develops resistance to current antibiotic treatments. S. pneumoniae pathogenicity is dependent on a cell-density communication mechanism, or quorum sensing (QS), termed the competence regulon. In this work, we set out to design signal-based QS modulators capable of affecting the two specificity groups found in S. pneumoniae. Through systematic analysis and rational design, we were able to construct peptide-based pan-group QS activators and inhibitors with activities in the nanomolar range. These novel analogues are privileged scaffolds for the development of anti-virulence therapeutics against S. pneumoniae infections.

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

Refining the Pneumococcal Competence Regulon by RNA Sequencing.

Competence for genetic transformation allows the opportunistic human pathogen Streptococcus pneumoniae to take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniae is an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

Unveiling the Importance of Amide Protons in CSP:ComD Interactions in Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic pathogen that can cause diseases ranging from mild respiratory infections to life-threatening conditions such as pneumonia, meningitis, and bacteremia. S. pneumoniae pathogenicity is dependent on the action of a 17-amino acid peptide pheromone, termed competence stimulating peptide (CSP) that controls the competence regulon, a quorum sensing (QS) circuit. Therefore, intercepting QS could have therapeutic implications in treating pneumococcal infections while avoiding emerging antimicrobial resistance. In this study, we set out to evaluate the impact of amide protons on CSP activity and metabolic stability through systematic N-methylation. Our results indicate that the majority of amide protons are critical for CSP activity, either through direct interactions with the cognate receptor or by stabilizing the bioactive conformation. Importantly, we identified several N-methyl CSP analogs, namely, CSP1(15)-N-Me-K6 and CSP1(15)-N-Me-F7, that retain their biological activity while exhibiting enhanced metabolic stability. These analogs are privileged scaffolds for the design of CSP-based QS modulators with drug-like properties.

AphA is a master regulator of natural competence in Vibrio cholerae

Vibrio cholerae is the etiological agent of cholera, a disease affecting 4.3 million people each year (WHO, 2015). The symptoms of cholera include profuse watery diarrhoea and vomiting, leading to coma and death in serious cases. The El Tor biotype of V. cholerae is globally predominant, and the success of this biotype to cause disease is thought to be due, in part, to the ability of the organism to successfully colonise two different environments; i) marine and brackish coastal waters where V. cholerae forms biofilms on the chitinous surfaces of shellfish, and ii) the human intestine. When bound to chitinous surfaces V. cholerae expresses genes required for biofilm formation, chitin utilisation and natural competence. Conversely, within the human host, V. cholerae switches on the expression of virulence genes. The transcription factor AphA was discovered as a regulator of genes encoding the toxin co-regulated pilus (TCP) of V. cholerae. It is also known that AphA is active when V. cholerae populations have a low cell density. Hence, AphA is generally considered a regulator of virulence and quorum sensing. We have mapped DNA binding by AphA across the entire V. cholerae genome. The majority of AphA target genes encode cell surface and cell envelope proteins. Unexpectedly, we show that AphA also targets key gene clusters within the natural competence regulon of V. cholerae. Using biochemistry and genetics we show that AphA is a master repressor of natural competence and acts to antagonise transcription activation by the cyclic-AMP receptor protein.

Hidden Gems in the Transcriptome Maps of Competent Streptococci.

Natural transformation is regarded as an important mechanism in bacteria that allows for adaptation to different environmental stressors by ensuring genome plasticity. Since the discovery of this phenomenon in Streptococcus pneumoniae, remarkable progress has been made in the understanding of the molecular mechanisms and pathways coordinating this process. Recently, the advent of high-throughput sequencing allows the posing of questions that address the system at a larger scale but also allow for the creation of high-resolution maps of transcription. Thus, while much is already known about genetic competence in streptococci, recent studies continue to reveal intricate novel regulation pathways and components. In this perspective article, we highlight the use of transcriptional profiling and mapping as a valuable resource in the identification and characterization of “hidden gems” pertinent to the natural transformation system. Such strategies have recently been employed in a variety of different species. In S. mutans, for example, genome editing combined with the power of promoter mapping and RNA-Seq allowed for the identification of a link between the ComCDE and the ComRS systems, a ComR positive feedback loop mediated by SigX, and the XrpA peptide, encoded within sigX, which inhibits competence. In S. pneumoniae, a novel member of the competence regulon termed BriC was found to be directly under control of ComE and to promote biofilm formation and nasopharyngeal colonization but not competence. Together these new technologies enable us to discover new links and to revisit old pathways in the compelling study of natural genetic transformation.