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Quorum sensing (QS) is a communication system used by bacteria to coordinate a wide panel of biological functions in a cell density-dependent manner. The Gram-negative Chromobacterium violaceum has previously been shown to use an acyl-homoserine lactone (AHL)-based QS to regulate various behaviors, including the production of proteases, hydrogen cyanide, or antimicrobial compounds such as violacein. By using combined metabolomic and proteomic approaches, we demonstrated that QS modulates the production of antimicrobial and toxic compounds in C. violaceum ATCC 12472. We provided the first evidence of anisomycin antibiotic production by this strain as well as evidence of its regulation by QS and identified new AHLs produced by C. violaceum ATCC 12472. Furthermore, we demonstrated that targeting AHLs with lactonase leads to major QS disruption yielding significant molecular and phenotypic changes. These modifications resulted in drastic changes in social interactions between C. violaceum and a Gram-positive bacterium (Bacillus cereus), a yeast (Saccharomyces cerevisiae), immune cells (murine macrophages), and an animal model (planarian Schmidtea mediterranea). These results underscored that AHL-based QS plays a key role in the capacity of C. violaceum to interact with micro- and macroorganisms and that quorum quenching can affect microbial population dynamics beyond AHL-producing bacteria and Gram-negative bacteria.

Organophosphorus compounds (OPs) are neurotoxic molecules developed as pesticides and chemical warfare nerve agents (CWNAs). Most of them are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in nervous systems, and are therefore responsible for numerous poisonings around the world. Many animal models have been studied over the years in order to decipher the toxicity of OPs and to provide insights for therapeutic and decontamination purposes. Environmental impact on wild animal species has been analyzed to understand the consequences of OP uses in agriculture. In complement, various laboratory models, from invertebrates to aquatic organisms, rodents and primates, have been chosen to study chronic and acute toxicity as well as neurobehavioral impact, immune response, developmental disruption, and other pathological signs. Several decontamination approaches were developed to counteract the poisoning effects of OPs. Among these, enzyme-based strategies are particularly attractive as they allow efficient external decontamination without toxicity or environmental impact and may be of interest for treatment. Approaches using bioscavengers for prophylaxis, treatment, and external decontamination are emphasized and their potential is discussed in the light of toxicological observations from various animal models. The relevance of animal models, regarding their cholinergic system and the abundance of naturally protecting enzymes, is also discussed for better extrapolation of results to human.

International audience; Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density-dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

Quorum Sensing (QS) is a communication system used by numerous bacteria to synchronize their behavior according to the cell density. In this way, bacteria secrete and sense small mediating molecules, called autoinducers (AI), which concentration increases in the environment proportionally to bacterial cell number. QS induces major physiological and phenotypic changes such as virulence induction and biofilm formation. Biofilm represents a physical barrier which shelters bacteria poorly sensitive to antimicrobial treatments and favors the apparition of resistance mechanisms. Disturbing QS is referred to as quorum quenching (QQ). This strategy is used by microorganisms themselves to prevent the development of specific group behaviors. Two strategies are mainly employed: the use of quorum sensing inhibitors (QSI) and of quorum quenching enzymes (QQE) that degrades AI. Many studies have been dedicated to identifying QSI (natural or synthetic) as well as QQE and demonstrating their anti-virulence and anti-biofilm effects on numerous bacterial species. Synergistic effects between QQ and traditional treatments such as antibiotherapy or with reemerging phage therapy have been put forward. The efficiency of numerous QSI and QQE was thereby demonstrated either with in vitro or in vivo animal models leading to the development of medical devices containing QSI and QQE to improve already existing treatments.

Quorum sensing (QS) is a molecular communication system that bacteria use to harmonize the regulation of genes in a cell density-dependent manner. In proteobacteria, QS is involved, among others, in virulence, biofilm formation or CRISPR-Cas gene regulation. Here, we report for the first time the effect of a QS-interfering enzyme to alter the regulation of CRISPR-Cas systems in model and clinical strains of Pseudomonas aeruginosa, as well as in the marine bacterium Chromobacterium violaceum CV12472. The expression of CRISPR-Cas genes decreased in most cases suggesting that enzymatic disruption of QS is promising for modulating phage-bacteria interactions.

