Molecular determinants of the DprA−RecA interaction for nucleation on ssDNA

International audienceNatural transformation is a major mechanism of horizontal gene transfer in bacteria that depends on DNA recombination. RecA is central to the homologous recombination pathway, catalyzing DNA strand invasion and homology search. DprA was shown to be a key binding partner of RecA acting as a specific mediator for its loading on the incoming exogenous ssDNA. Although the 3D structures of both RecA and DprA have been solved, the mechanisms underlying their cross-talk remained elusive. By combining molecular docking simulations and experimental validation, we identified a region on RecA, buried at its self-assembly interface and involving three basic residues that contact an acidic triad of DprA previously shown to be crucial for the interaction. At the core of these patches, DprA M238 and RecA F230 are involved in the interaction. The other DprA binding regions of RecA could involve the N-terminal ␣-helix and a DNA-binding region. Our data favor a model of DprA acting as a cap of the RecA filament, involving a DprA−RecA interplay at two levels: their own oligomeric states and their respective interaction with DNA. Our model forms the basis for a mech-anistic explanation of how DprA can act as a mediator for the loading of RecA on ssDNA

The RstAB System Impacts Virulence, Motility, Cell Morphology, Penicillin Tolerance and Production of Type II Secretion System-Dependent Factors in the Fish and Human Pathogen Photobacterium damselae subsp. damselae

The RstB histidine kinase of the two component system RstAB positively regulates the expression of damselysin (Dly), phobalysin P (PhlyP) and phobalysin C (PhlyC) cytotoxins in the fish and human pathogen Photobacterium damselae subsp. damselae, a marine bacterium of the family Vibrionaceae. However, the function of the predicted cognate response regulator RstA has not been studied so far, and the role of the RstAB system in other cell functions and phenotypes remain uninvestigated. Here, we analyzed the effect of rstA and rstB mutations in cell fitness and in diverse virulence-related features. Both rstA and rstB mutants were severely impaired in virulence for sea bream and sea bass fish. Mutants in rstA and rstB genes were impaired in hemolysis and in Dly-dependent phospholipase activity but had intact PlpV-dependent phospholipase and ColP-dependent gelatinase activities. rstA and rstB mutants grown at 0.5% NaCl exhibited impaired swimming motility, enlarged cell size and impaired ability to eparate after cell division, whereas at 1% NaCl the mutants exhibited normal henotypes. Mutation of any of the two genes also impacted tolerance to benzylpenicillin. Notably, rstA and rstB mutants showed impaired secretion of a number of type II secretion system (T2SS)-dependent proteins, which included the three major cytotoxins Dly, PhlyP and PhlyC, as well as a putative delta-endotoxin and three additional uncharacterized proteins which might constitute novel virulence factors of this pathogenic bacterium. The analysis of the T2SS-dependent secretome of P. damselae subsp. damselae also led to the identification of RstAB-independent potential virulence factors as lipoproteins, Frontiers in sialidases and proteases. The RstAB regulon included plasmid, chromosome I and chromosome II-encoded genes that showed a differential distribution among isolates of this subspecies. This study establishes RstAB as a major regulator of virulence and diverse cellular functions in P. damselae subsp. damselaeThis work has been supported by grant AGL2016-79738-R (AEI/FEDER, EU) from the State Agency for Research (AEI) of Spain, and co-funded by the FEDER Programme from the European Union. The support of Xunta de Galicia (Spain) with grant ED431C 2018/18 is also acknowledged. MT thanks the Brazilian Ministry of Education and CAPES (Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior) for a predoctoral fellowship. XM thanks Xunta de Galicia for a predoctoral fellowship. AdV was supported by the FCT fellowship SFRH/BPD/95777/2013. The mass spectrometry technique was performed at the Proteomics i3S Scientific Platform with the assistance of Hugo Osório. This work had support from the Portuguese Mass Spectrometry Network, integrated in the National Roadmap of Research Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125)S

A Secreted NlpC/P60 Endopeptidase from Photobacterium damselae subsp. piscicida Cleaves the Peptidoglycan of Potentially Competing Bacteria

