Homologous recombination (HR) is a universally conserved mechanism of DNA strand exchange between homologous sequences, driven in bacteria by the RecA recombinase. HR is key for the maintenance of bacterial genomes via replication fork restart and DNA repair, as well as for their plasticity via the widespread mechanism of natural transformation. Transformation involves the capture and internalization of exogenous DNA in the form of single strands, followed by HR-mediated chromosomal integration. In the human pathogen Streptococcus pneumoniae, transformation occurs during a transient, stress-induced differentiation state called competence. RecA and its partner DNA branch migration translocase RadA are both well conserved and cooperate in HR during transformation and certain genome maintenance pathways. Both recA and radA genes are basally expressed and transcriptionally induced during competence. In this study, we explored the importance of competence induction of recA and radA in transformation and genome maintenance. We confirmed that competence induction of recA, but not radA, was important for transformation. In contrast, we uncovered that the competence induction of both genes was required for optimal tolerance faced with transient exposure to the lethal genotoxic agent methyl methanesulfonate. However, this was not the case for another DNA-damaging agent, norfloxacin. These results show that competence induction of HR effectors is important for the increased tolerance to genotoxic stress provided to competent pneumococci. This reinforces the finding that pneumococcal competence is a stress-sensing mechanism, transiently increasing the expression of some genes not to optimize transformation but to improve survival faced with specific lethal stresses.IMPORTANCEHomologous recombination (HR) is a mechanism of DNA strand exchange important for both the maintenance and plasticity of bacterial genomes. Bacterial HR is driven by the recombinase RecA along with many accessory partner proteins, which define multiple dedicated pathways crucial to genome biology. Thus, a main mechanism of genome plasticity in bacteria is natural genetic transformation, which involves uptake and chromosomal integration of exogenous DNA via HR. In the human pathogen Streptococcus pneumoniae, transformation occurs during a transient, stress-induced physiological state called competence. RecA and the helicase RadA are key for both genome maintenance and transformation, and both are over-produced during competence. Here, we explore the importance of this over-production for transformation and genome maintenance, quantified by tolerance to genotoxic stress. While over-production of RecA was important for both processes, over-production of RadA was required only for genotoxic stress tolerance. This highlights the importance of competence as a stress-responsive mechanism, with induction of HR genes important for genotoxic stress tolerance.

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.

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 ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.

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 corn 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.

Homologous recombination (HR) is a central process of genome biology driven by a conserved recombinase, which catalyses the pairing of single-stranded DNA (ssDNA) with double-stranded DNA to generate a D-loop intermediate. Bacterial RadA is a conserved HR effector acting with RecA recombinase to promote ssDNA integration. The mechanism of this RadA-mediated assistance to RecA is unknown. Here, we report functional and structural analyses of RadA from the human pathogen Streptococcus pneumoniae . RadA is found to facilitate RecA-driven ssDNA recombination over long genomic distances during natural transformation. RadA is revealed as a hexameric DnaB-type helicase, which interacts with RecA to promote orientated unwinding of branched DNA molecules mimicking D-loop boundaries. These findings support a model of DNA branch migration in HR, relying on RecA-mediated loading of RadA hexamers on each strand of the recipient dsDNA in the D-loop, from which they migrate divergently to facilitate incorporation of invading ssDNA.

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.

Significance This article concerns the control of competence for bacterial genetic transformation. Competence is transient in the human pathogen Streptococcus pneumoniae , involving the specific expression of ∼100 genes that are turned ON suddenly and OFF almost as abruptly. Although the mechanism rendering all cells in a culture simultaneously competent is well understood, how competence stops has remained unknown. Here, we unravel the mechanism of shut-off, describing the discovery that a key recombination protein, DprA, exerts a negative control on competence through direct, physical interaction with the master regulator of competence, the response regulator ComE, to abolish transcription from ComE-activated promoters.

Objective The gut microbiota, which is considered a causal factor in metabolic diseases as shown best in animals, is under the dual influence of the host genome and nutritional environment. This study investigated whether the gut microbiota per se, aside from changes in genetic background and diet, could sign different metabolic phenotypes in mice. Methods The unique animal model of metabolic adaptation was used, whereby C57Bl/6 male mice fed a high-fat carbohydrate-free diet (HFD) became either diabetic (HFD diabetic, HFD-D) or resisted diabetes (HFD diabetes-resistant, HFD-DR). Pyrosequencing of the gut microbiota was carried out to profile the gut microbial community of different metabolic phenotypes. Inflammation, gut permeability, features of white adipose tissue, liver and skeletal muscle were studied. Furthermore, to modify the gut microbiota directly, an additional group of mice was given a gluco-oligosaccharide (GOS)-supplemented HFD (HFD+GOS). Results Despite the mice having the same genetic background and nutritional status, a gut microbial profile specific to each metabolic phenotype was identified. The HFD-D gut microbial profile was associated with increased gut permeability linked to increased endotoxaemia and to a dramatic increase in cell number in the stroma vascular fraction from visceral white adipose tissue. Most of the physiological characteristics of the HFD-fed mice were modulated when gut microbiota was intentionally modified by GOS dietary fibres. Conclusions The gut microbiota is a signature of the metabolic phenotypes independent of differences in host genetic background and diet.

