RecA and DNA recombination: a review of molecular mechanisms

International audienceRecombinases are responsible for homologous recombination and maintenance of genome integrity. In Escherichia coli, the recombinase RecA forms a nucleoprotein filament with the ssDNA present at a DNA break and searches for a homologous dsDNA to use as a template for break repair. During the first step of this process, the ssDNA is bound to RecA and stretched into a Watson-Crick base-paired triplet conformation. The RecA nucleoprotein filament also contains ATP and Mg 2+ , two cofactors required for RecA activity. Then, the complex starts a homology search by interacting with and stretching dsDNA. Thanks to supercoiling, intersegment sampling and RecA clustering, a genome-wide homology search takes place at a relevant metabolic timescale. When a region of homology 8 to 20 base pairs in length is found and stabilized, DNA strand exchange proceeds, forming a heteroduplex complex that is resolved through a combination of DNA synthesis, ligation and resolution. RecA activities can take place without ATP hydrolysis, but this latter activity is necessary to improve and accelerate the process. Protein flexibility and monomer-monomer interactions are fundamental for RecA activity, which functions cooperatively. A structure/function relationship analysis suggests that the recombinogenic activity can be improved and that recombinases have an inherently large recombination potential. Understanding this relationship is essential for designing RecA derivatives with enhanced activity for biotechnology applications. For example, this protein is a major actor in the recombinase polymerase isothermal amplification (RPA) used in point-of-care diagnostics

The nucleoid-associated proteins H-NS and FIS modulate the DNA supercoiling response of the pel genes, the major virulence factors in the plant pathogen bacterium Dickeya dadantii

Dickeya dadantii is a pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that can destroy the plant cell walls. Previously we found that the pel gene expression is modulated by H-NS and FIS, two nucleoid-associated proteins (NAPs) modulating the DNA topology. Here, we show that relaxation of the DNA in growing D. dadantii cells decreases the expression of pel genes. Deletion of fis aggravates, whereas that of hns alleviates the negative impact of DNA relaxation on pel expression. We further show that H-NS and FIS directly bind the pelE promoter and that the response of D. dadantii pel genes to stresses that induce DNA relaxation is modulated, although to different extents, by H-NS and FIS. We infer that FIS acts as a repressor buffering the negative impact of DNA relaxation on pel gene transcription, whereas H-NS fine-tunes the response of virulence genes precluding their expression under suboptimal conditions of supercoiling. This novel dependence of H-NS effect on DNA topology expands our understanding of the role of NAPs in regulating the global bacterial gene expression and bacterial pathogenicity

lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. [br/] Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. [br/] Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria

Quorum Sensing Signaling Molecules Produced by Reference and Emerging Soft-Rot Bacteria (Dickeya and Pectobacterium spp.)

International audienceBACKGROUND: Several small diffusible molecules are involved in bacterial quorum sensing and virulence. The production of autoinducers-1 and -2, quinolone, indole and γ-amino butyrate signaling molecules was investigated in a set of soft-rot bacteria belonging to six Dickeya or Pectobacterium species including recent or emerging potato isolates. METHODOLOGY/PRINCIPAL FINDINGS: Using bacterial biosensors, immunoassay, and chromatographic analysis, we showed that soft-rot bacteria have the common ability to produce transiently during their exponential phase of growth the N-3-oxo-hexanoyl- or the N-3-oxo-octanoyl-l-homoserine lactones and a molecule of the autoinducer-2 family. Dickeya spp. produced in addition the indole-3-acetic acid in tryptophan-rich conditions. All these signaling molecules have been identified for the first time in the novel Dickeya solani species. In contrast, quinolone and γ-amino butyrate signals were not identified and the corresponding synthases are not present in the available genomes of soft-rot bacteria. To determine if the variations of signal production according to growth phase could result from expression modifications of the corresponding synthase gene, the respective mRNA levels were estimated by reverse transcriptase-PCR. While the N-acyl-homoserine lactone production is systematically correlated to the synthase expression, that of the autoinducer-2 follows the expression of an enzyme upstream in the activated methyl cycle and providing its precursor, rather than the expression of its own synthase. CONCLUSIONS/SIGNIFICANCE: Despite sharing the S-adenosylmethionine precursor, no strong link was detected between the production kinetics or metabolic pathways of autoinducers-1 and -2. In contrast, the signaling pathway of autoinducer-2 seems to be switched off by the indole-3-acetic acid pathway under tryptophan control. It therefore appears that the two genera of soft-rot bacteria have similarities but also differences in the mechanisms of communication via the diffusible molecules. Our results designate autoinducer-1 lactones as the main targets for a global biocontrol of soft-rot bacteria communications, including those of emerging isolates

The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity

Les vésiculites chez le taureau reproducteur, mise au point d'une technique d'ablation des vésicules séminales et conséquences sur le spermogramme

Les vésiculites constituent chez le taureau d'insémination une des affections majeures du tractus génital en terme de fréquence et de gravité. Elles provoquent abattement, fièvre et douleur abdominale qui peuvent inhiber la monte et l'éjaculation. Les vésiculites entrainent également des altérations de la qualité de la semence (présence de pus, modification du pH, diminution du volume de l'éjaculat). L'issue peut aller jusqu'à la réforme de l'animal. Les traitements médicaux, souvent très longs et coûteux, se soldent fréquemment par des échecs. L'impact économique, zootechnique et génétique des vésiculites peut donc être important. Cette thèse s'attache à décrire et évaluer une technique chirurgicale d'ablation des vésicules séminales du taureau. Une première partie bibliographique présente l'anatomie des vésicules séminales, leur rôle, leur physiologie, leurs affections et les traitements associés. L'étude expérimentale, développée dans une seconde partie, présente une technique chirurgicale d'ablation des vésicules séminales par abord rectal ventral sur deux taureaux de race Prim' Holstein. L'évolution post-opératoire, en particulier celle du spermogramme, est décrite.MAISONS-ALFORT-Ecole Vétérin (940462302) / SudocSudocFranceF

Les acteurs du 'quorum sensing' chez la bactérie phytopathogène à Gram négatif Erwinia chrysanthemi

E. chrysanthemi est une bactérie phytopathogène à Gram négatif. La synthèse des pectinases, ses principaux facteurs de virulence, est fortement induite en fin de phase exponentielle de croissance. Cette induction semble impliquer un système de régulation de type quorum sensing' (QS). Chez les bactéries à Gram négatif, le système de QS le plus répandu est celui répondant aux acyl-homosérine lactones (acyl-HSLs). Ce contrôle fait intervenir chez E. chrysanthemi un couple de protéines ExpI-ExpR homologues au couple LuxI/LuxR de la bactérie Vibrio fischeri, première bactérie chez laquelle le phénomène de QS a été découvert. Afin de comprendre le processus d'activation des régulateurs de type LuxR par les acyl-HSLs, nous avons engagé une étude structurale et fonctionnelle du régulateur ExpR. Cette étude a permis de montrer que : i) ExpR réprime directement l'expression de son propre gène ; ii) cette répression se fait par une inhibition de l'initiation de la transcription par l'ARN polymérase et est modulée par la présence du signal de QS, la 3-oxo-C6-HSL ; iii) la 3-oxo-C6-HSL provoque la dimérisation du domaine N-terminal de ExpR (ExpR-N). Des expériences de mutagénèse dirigée ont permis d'identifier des acides aminés du domaine N-terminal de ExpR impliqués dans la cascade de réarrangements structuraux induite par la fixation de la 3-oxo-C6-HSL jusqu'au domaine C-terminal de fixation à l'ADN. Une étude structurale de ExpR-N en complexe avec la 3-oxo-C6-HSL par cristallographie aux rayons X a été réalisée. Des cristaux diffractant à 3Å ont été obtenus. Cependant, nos tentatives pour résoudre le problème des phases n'ont pas abouti et la structure 3D de ExpR-N n'a pu être déterminée. Afin d'interférer dans le processus de QS, des analogues structuraux des acyl-HSLs ont été synthétisés. Les N-sulfonylhomosérine lactones et les urées dérivées de l'homosérine lactone se sont révélées être des inhibiteurs compétitifs du QS. Une deuxième partie de mon travail de thèse a consisté à caractériser les autres systèmes de QS présents chez E. chrysanthemi. L'analyse du génome de la souche d'E. chrysanthemi 3937 a révélé l'existence d'un deuxième régulateur de type LuxR, appelé CanR et la présence du gène luxS potentiellement responsable de la synthèse d'AI-2, un autre signal de QS retrouvé chez les bactéries. Les mutants canR et luxS ont donc été construits et caractérisés. Ainsi, E. chrysanthemi 3937 produit bien le signal AI-2 et celui-ci n'est pas détecté dans un mutant luxS. Bien qu'un retard dans l'apparition des symptômes a pu être observé dans un mutant luxS, la virulence ne semble pas significativement affectée par rapport à la souche sauvage, suggérant que le système de QS dépendant de AI-2 chez E. chrysanthemi ne régule pas de facteurs de virulence majeurs. Un mutant canR n'est pas affecté pour la synthèse de pectinases mais sa virulence est fortement réduite. L'analyse de fusions transcriptionnelles de plusieurs protéines extracellulaires d'E. chrysanthemi dans les mutants expI, expR, canR et dans le double mutant canR-expR a permis de montrer que la synthèse de la protéase PrtC, est diminuée à la fois dans un mutant expI et dans un mutant canRLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Quorum Sensing Regulation in Phytopathogenic Bacteria

International audienceQuorum sensing is a type of chemical communication by which bacterial populations control expression of their genes in a coordinated manner. This regulatory mechanism is commonly used by pathogens to control the expression of genes encoding virulence factors and that of genes involved in the bacterial adaptation to variations in environmental conditions. In phytopathogenic bacteria, several mechanisms of quorum sensing have been characterized. In this review, we describe the different quorum sensing systems present in phytopathogenic bacteria, such as those using the signal molecules named N-acyl-homoserine lactone (AHL), diffusible signal factor (DSF), and the unknown signal molecule of the virulence factor modulating (VFM) system. We focus on studies performed on phytopathogenic bacteria of major importance, including Pseudomonas, Ralstonia, Agrobacterium, Xanthomonas, Erwinia, Xylella,Dickeya, and Pectobacterium spp. For each system, we present the mechanism of regulation, the functions targeted by the quorum sensing system, and the mechanisms by which quorum sensing is regulated

Plant-phytopathogen interactions: bacterial responses to environmental and plant stimuli

International audiencePlant pathogenic bacteria attack numerous agricultural crops, causing devastating effects on plant productivity and yield. They survive in diverse environments, both in plants, as pathogens, and also outside their hosts as saprophytes. Hence, they are confronted with numerous changing environmental parameters. During infection, plant pathogens have to deal with stressful conditions, such as acidic, oxidative and osmotic stresses; anaerobiosis; plant defenses; and contact with antimicrobial compounds. These adverse conditions can reduce bacterial survival and compromise disease initiation and propagation. Successful bacterial plant pathogens must detect potential hosts and also coordinate their possibly conflicting programs for survival and virulence. Consequently, these bacteria have a strong and finely tuned capacity for sensing and responding to environmental and plant stimuli. This review summarizes our current knowledge of the signals and genetic circuits that affect survival and virulence factor expression in three important and well-studied plant pathogenic bacteria with wide host ranges and the capacity for long-term environmental survival. These are: Ralstonia solanacerarum, a vascular pathogen that causes wilt disease; Agrobacterium tumefaciens, a biotrophic tumorigenic pathogen responsible for crown gall disease and Dickeya, a brute force apoplastic pathogen responsible for soft-rot disease

Characterization of Indigoidine Biosynthetic Genes in Erwinia chrysanthemi and Role of This Blue Pigment in Pathogenicity

In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response