Peptidoglycan (PG) is a major component of the bacterial cell wall, forming a mesh-like structure enwrapping the bacteria that is essential for maintaining structural integrity and providing support for anchoring other components of the cell envelope. PG biogenesis is highly dynamic and requires multiple enzymes, including several hydrolases that cleave glycosidic or amide bonds in the PG. This work describes the structural and functional characterization of an NlpC/P60-contain-ing peptidase from Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes high mortality of warm-water marine fish with great impact for the aquaculture industry. PnpA (Photobacterium NlpC-like protein A) has a four-domain structure with a hydrophobic and narrow access to the catalytic center and specificity for the γ-D-glutamyl-meso-diaminopimelic acid bond. However, PnpA does not cleave the PG of Phdp or PG of several Gram-negative and Gram-positive bacterial species. Interestingly, it is secreted by the Phdp type II secretion system and degrades the PG of Vibrio anguillarum and Vibrio vulnificus. This suggests that PnpA is used by Phdp to gain an advantage over bacteria that compete for the same resources or to obtain nutrients in nutrient-scarce environments. Comparison of the muropeptide composition of PG susceptible and resistant to the catalytic activity of PnpA showed that the global content of muropeptides is similar, suggesting that susceptibility to PnpA is determined by the three-dimensional organization of the muropeptides in the PG. IMPORTANCE Peptidoglycan (PG) is a major component of the bacterial cell wall formed by long chains of two alternating sugars interconnected by short peptides, generating a mesh-like structure that enwraps the bacterial cell. Although PG provides structural integrity and support for anchoring other components of the cell envelope, it is constantly being remodeled through the action of specific enzymes that cleave or join its components. Here, it is shown that Photobacterium damselae subsp. piscicida, a bacterium that causes high mortality in warm-water marine fish, produces PnpA, an enzyme that is secreted into the environment and is able to cleave the PG of potentially competing bacteria, either to gain a competitive advantage and/or to obtain nutrients. The specificity of PnpA for the PG of some bacteria and its inability to cleave others may be explained by differences in the structure of the PG mesh and not by different muropeptide composition.Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 Operacional Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior and Fundação para a Ciência e a Tecnologia (FCT), I.P., within the scope of the Norma Transitória - DL57/2016/CP1355/CT0010. This work had also support from the State Agency for Research (AEI) of Spain cofunded by the FEDER Program from the European Union (grants AGL2016-79738-R and BIO2016-77639-P

MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence

The foodborne pathogen Listeria monocytogenes (Lm) causes invasive infection in susceptible ani- mals and humans. To survive and proliferate within hosts, this facultative intracellular pathogen tightly coordinates the expression of a complex regulatory network that controls the expression of virulence fac- tors. Here, we identified and characterized MouR, a novel virulence regulator of Lm. Through RNA-seq transcriptomic analysis, we determined the MouR regulon and demonstrated how MouR positively con- trols the expression of the Agr quorum sensing sys- tem (agrBDCA) of Lm. The MouR three-dimensional structure revealed a dimeric DNA-binding transcrip- tion factor belonging to the VanR class of the GntR superfamily of regulatory proteins. We also showed that by directly binding to the agr promoter region, MouR ultimately modulates chitinase activity and biofilm formation. Importantly, we demonstrated by in vitro cell invasion assays and in vivo mice infec- tions the role of MouR in Lm virulence.Peer reviewe

Structural and functional studies of DprA and its partners involved in the natural genetic transformation

La transformation génétique naturelle est un mode de transfert horizontal de gènes chez les bactéries, qui contribue au maintien et à l'évolution de leurs génomes. C’est un mécanisme clé pour l’adaptation des bactéries, qui pourrait être responsable de la transmission des résistances aux antibiotiques observée en clinique chez certaines espèces pathogènes (S. pneumoniae, H. pylori,…). La transformation naturelle s’effectue par l’internalisation d’ADN exogène à travers la membrane, puis par sa prise en charge jusqu’à son intégration dans le chromosome bactérien par recombinaison homologue. Le processus de prise en charge fait intervenir la protéine DprA, très conservée dans le monde bactérien, impliquée dans la protection de l’ADN entrant contre les nucléases, et dans le recrutement de la recombinase universelle RecA sur l’ADNsb. DprA joue donc un rôle majeur et a récemment été décrite comme étant impliquée dans d’autres aspects de la transformation génétique naturelle, comme la fermeture de la compétence via une interaction directe avec le régulateur de réponse ComE, ou la levée de la barrière du système de restriction-modification afin de faciliter la transformation. Chez H. pylori, DprA est en opéron avec DprB, suggérant l’implication de ces 2 protéines dans une même voie et une interaction directe entre elles. DprA apparaît donc comme étant au cœur d’un véritable réseau d’interaction, protéique et nucléique.The natural genetic transformation is a mode of horizontal gene transfer that contributes to the maintenance and to the evolution of the genomes in bacteria. It is a key mechanism for their adaptation which could be responsible for the transmission of antibiotic resistances observed clinically for some pathogenic species (S. pneumoniae, H. pylori...). Natural transformation is performed by internalizing exogenous DNA followed by its processing and its integration into the bacterial chromosome by homologous recombination. The DNA processing involves the highly conserved DprA protein for the protection of the incoming DNA against nucleases and the recruitment of the universal recombinase RecA on ssDNA. DprA plays a key role and has recently been suggested to be involved in other aspects of the natural genetic transformation, such as the shut-off of the competence via a direct interaction with the response regulator ComE, or removal of the restriction-modification barrier system in order to facilitate the processing. In H. pylori, the dprA gene is in operon with dprB, whose function is unknown, suggesting their involvement in the same pathway and their likely direct interaction. DprA appears to be central in protein/nucleic acid interactions network