Background: Pseudomonas aeruginosa orchestrates the expression of many genes by using quorum sensing (QS) communication process. QS relies on an intricate molecular network including two acyl-homoserine lactones (AHL), involved in the modulation of virulence factors production, biofilm formation or antimicrobial sensitivity. Disrupting QS is a promising approach to modulate virulence while not challenging bacterial survival and can be achieved with AHL-degrading lactonases. Methods: Here, we used two lactonases both targeting the signal molecules N-(3-oxododecanoyl)-L-Homoserine lactone (3-oxo-C12 HSL) and butyryl-homoserine lactone (C4 HSL) albeit with different efficacy on C4 HSL. We used phenotypic analyses to characterize the specific impact of each lactonases on P. aeruginosa virulence. We further used molecular approaches for finely determine the impact of lactonases on gene regulation and protein expressions of P. aeruginosa. Findings: Although, both lactonases similarly impacted AHL-based circuits, strong variations were however observed in Pseudomonas Quinolone Signal regulation yielding in different impacts on phenotypic traits related to virulence, biofilm, pathogenicity or antibiotic susceptibility. Proteomic analysis further allowed to identify the specific impacts of lactonases on P. aeruginosa behavior. Interpretation: This global analysis provides the first evidence that QQ enzyme specificity is crucial to modulate QS-associated behavior in P. aeruginosa PA14 and will help to develop efficient quorum quenching strategies. Funding Statement: This work was supported by a project RAPID (LACTO-TEX) from Direction Generale de l'Armement (DGA, France), "Investissements d'avenir" program (Mediterranee Infection 10-IAHU-03) of the French Agence Nationale de la Recherche (ANR), "Emplois Jeunes Doctorants" program of Region Provence-Alpes-Cote d'Azur (PACA, France) and MnDrive initiative. Declaration of Interests: ME and EC have a patent WO2014167140 A1 licensed to Gene&GreenTK. LP, DD, BR, ME and EC report personal fees from Gene&GreenTK during the conduct of the study. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics Approval Statement: Not required.

International audience; Most bacteria use a communication system known as quorum sensing which relies on the secretion and perception of small molecules called autoinducers enabling bacteria to adapt their behavior according to the population size and synchronize the expression of genes involved in virulence, antimicrobial resistance and biofilm formation. Methods have emerged to inhibit bacterial communication and limit their noxious traits. Chemical inhibitors, sequestering antibodies and degrading enzymes have been developed and proved efficient to decrease bacterial virulence both in vitro and in vivo. This strategy, named quorum quenching, also showed synergistic effects with traditional antibacterial treatments by increasing bacterial susceptibility to antibiotics. Thereby quorum quenching constitutes an interesting therapeutic strategy to fight against bacterial infections and limit the consequences of antibiotic resistance.; La plupart des bactéries utilisent un système de communication, le quorum sensing, fondé sur la sécrétion et la perception de petites molécules appelées autoinducteurs qui leur permettent d’adapter leur comportement en fonction de la taille de la population. Les bactéries mutualisent ainsi leurs efforts de survie en synchronisant entre elles la régulation de gènes impliqués notamment dans la virulence, la résistance aux antimicrobiens ou la formation du biofilm. Des méthodes ont vu le jour pour inhiber cette communication entre bactéries et limiter leurs effets nocifs. Des inhibiteurs chimiques, des anticorps ou encore des enzymes capables d’interférer avec les autoinducteurs ont été développés et se sont montrés efficaces pour diminuer la virulence des bactéries à la fois in vitro et in vivo. Cette stratégie, appelée quorum quenching, a également montré des effets synergiques avec des traitements antibactériens classiques. Il permettrait notamment d’augmenter la sensibilité des bactéries aux antibiotiques. Ceci constitue une piste thérapeutique prometteuse pour lutter contre les infections bactériennes et limiter les conséquences de l’antibiorésistance.

Many bacteria use extracellular signaling molecules to coordinate group behavior, a process referred to as quorum sensing (QS). However, some QS molecules are hydrophobic in character and are probably unable to diffuse across the bacterial cell envelope. How these molecules are disseminated between bacterial cells within a population is not yet fully understood. Here, we show that the marine pathogen Vibrio harveyi packages the hydrophobic QS molecule CAI-1, a long-chain amino ketone, into outer membrane vesicles. Electron micrographs indicate that outer membrane vesicles of variable size are predominantly produced and released into the surroundings during the stationary phase of V. harveyi, which correlates with the timing of CAI-1-dependent signaling. The large vesicles (diameter

Quorum sensing (QS) is a communication process that enables a bacterial population to coordinate and synchronize specific behaviors. The bioluminescent marine bacterium Vibrio harveyi integrates three autoinducer (AI) signals into one quorum-sensing cascade comprising a phosphorelay involving three hybrid sensor kinases: LuxU; LuxO, an Hfq/small RNA (sRNA) switch; and the transcriptional regulator LuxR. Using a new set of V. harveyi mutants lacking genes for the AI synthases and/or sensors, we assayed the activity of the quorum-sensing cascade at the population and single-cell levels, with a specific focus on signal integration and noise levels. We found that the ratios of kinase activities to phosphatase activities of the three sensors and, hence, the extent of phosphorylation of LuxU/LuxO are important not only for the signaling output but also for the degree of noise in the system. The pools of phosphorylated LuxU/LuxO per cell directly determine the amounts of sRNAs produced and, consequently, the copy number of LuxR, generating heterogeneous quorum-sensing activation at the single-cell level. We conclude that the ability to drive the heterogeneous expression of QS-regulated genes in V. harveyi is an inherent feature of the architecture of the QS cascade. IMPORTANCE V. harveyi possesses one of the most complex quorum-sensing (QS) cascades known, using three different autoinducers (AIs) to control the induction of, e.g., bioluminescence, virulence factors, and biofilm and exoprotease production. We constructed various V. harveyi mutants to study the impact of each component and subsystem of the QS signaling cascade on QS activation at the population and single-cell levels. We found that the output was homogeneous only in the presence of all AIs. In the absence of any one AI, QS activation varied from cell to cell, resulting in phenotypic heterogeneity. This study elucidates a molecular design principle which enables a tightly integrated signaling cascade to control the expression of diverse phenotypes within a genetically homogeneous population.

1st International Conference on Chemical Biological Radiological and Nuclear, Research and Innovation (CBRN-RI), Antibes Juan les Pins, FRANCE, MAR 16-18, 2015; International audience; Numerous bacteria use quorum sensing (QS) to synchronize their behavior and monitor their population density. They use signaling molecules known as autoinducers (AI's) that are synthesized and secreted into their local environment to regulate QS-dependent gene expression. Among QS-regulated pathways, biofilm formation and virulence factor secretion are particularly problematic as they are involved in surface -attachment, antimicrobial agent resistance, toxicity, and pathogenicity. Targeting QS represents a promising strategy to inhibit undesirable bacterial traits. This strategy, referred to as quorum quenching (QQ), includes QS-inhibitors and QQ enzymes. These approaches are appealing because they do not directly challenge bacterial survival, and consequently selection pressure may be low, yielding a lower occurrence of resistance. QQ enzymes are particularly promising because they act extracellularly to degrade AI's and can be used in catalytic quantities. This review draws an overview of QQ enzyme related applications, covering several economically important fields such as agriculture, aquaculture, biofouling and health issues. Finally, the possibility of resistance mechanism occurrence to QQ strategies is discussed. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Extremozymes have gained considerable interest as they could meet industrial requirements. Among these, SsoPox is a hyperthermostable enzyme isolated from the archaeon Sulfolobus solfataricus. This enzyme is a lactonase catalyzing the hydrolysis of acyl-homoserine lactones; these molecules are involved in Gram-negative bacterial communication referred to as quorum sensing. SsoPox exhibits promiscuous phosphotriesterase activity for the degradation of organophosphorous chemicals including insecticides and chemical warfare agents. Owing to its bi-functional catalytic abilities as well as its intrinsic stability, SsoPox is appealing for many applications, having potential uses in the agriculture, defense, food and health industries. Here we investigate the biotechnological properties of the mutant SsoPox-W263I, a variant with increased lactonase and phosphotriesterase activities. We tested enzyme resistance against diverse process-like and operating conditions such as heat resistance, contact with organic solvents, sterilization, storage and immobilization. Bacterial secreted materials from both Gram-negative and positive bacteria were harmless on SsoPox-W263I activity and could reactivate heat-inactivated enzyme. SsoPox showed resistance to harsh conditions demonstrating that it is an extremely attractive enzyme for many applications. Finally, the potential of SsoPox-W263I to be active at subzero temperature is highlighted and discussed in regards to the common idea that hyperthermophile enzymes are nearly inactive at low temperatures.

Phages are bacteria targeting viruses and represent the most abundant biological entities on earth. Marine environments are exceptionally rich in bacteriophages, harboring a total of 4x10(30) viruses. Nevertheless, marine phages remain poorly characterized. Here we describe the identification of intact prophage sequences in the genome of the marine gamma-proteobacterium Vibrio campbellii ATCC BAA-1116 (formerly known as V. harveyi ATCC BAA-1116), which presumably belong to the family of Myoviridae. One prophage was found on chromosome I and shows significant similarities to the previously identified phage Phi HAP-1. The second prophage region is located on chromosome II and is related to Vibrio phage kappa. Exposure of V. campbellii to mitomycin C induced the lytic cycle of two morphologically distinct phages and, as expected, extracellular DNA from induced cultures was found to be specifically enriched for the sequences previously identified as prophage regions. Heat stress (50 degrees C, 30 min) was also found to induce phage release in V. campbellii. Notably, promoter activity of two representative phage genes indicated heterogeneous phage induction within the population.

Summary Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most impor- tant bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R. solanacearum 'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this dele- tion is responsible for almost complete loss of patho- genicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional comple- mentation approaches which identified the methion- ine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine aux- otrophy nor reduced ability to multiply in planta .I n addition, this study indicated that three type III secre- tion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.

International audience; Background: Ralstonia solanacearum is a soil-borne beta-proteobacterium that causes bacterial wilt disease in many food crops and is a major problem for agriculture in intertropical regions. R. solanacearum is a heterogeneous species, both phenotypically and genetically, and is considered as a species complex. Pathogenicity of R. solanacearum relies on the Type III secretion system that injects Type III effector (T3E) proteins into plant cells. T3E collectively perturb host cell processes and modulate plant immunity to enable bacterial infection. Results: We provide the catalogue of T3E in the R. solanacearum species complex, as well as candidates in newly sequenced strains. 94 T3E orthologous groups were defined on phylogenetic bases and ordered using a uniform nomenclature. This curated T3E catalog is available on a public website and a bioinformatic pipeline has been designed to rapidly predict T3E genes in newly sequenced strains. Systematical analyses were performed to detect lateral T3E gene transfer events and identify T3E genes under positive selection. Our analyses also pinpoint the RipF translocon proteins as major discriminating determinants among the phylogenetic lineages. Conclusions: Establishment of T3E repertoires in strains representatives of the R. solanacearum biodiversity allowed determining a set of 22 T3E present in all the strains but provided no clues on host specificity determinants. The definition of a standardized nomenclature and the optimization of predictive tools will pave the way to understanding how variation of these repertoires is correlated to the diversification of this species complex and how they contribute to the different strain pathotypes.

Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most important bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R. solanacearum'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this deletion is responsible for almost complete loss of pathogenicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional complementation approaches which identified the methionine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine auxotrophy nor reduced ability to multiply in planta. In addition, this study indicated that three type III secretion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.

Janthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes. peerReviewed