A series of experiments has been carried out to determine the Log removal value (LRV) of MS2 bacteriophages suspended in various buffers (osmosed water, tap water, aqueous solutions of NaCl and phosphate buffer solution) during filtration through hollow fiber membranes made of cellulose acetate. Viral concentrations in permeate and retentate were determined using two different methods, namely plaque forming unit (PFU) counting, which reveals only infectious particles and quantitative RT-PCR which detects the total (infectious+inactivated) number of viral genomes regardless of their infectivity.From this experimental study, we propose guidelines for preparing the challenging solutions and measuring their concentration which ensure a reliable assessment of the membrane performance. (C) 2011 Elsevier B.V. All rights reserved.

Un régime riche en graisses modifie le microbiote intestinal, initie une inflammation à bas bruit, une résistance à l'insuline et un diabète de type 2. Mais les mécanismes associant microbiote et inflammation métabolique ne sont pas connus. Nos travaux démontrent qu'après une semaine de régime gras (avant l'apparition du diabète), le nombre de bactéries commensales intestinales viables augmente de plus de 100 fois dans le tissu adipeux et le sang des souris. Cette translocation de bactéries vivantes est prévenue chez des souris dont les récepteurs aux motifs bactériens Nod1 ou CD14 ont été invalidés. Ces souris ne développent ensuite pas de diabète. A l'inverse chez la souris ob/ob, la translocation de bactéries viables vers les tissus est spontanément très augmentée. Cette « bactériémie métabolique » est notamment associée à une augmentation de l'adhérence d'Escherichia coli à la muqueuse intestinale et à sa colocalisation avec les cellules dendritiques de la lamina propria. Afin de diminuer l'adhérence et la translocation bactérienne nous avons traité pendant six semaines des souris diabétiques avec le probiotique Bifidobacterium animalis subsp. lactis 420. Ce traitement a réduit l'inflammation métabolique, l'intolérance au glucose et la sensibilité à l'insuline. Nos résultats démontrent que le développement d'un état diabétique sous régime gras chez la souris, est précédé par une augmentation de la translocation bactérienne depuis l'intestin vers les tissus, alimentant ainsi en continu bactériémie et inflammation métaboliques. De nouvelles perspectives thérapeutiques sont ainsi envisagées pour le traitement du diabète de type 2.

A fat‐enriched diet modifies intestinal microbiota and initiates a low‐grade inflammation, insulin resistance and type‐2 diabetes. Here, we demonstrate that before the onset of diabetes, after only one week of a high‐fat diet (HFD), live commensal intestinal bacteria are present in large numbers in the adipose tissue and the blood where they can induce inflammation. This translocation is prevented in mice lacking the microbial pattern recognition receptors Nod1 or CD14, but overtly increased in Myd88 knockout and ob/ob mouse. This ‘metabolic bacteremia’ is characterized by an increased co‐localization with dendritic cells from the intestinal lamina propria and by an augmented intestinal mucosal adherence of non‐pathogenic Escherichia coli. The bacterial translocation process from intestine towards tissue can be reversed by six weeks of treatment with the probiotic strain Bifidobacterium animalis subsp. lactis 420, which improves the animals' overall inflammatory and metabolic status. Altogether, these data demonstrate that the early onset of HFD‐induced hyperglycemia is characterized by an increased bacterial translocation from intestine towards tissues, fuelling a continuous metabolic bacteremia, which could represent new therapeutic targets.

Bacteriophage MS2 is widely used as a surrogate to estimate pathogenic virus elimination by membrane filtration processes used in water treatment. Given that this water technology may be conducted with different types of waters, we focused on investigating the effects of ionic strength on MS2 behavior. For this, MS2 was analyzed while suspended in solutions of various ionic strengths, first in a batch experiment and second during membrane ultrafiltration, and quantified using (i) quantitative reverse transcriptase PCR (qRT-PCR), which detects the total number of viral genomes, (ii) qRT-PCR without the RNA extraction step, which reflects only particles with a broken capsid (free RNA), and (iii) the PFU method, which detects only infectious viruses. At the beginning of the batch experiments using solutions containing small amounts of salts, losses of MS2 infectivity (90%) and broken particles (20%) were observed; these proportions did not change during filtration. In contrast, in high-ionic-strength solutions, bacteriophage kept its biological activity under static conditions, but it quickly lost its infectivity during the filtration process. Increasing the ionic strength decreased both the inactivation and the capsid breakup in the feed suspension and increased the loss of infectivity in the filtration retentate, while the numbers of MS2 genomes were identical in both experiments. In conclusion, the effects of ionic strength on MS2 behavior may significantly distort the results of membrane filtration processes, and therefore, the combination of classical and molecular methods used here is useful for an effective validation of the retention efficiency of ultrafiltration membranes.

Biofilms formed in aerobic seawater on stainless steel are known to be efficient catalysts of the electrochemical reduction of oxygen. Based on their genomic analysis, seven bacterial isolates were selected and a cyclic voltammetry (CV) procedure was implemented to check their electrocatalytic activity towards oxygen reduction. All isolates exhibited close catalytic characteristics. Comparison between CVs recorded with glassy carbon and pyrolytic graphite electrodes showed that the catalytic effect was not correlated with the surface area covered by the cells. The low catalytic effect obtained with filtered isolates indicated the involvement of released redox compounds, which was confirmed by CVs performed with adsorbed iron–porphyrin. None of the isolates were able to form electro-active biofilms under constant polarization. The capacity to catalyze oxygen reduction is shown to be a widespread property among bacteria, but the property detected by CV does not necessarily confer the ability to achieve stable oxygen reduction under constant polarization.

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm−2) or 5.34 ± 0.33 log (gu cm−2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ∼300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.

Pseudomonas aeruginosa has already been shown to catalyze oxidation processes in the anode compartment of a microbial fuel cell. The present study focuses on the reverse capacity of the bacterium, i.e. reduction catalysis. Here we show that P. aeruginosa is able to catalyze the electrochemical reduction of oxygen. The use of cyclic voltammetry showed that, for a given range of potential values, the current generated in the presence of bacteria could reach up to four times the current obtained without bacteria. The adhesion of bacteria to the working electrode was necessary for the catalysis to be observed but was not sufficient. The electron transfer between the working electrode and the bacteria did not involve mediator metabolites like phenazines. The transfer was by direct contact. The catalysis required a certain contact duration between electrodes and live bacteria but after this delay, the metabolic activity of cells was no longer necessary. Membrane-bound proteins, like catalase, may be involved. Various strains of P. aeruginosa, including clinical isolates, were tested and all of them, even catalase-defective mutants, presented the same catalytic property. P. aeruginosa offers a new model for the analysis of reduction catalysis and the protocol designed here may provide a basis for developing an interesting tool in the field of bacterial adhesion.

The discovery of quorum sensing (QS) communication systems regulating bacterial virulence has afforded a novel opportunity for controlling infectious bacteria by interfering with QS. Pseudomonas aeruginosa is an example of an opportunistic human pathogen for which N-acyl homoserine lactone (AHL)-related compounds have been described as potent inhibitors of biofilm formation and virulence factors, given their similarity to the natural QS autoinducers (AHLs). Our purpose was to design potent analogs of N-butanoyl-L-homoserine lactone (C4-HSL) and to screen them for biological activity. Eleven original compounds characterized by the modification of the lactone moiety were screened for their ability to impair biofilm formation. Among them, compound 11 was able to modify the growth kinetics and to restrict the number of adherent cells when added from the early stages of biofilm formation (i.e., adhesion and microcolony formation) in a dose-dependent manner. To demonstrate antagonism with C4-HSL, we showed that the inhibition of biofilm formation by compound 11 was impaired when C4-HSL was added. Structure-activity relationships are discussed with respect to the results obtained.

Most bacteria known to be electrochemically active have been harvested in the anodic compartments of microbial fuel cells (MFCs) and are able to use electrodes as electron acceptors. The reverse phenomenon, i.e. using solid electrodes as electron donors, is not so widely studied. To our knowledge, most of the electrochemically active bacteria are Gram-negative. The present study implements a transitory electrochemical technique (cyclic voltammetry) to study the microbial catalysis of the electrochemical reduction of oxygen. It is demonstrated that a wide range of aerobic and facultative anaerobic bacteria are able to catalyze oxygen reduction. Among these electroactive bacteria, several were Gram-positive. The transfer of electrons was direct since no activity was obtained with the filtrate. These findings, showing a widespread property among bacteria including Gram-positive ones, open new and interesting routes in the field of electroactive bacteria research.

Il a été récemment démontré que les facteurs environnementaux tels que régime riche en graisses et flore intestinale interagissent pour induire une obésité. Cependant, le rôle de ces facteurs sur le diabète de type 2 (DT2) reste inconnu. Pour démontrer si le DT2 est associé avec un profil métagénomique indépendant du régime gras nous avons nourri des souris C57bl6 avec ce régime gras diabétogène (sans obésité) pendant 3 mois et sélectionné les souris sensibles (D) et celles résistantes (DR) à l’effet diabétogène du régime. Les profils métagénomiques (DGGE) montrent des différences importantes entre les 2 groupes de souris. Le métagénotype des souris D était associé à une augmentation de la perméabilité paracellulaire du caecum, une altération de l’expression des adipokines dans les tissus adipeux, et une augmentation du nombre des lymphocytes dans les dépôts adipeux. Nous avons ensuite nourri des souris diabétiques par des glucooligosaccharides prébiotiques impactant l’écologie microbienne. Dans ces conditions, les profils métagénomiques étaient profondément altérés, la perméabilité intestinale était réduite, la tolérance au glucose et le métabolisme lipidique des adipocytes pratiquement normalisés. Dans l’ensemble, ces données montrent que le phénotype diabétique est associé à une flore intestinale particulière indépendamment du statut nutritionnel. L’utilisation de prébiotiques peut contrôler la flore intestinale et secondairement le diabète.