Etude structurale et fonctionnelle de DprA et de ses partenaires au cours de la transformation génétique naturelle

La transformation génétique naturelle est un mode de transfert horizontal de gènes chez les bactéries, qui contribue au maintien et à l'évolution de leurs génomes. C est un mécanisme clé pour l adaptation des bactéries, qui pourrait être responsable de la transmission des résistances aux antibiotiques observée en clinique chez certaines espèces pathogènes (S. pneumoniae, H. pylori, ). La transformation naturelle s effectue par l internalisation d ADN exogène à travers la membrane, puis par sa prise en charge jusqu à son intégration dans le chromosome bactérien par recombinaison homologue. Le processus de prise en charge fait intervenir la protéine DprA, très conservée dans le monde bactérien, impliquée dans la protection de l ADN entrant contre les nucléases, et dans le recrutement de la recombinase universelle RecA sur l ADNsb. DprA joue donc un rôle majeur et a récemment été décrite comme étant impliquée dans d autres aspects de la transformation génétique naturelle, comme la fermeture de la compétence via une interaction directe avec le régulateur de réponse ComE, ou la levée de la barrière du système de restriction-modification afin de faciliter la transformation. Chez H. pylori, DprA est en opéron avec DprB, suggérant l implication de ces 2 protéines dans une même voie et une interaction directe entre elles. DprA apparaît donc comme étant au cœur d un véritable réseau d interaction, protéique et nucléique.The natural genetic transformation is a mode of horizontal gene transfer that contributes to the maintenance and to the evolution of the genomes in bacteria. It is a key mechanism for their adaptation which could be responsible for the transmission of antibiotic resistances observed clinically for some pathogenic species (S. pneumoniae, H. pylori...). Natural transformation is performed by internalizing exogenous DNA followed by its processing and its integration into the bacterial chromosome by homologous recombination. The DNA processing involves the highly conserved DprA protein for the protection of the incoming DNA against nucleases and the recruitment of the universal recombinase RecA on ssDNA. DprA plays a key role and has recently been suggested to be involved in other aspects of the natural genetic transformation, such as the shut-off of the competence via a direct interaction with the response regulator ComE, or removal of the restriction-modification barrier system in order to facilitate the processing. In H. pylori, the dprA gene is in operon with dprB, whose function is unknown, suggesting their involvement in the same pathway and their likely direct interaction. DprA appears to be central in protein/nucleic acid interactions network.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Susceptibility of Sea Bream (Sparus aurata) to AIP56, an AB-Type Toxin Secreted by Photobacterium damselae subsp. piscicida

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative bacterium that infects a large number of marine fish species in Europe, Asia, and America, both in aquacultures and in the natural environment. Among the affected hosts are economically important cultured fish, such as sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), yellowtail (Seriola quinqueradiata), and cobia (Rachycentron canadum). The best characterized virulence factor of Phdp is the Apoptosis-Inducing Protein of 56 kDa (AIP56), a secreted AB-type toxin that has been shown to induce apoptosis of sea bass phagocytes during infection. AIP56 has an A subunit that displays metalloprotease activity against NF-kB p65 and a B subunit that mediates binding and internalization of the A subunit in susceptible cells. Despite the fact that the aip56 gene is highly prevalent in Phdp isolates from different fish species, the toxicity of AIP56 has only been studied in sea bass. In the present study, the toxicity of AIP56 for sea bream was evaluated. Ex vivo assays showed that sea bream phagocytes are resistant to AIP56 cytotoxicity and that resistance was associated with an inefficient internalization of the toxin by those cells. Accordingly, in vivo intoxication assays revealed that sea bream is much more resistant to AIP56-induced lethality than sea bass. These findings, showing that the effect of AIP56 is different in sea bass and sea bream, set the basis for future studies to characterize the effects of AIP56 and to fully elucidate its virulence role in different Phdp susceptible hosts

PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria

International audiencePprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation

The Carboxy-Terminal αN Helix of the Archaeal XerA Tyrosine Recombinase Is a Molecular Switch to Control Site-Specific Recombination

International audienceTyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers