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.

The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in the pneumococcus.

Homologous recombination (HR) is a crucial mechanism of DNA strand exchange that promotes genetic repair and diversity in all kingdoms of life. Bacterial HR is driven by the universal recombinase RecA, assisted in the early steps by dedicated mediators that promote its polymerization on single-stranded DNA (ssDNA). In bacteria, natural transformation is a prominent HR-driven mechanism of horizontal gene transfer specifically dependent on the conserved DprA recombination mediator. Transformation involves internalization of exogenous DNA as ssDNA, followed by its integration into the chromosome by RecA-directed HR. How DprA-mediated RecA filamentation on transforming ssDNA is spatiotemporally coordinated with other cellular processes remains unknown. Here, we tracked the localization of fluorescent fusions to DprA and RecA in Streptococcus pneumoniae and revealed that both accumulate in an interdependent manner with internalized ssDNA at replication forks. In addition, dynamic RecA filaments were observed emanating from replication forks, even with heterologous transforming DNA, which probably represent chromosomal homology search. In conclusion, this unveiled interaction between HR transformation and replication machineries highlights an unprecedented role for replisomes as landing pads for chromosomal access of tDNA, which would define a pivotal early HR step for its chromosomal integration.

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

The unusual structure of Ruminococcin C1 antimicrobial peptide confers clinical properties.

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.

Fine-tuning cellular levels of DprA ensures transformant fitness in the human pathogen Streptococcus pneumoniae.

Natural genetic transformation is a widespread mechanism of horizontal gene transfer. It involves the internalization of exogenous DNA as single strands and chromosomal integration via homologous recombination, promoting acquisition of new genetic traits. Transformation occurs during a distinct physiological state called competence. In Streptococcus pneumoniae, competence is controlled by ComDE, a two-component system induced by an exported peptide pheromone. DprA is universal among transformable species, strongly induced during pneumococcal competence, and crucial for pneumococcal transformation. Pneumococcal DprA plays three crucial roles in transformation and competence. Firstly, DprA protects internalized DNA from degradation. Secondly, DprA loads the homologous recombinase RecA onto transforming DNA to promote transformation. Finally, DprA interacts with the response regulator ComE to shut-off competence. Here, we explored the effect of altering the cellular levels of DprA on these three roles. High cellular levels of DprA were not required for the primary role of DprA as a transformation-dedicated recombinase loader or for protection of transforming DNA. In contrast, full expression of dprA was required for optimal competence shut-off and transformant fitness. High cellular levels of DprA thus ensure the fitness of pneumococcal transformants by mediating competence shut-off. This promotes survival and propagation of transformants, maximizing pneumococcal adaptive potential.

Pneumococcal Competence Coordination Relies on a Cell-Contact Sensing Mechanism.

Bacteria have evolved various inducible genetic programs to face many types of stress that challenge their growth and survival. Competence is one such program. It enables genetic transformation, a major horizontal gene transfer process. Competence development in liquid cultures of Streptococcus pneumoniae is synchronized within the whole cell population. This collective behavior is known to depend on an exported signaling Competence Stimulating Peptide (CSP), whose action generates a positive feedback loop. However, it is unclear how this CSP-dependent population switch is coordinated. By monitoring spontaneous competence development in real time during growth of four distinct pneumococcal lineages, we have found that competence shift in the population relies on a self-activated cell fraction that arises via a growth time-dependent mechanism. We demonstrate that CSP remains bound to cells during this event, and conclude that the rate of competence development corresponds to the propagation of competence by contact between activated and quiescent cells. We validated this two-step cell-contact sensing mechanism by measuring competence development during co-cultivation of strains with altered capacity to produce or respond to CSP. Finally, we found that the membrane protein ComD retains the CSP, limiting its free diffusion in the medium. We propose that competence initiator cells originate stochastically in response to stress, to form a distinct subpopulation that then transmits the CSP by cell-cell contact.

RecFOR is not required for pneumococcal transformation but together with XerS for resolution of chromosome dimers frequently formed in the process.

Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR machineries exist for genome maintenance and transformation in pneumococci. These observations presumably apply to most naturally transformable species.

Fine-tuning of choline metabolism is important for pneumococcal colonization.

The human pathogen Streptococcus pneumoniae (the pneumococcus) is rare in having a strict requirement for the amino alcohol choline, which decorates pneumococcal teichoic acids. This process relies on the lic locus, containing the lic1 and lic2 operons. These operons produce eight proteins that import and metabolize choline, generate teichoic acid precursors and decorate these with choline. Three promoters control expression of lic operons, with Plic1P1 and Plic1P2 controlling lic1 and Plic2 controlling lic2. To investigate the importance of lic regulation for pneumococci, we assayed the activity of transcriptional fusions of the three lic promoters to the luciferase reporter gene. Plic1P1 , whose activity depends on the response regulator CiaR, responded to fluctuations in extracellular choline, with activity increasing greatly upon choline depletion. We uncovered a complex regulatory mechanism controlling Plic1P1 , involving activity driven by CiaR, repression by putative repressor LicR in the presence of choline, and derepression upon choline depletion mediated by LicC, a choline metabolism enzyme. Finally, the ability to regulate Plic1P1 in response to choline was important for pneumococcal colonization. We suggest that derepression of Plic1P1 upon choline depletion maximizing choline internalization constitutes an adaptive response mechanism allowing pneumococci to optimize growth and survival in environments where choline is scarce.

Natural genetic transformation generates a population of merodiploids in Streptococcus pneumoniae.

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ~3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ~100 to ~900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.

Direct involvement of DprA, the transformation-dedicated RecA loader, in the shut-off of pneumococcal competence.

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 σ(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 σ(X) control, interacts with ComE∼P to block ComE-driven transcription, chiefly impacting σ(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.

Programmed protection of foreign DNA from restriction allows pathogenicity island exchange during pneumococcal transformation.

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through programmed protection and incorporation of foreign DNA.

Midcell recruitment of the DNA uptake and virulence nuclease, EndA, for pneumococcal transformation.

Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen Streptococcus pneumoniae, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under 'static' binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60-70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10-11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.

Structure-function analysis of pneumococcal DprA protein reveals that dimerization is crucial for loading RecA recombinase onto DNA during transformation.

Transformation promotes genome plasticity in bacteria via RecA-driven homologous recombination. In the gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-to-tail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprA-RecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.

ComE/ComE~P interplay dictates activation or extinction status of pneumococcal X-state (competence).

Since 1996, induction of competence for genetic transformation of Streptococcus pneumoniae is known to be controlled by the ComD/ComE two-component regulatory system. The mechanism of induction is generally described as involving ComD autophosphorylation, transphosphorylation of ComE and transcriptional activation by ComE~P of the early competence (com) genes, including comX which encodes the competence-specific σ(X) . However, none of these features has been experimentally established. Here we document the autokinase activity of ComD proteins in vitro, and provide an estimate of the stoichiometry of ComD and ComE in vivo. We report that a phosphorylmimetic mutant, ComE(D58E), constructed because of the failure to detect transphosphorylation of purified ComE in vitro, displays full spontaneous competence in ΔcomD cells, an that in vitro ComE(D58E) exhibits significantly improved binding affinity for P(comCDE). We also provide evidence for a differential transcriptional activation and repression of P(comCDE) and P(comX). Altogether, these data support the model of ComE~P-dependent activation of transcription. Finally, we establish that ComE antagonizes expression of the early com genes and propose that the rapid deceleration of transcription from P(comCDE) observed even in cells lacking σ(X) is due to the progressive accumulation of ComE, which outcompetes ComE~P.

Role of the single-stranded DNA-binding protein SsbB in pneumococcal transformation: maintenance of a reservoir for genetic plasticity.

Bacteria encode a single-stranded DNA (ssDNA) binding protein (SSB) crucial for genome maintenance. In Bacillus subtilis and Streptococcus pneumoniae, an alternative SSB, SsbB, is expressed uniquely during competence for genetic transformation, but its precise role has been disappointingly obscure. Here, we report our investigations involving comparison of a null mutant (ssbB(-)) and a C-ter truncation (ssbBΔ7) of SsbB of S. pneumoniae, the latter constructed because SSBs' acidic tail has emerged as a key site for interactions with partner proteins. We provide evidence that SsbB directly protects internalized ssDNA. We show that SsbB is highly abundant, potentially allowing the binding of ~1.15 Mb ssDNA (half a genome equivalent); that it participates in the processing of ssDNA into recombinants; and that, at high DNA concentration, it is of crucial importance for chromosomal transformation whilst antagonizing plasmid transformation. While the latter observation explains a long-standing observation that plasmid transformation is very inefficient in S. pneumoniae (compared to chromosomal transformation), the former supports our previous suggestion that SsbB creates a reservoir of ssDNA, allowing successive recombination cycles. SsbBΔ7 fulfils the reservoir function, suggesting that SsbB C-ter is not necessary for processing protein(s) to access stored ssDNA. We propose that the evolutionary raison d'être of SsbB and its abundance is maintenance of this reservoir, which contributes to the genetic plasticity of S. pneumoniae by increasing the likelihood of multiple transformation events in the same cell.

Pneumococcal competence is a populational health sensor driving multilevel heterogeneity in response to antibiotics.

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.

The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter)) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter) interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter) deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter) acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

Mechanistic and functional aspects of the Ruminococcin C sactipeptide isoforms.

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

The dynamic protein partnership of RNA polymerase in Bacillus subtilis.

In prokaryotes, transcription results from the activity of a 400 kDa RNA polymerase (RNAP) protein complex composed of at least five subunits (2α, ß, ß', ω). To ensure adequate responses to changing environmental cues, RNAP activity is tightly controlled by means of interacting regulatory proteins. Here, we report the affinity-purification of the Bacillus subtilis RNAP complexes from cells in different growth states and stress conditions, and the quantitative assessment by mass spectrometry of the dynamic changes in the composition of the RNAP complex. The stoichiometry of RNA polymerase was determined by a comparison of two mass spectrometry-based quantification methods: a label-based and a label-free method. The validated label-free method was then used to quantify the proteins associated with RNAP. The levels of sigma factors bound to RNAP varied during growth and exposure to stress. Elongation factors, helicases such as HelD and PcrA, and novel unknown proteins were also associated with RNAP complexes. The content in 6S RNAs of purified RNAP complexes increased at the onset of the stationary phase. These quantitative variations in the protein and RNA composition of the RNAP complexes well correlate with the known physiology of B. subtilis cells under different conditions.

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.