Les PLLs sont une famille d’enzyme dont l’activité catalytique est double. L’activité lactonase permet entre autre de détruire les AHLs, molécules médiatrices de la communication chez les bactéries Gram négatives, empêchant ainsi la synchronisation à l’échelle de la population de la sécrétion de facteurs de virulence ou la formation de biofilm. Elles sont non seulement capables d’hydrolyser les molécules possédant un noyau lactone, mais aussi les phosphotriésters par promiscuité de substrat. L’activité phosphotriestérase permet de dégrader les composés organophosphorés (OPs) hautement toxiques, que ce soit les insecticides (Paraoxon) ou les agents neurotoxiques de guerre (Sarin, VX). Les travaux effectués ont consisté à évaluer l’efficacité de la PLL SsoPox, provenant de l’archée extrêmophile Sulfolobus solfataricus, pour empêcher la mise en place de la virulence et du biofilm chez Pseudomonas aeruginosa PAO1. Cette évaluation a été effectuée in vitro et in vivo via un modèle d’infection pulmonaire chez le rat. D’autre part, une étude in vitro a été réalisée sur 73 souches cliniques de P. aeruginosa isolés sur des patients atteints de pied diabétique, en évaluant la quantité de pyocyanine et l’activité protéolytique. Enfin, une phase prospective pour identifier de nouvelles PLLs a permis la caractérisation enzymatique et structurale de 2 nouvelles enzymes, SacPox et VmoLac, contribuant ainsi à affiner la connaissance sur cette famille et leur potentiel d’amélioration par ingénierie protéique. Au final, les PLLs offrent un intérêt biotechnologique majeur et peuvent mener à une valorisation concrète pour la santé humaine mais également pour la bioremédiation des OPs. / Phosphotriesterase-Like Lactonases (PLLs) are a family of enzyme displaying a dual catalytic activity. Lactonase activity allows for, among others, the destruction of Acyl Homoserine Lactones (AHLs). These molecules mediate the communication in Gram negative bacteria, allowing them to synchronize group behavior like virulence factor secretion or biofilm formation. Beside their ability to hydrolyze molecules with a lactones moiety, PLL also show substrate promiscuity for hydrolyzing highly toxic organophosphate compounds (OPs), such as pesticides (i.e. Paraoxon) or Chemical Warfare Nerve Agents (CWNAs) (e.g. Sarin & VX). The work described here consisted in evaluating the efficacy of the PLL SsoPox, originating from the extremophile Archaea Sulfolobus solfataricus, to prevent virulence and biofilm formation on the model strain Pseudomonas aeruginosa PAO1. This evaluation was performed both in vitro and in vivo, by using a rat pulmonary infection model. A study has been performed on a strain collection of 73 clinical strains of P. aeruginosa isolated from diabetic foot, to assess the enzyme effects on pyocyanin secretion and protease activity. Finally, a prospective phase to identify new PLLs allowed for the enzymatic and structural characterization of two new enzymes, SacPox and VmoLac, thus contributing to refine the knowledge about this enzyme family and their engineering potential. In conclusion, PLLs are of prime biotechnological interest and could lead to developments in human health, but also for OPs bioremediation with a non aggressive decontamination solution.

Gram-negative bacteria use N-acyl homoserine lactones (AHLs) to sense population density and regulate gene expression, including virulent phenotypes. The quorum-quenching AHL lactonase from Bacillus thuringiensis cleaves the lactone ring of AHLs, disabling this mode of gene regulation. Despite the potential applications of this enzyme as an antibacterial weapon, little was known about it's lactone ring-opening mechanism. As a member of the metallo-beta-lactamase superfamily, AHL lactonase requires two divalent metal ions for catalysis. NMR experiments confirm that these metal ions are also involved in proper enzyme folding. The chemical mechanism of ring opening was explored using isotope incorporation studies, and hydrolysis was determined to proceed via a nucleophilic attack by a solvent-derived hydroxide at the carbonyl of the lactone ring. A transient, kinetically significant metal-leaving group interaction was detected in steady-state kinetic assays with AHL lactonase containing alternative divalent metal ions hydrolyzing a sulfur-containing substrate. High-resolution crystal structures implicated two residues in substrate binding and hydrolysis, Tyr194 and Asp108. Site-directed mutagenesis of these residues followed by steady-state kinetic studies with wild-type and mutant enzymes hydrolyzing a spectrum of AHL substrates revealed that mutations Y194F and D108N significantly affect catalysis. Combining these results allows the proposal of a detailed hydrolytic mechanism. The binding site for the N-acyl hydrophobic moiety was probed using steady-state kinetics with a variety of naturally occurring and non-natural AHL substrates, and these studies indicate that AHL lactonase will accept a broad range of homoserine lactone containing substrates. Crystal structures with AHL substrates and non-hydrolyzable analogs reveal two distinct binding sites for this N-acyl group. Based on the ability of this enzyme to accommodate a variety of substrates, AHL lactonase was shown to have the ability to quench quorum sensing regulated by a newly discovered class of homoserine lactone signal molecules possessing an N-aryl group using a bioassay. Steady-state kinetic studies confirm that this class of signal molecules are indeed substrates for AHL lactonase. / text

Swimming bacteria are ubiquitous in aqueous environments ranging from oceans to fluidic environments within a living host. Furthermore, engineered bacteria are being increasingly utilized for a host of applications including environmental bioremediation, biosensing, and for the treatment of diseases. Often driven by chemotaxis (i.e. biased migration in response to gradients of chemical effectors) and quorum sensing (i.e. number density dependent regulation of gene expression), bacterial population dynamics and emergent behavior play a key role in regulating their own life and their impact on their immediate environment. Computational models that accurately and robustly describe bacterial population behavior and response to environmental stimuli are crucial to both understanding the dynamics of microbial communities and efficiently utilizing engineered microbes in practice. Many existing computational frameworks are finely-detailed at the cellular level, leading to extended computational time requirements, or are strictly population scale models, which do not permit population heterogeneities or spatiotemporal variability in the environment. To bridge this gap, we have created and experimentally validated a scalable, computationally-efficient, agent-based model of bacterial chemotaxis and quorum sensing (QS) which robustly simulates the stochastic behavior of each cell across a wide range of bacterial populations, ranging from a few to several hundred cells. We quantitatively and accurately capture emergent behavior in both isogenic QS populations and the altered QS response in a mixed QS and quorum quenching (QQ) microbial community. Finally, we show that the model can be used to predictively design synthetic genetic components towards programmed microbial behavior. / Master of Science / Bacteria are an integral part of life and possess a host of characteristics that make them a powerful tool with which to confront many modern day problems. Using genetic engineering and the burgeoning field of synthetic biology, these single-celled organisms can be manipulated to perform many useful tasks such as detecting oil spills or other environmental pollutants, producing pharmaceuticals such as insulin, and even invading and killing cancer cells. Accurate computational simulations of microbial behavior will aid in the efficient design of such synthetic bacteria-based systems and reduce dependency on the current time-intensive “guess and check” paradigm. Towards this goal, we have built a comprehensive computer simulation of bacterial swimming behavior, response to chemo-effector concentration gradients called chemotaxis, a form of microbial communication called quorum sensing (QS), and a form of communication disruption called quorum quenching (QQ). Not only do we demonstrate an unprecedented level of accuracy in predicting experimental results, but we also couple the simulation with synthetic biology to precisely tune bacteria QS behavior, neither of which have previously been reported in literature. The overarching outcome of this thesis is a tool that could be used to improve the design process of useful bacteria-based systems in diverse areas of biotechnology.

With the increase of agricultural production, there is an improvement in the use of mineral fertilizers, which may cause different environmental problems, besides the soil salinization. A possible alternative for reducing the application of these products is the use of plant growth-promoting bacteria (PGPB), that can be used alone or in co-inoculation, resulting in an alternative environmentally and economically feasible. Better results can be obtained if the interaction among bacteria-bacteria and bacteria-plant be elucidated, and strategy developed to optimize these interactions. Thus, the plant growth-promoting Bacillus sp. RZ2MS9, previous described as a potential PGPB in maize and soybean, was GFP-tagged and monitored alone and co-inoculated with Azospirillum brasilense (Ab-v5::pWM1013) during maize colonization. The interaction of tagged strains in maize were monitored by fluorescent microscopy (FM) and quantitative PCR (qPCR), demonstrating an endophytic behavior of Bacillus sp. RZ2MS9. Although the non-detection of Ab-v5::pWM1013, the co-inoculation resulted in the best increase in root and shoot dried weight, root volume and in root diameter, showing that inoculation with more than one strain can be a good choice to development of bio-fertilizers. One important system to bacterial interaction is the quorum sensing (QS). The QS is an important cell-cell communication system that allows bacterial cells to recognize their own population and modulate their gene expression. This, system is also involved in the interspecific communication, including other bacterial species and plants. In the other hand, enzymes able to detect and degrade these molecules evolved, the called quorum quenching (QQ) system, that has been evolved in some bacteria as competitive advantage for niches colonization. The aiiA gene, was one of the first gene related with the QQ in Bacillus. The aiiA was found in Bacillus sp. RZ2MS9 genome. Through construction of a new QQ biosensor, Agrobacterium tumefaciens At11006, and validated by A. tumefaciens NTL4, the ability of RZ2MS9 to degrade QS molecules was confirmed. The knockout of aiiA gene was performed using the CRISPR-Cas9 system, confirming this gene function. By these results, the influence of QQ system of Bacillus sp. RZ2MS9 during maize colonization and RZ2MS9 - A. brasilense - maize can be better investigated, opens the possibility to better understand the role of QQ system in the interaction among PGPB and plants. / Concomitantemente ao aumento da produção agrícola, há o aumento do uso de fertilizantes minerais, que pode acarretar no desenvolvimento de diferentes problemas ambientais, além de causar a salinização dos solos. Uma possível alternativa para tentar reduzir a aplicação desses produtos é o uso de bactérias promotoras de crescimento de plantas (BPCPs), que podem ser usadas isoladamente ou em co-inoculação com outras bactérias, tornando-as uma alternativa ambientalmente e economicamente viável. Melhores resultados podem ser obtidos se a interação bactéria-bactéria e bactéria-planta for elucidada, permitindo que estratégias sejam desenvolvidas para otimizar essas interações. Em vista disso, a bactéria Bacillus sp. RZ2MS9, previamente descrita como uma potencial BPCP em milho e soja, foi marcada com GFP e monitorada durante a colonização de milho inoculada sozinha, bem como em co-inoculação com Azospirillum brasilense (Ab-v5::pWM1013). A interação dessas linhagens marcadas em milho, foi monitorada por microscopia de fluorescência (FM) e PCR quantitativo (qPCR), revelando um comportamento endofítico de Bacillus sp. RZ2MS9. Em plantas co-inoculadas, apesar da linhagem Ab-v5::pWM1013 não ter sido detectada por qPCR, a co-inoculação resultou no aumento do peso seco das raízes e da parte aérea, no volume e no diâmetro do sistema radicular, demonstrando que a inoculação com mais de uma linhagem bacteriana pode ser uma boa alternativa para o desenvolvimento de bio-fertilizantes. O quorum sensing (QS) é um importante sistema de comunicação célula-célula que permite que as bactérias reconheçam sua própria população e modulem sua expressão gênica. Este sistema também está envolvido na comunicação interespecífica, incluindo outras espécies bacterianas e plantas. Co-evolutivamente, enzimas capazes de detectar e degradar essas moléculas evoluíram, dando origem ao chamado quorum quenching (QQ), sistema que evoluiu em algumas bactérias como uma vantagem competitiva para a colonização de nichos. O gene aiiA, foi um dos primeiros genes relacionados ao sistema QQ descrito no gênero Bacillus, gene este que foi anotado no genoma de RZ2MS9. Através da construção de uma nova linhagem biossensora de QQ, Agrobacterium tumefaciens At11006, e validada através da linhagem A. tumefaciens NTL4, a capacidade de RZ2MS9 de degradar moléculas de QS foi confirmada. O knockout do gene aiiA foi realizado utilizando o sistema CRISPR-Cas9, confirmando a função desse gene. Através dos resultados obtidos neste trabalho, a influência do sistema QQ de Bacillus sp. RZ2MS9 durante a colonização do milho, bem como a interação RZ2MS9 - A. brasilense - milho pode ser melhor investigada, abrindo a possibilidade de uma melhor compreensão do papel do sistema QQ na interação entre bactérias promotoras de crescimento e plantas.

L’identification, chez divers organismes, d’enzymes de dégradation des N-acyl homosérineslactones (NAHLs) impliquées dans la signalisation QS pose la question de leurs rôles dans lesinteractions bactéries-eucaryotes. Dans une première partie, une synthèse bibliographique analyse lesconnaissances acquises sur ces enzymes dégradant les NAHLs. Dans une seconde partie, la croissancedes bactéries dégradant les signaux NAHLs a été stimulée par l’application de g-caprolactone (GCL)dans la rhizosphère de plants de pommes de terre à des fins de phytoprotection. L’effet de cetraitement sur la diversité des communautés bactériennes rhizosphériques a été évalué en combinantdifférentes approches d’écologie microbienne moléculaire comme la DGGE, le pyroséquençaged’amplicons rrs, et la métagénomique fonctionnelle. Cette dernière approche appliquée à une banquede 30 000 clones environ a conduit à l’identification d’un gène qsdB codant la dégradation des signauxNAHL. Ce travail révèle ainsi l’existence d’une nouvelle classe d’enzymes de dégradation des NAHLsappartenant à la famille des enzymes possédant une signature amidase (AS) dont des membres sontpar ailleurs impliqués dans la dégradation de composés xénobiotiques. Dans une troisième partie, unsystème expérimental a été développé afin de mesurer le transfert conjugatif du plasmide de virulenceTi (tumor inducing) chez des dérivés du pathogène Agrobacterium tumefaciens, appelés «tricheurs»,incapables de produire des signaux NAHLs mais utilisateurs de ceux produits par les autres bactéries.Ce modèle a permis de montrer l’effet modérateur de lactonases dégradant les NAHLs exprimées chezdes agrobactéries produisant les NAHLs, chez des bactéries réceptrices du plasmide Ti, ou des planteshôtes des agrobactéries sur le transfert conjugatif initié par les tricheurs. L’ensemble de ce travailrévèle à la fois une nouvelle famille d’enzymes impliquées dans la dégradation des NAHLs, ainsiqu’un nouveau rôle de ces enzymes dans la modulation des flux de gènes entre bactériesphytopathogènes en interaction avec une plante hôte. / Identification of bacterial and eukaryotic enzymes that degrade N-acyl homoserine lactones(NAHLs) involved in QS signaling raises the question of their roles in bacteria-eucaryotesinteractions. In a first part of this study, a bibliographic report analyzes the current data on thoseNAHL-degrading enzymes. In a second part, the growth of NAHL-degrading bacteria was stimulatedby g-caprolactone (GCL) amendment in potato rhizosphere to protect this plant against the soft-rotpathogen Pectobacterium. The effect of the GCL treatment on rhizospheric bacterial communities wasevaluated by a combination of different molecular microbial ecology techniques such as DGGE,pyrosequencing and functional metagenomic. This last approach was applied to generate ametagenomic library of ca. 30,000 clones and lead to the identification of the qsdB gene that encodesNAHL degradation, This work revealed the occurrence of a novel class of NAHL-degrading enzymesthat belong to the amidase signature (AS) family, some members of which being involved inxenobiotic compound degradation. In a third part, an experimental system was developed to measurethe conjugative transfer of Ti plasmid in various strains of the pathogen Agrobacterium tumefaciens,including “cheaters”, i.e. bacteria unable to produce NAHL signals but capable to use signals producedby other bacteria. Using this model; variations of the plasmid transfer of cheaters were measured whenNAHL-degrading lactonases were expressed in agrobacteria that produce NAHL signals, in recipientbacteria of Ti plasmid, or in agrobacterial host plant. Taken together, thesis experiments revealed anovel class of enzymes involved in NAHL-degradation and a new role for thoses enzymes in themodulation of gene transfer between pathogenic bacteria interacting with host plants.

Muitos genes bacterianos são regulados pelo mecanismo de comunicação denominado quorum sensing (QS). Neste sistema, moléculas sinalizadoras ativam um comportamento de grupo, conforme a densidade celular, permitindo o controle da expressão gênica. Estudos sugerem o potencial de compostos extraídos de plantas sobre o QS, a exemplo da quercetina, um flavonol presente em concentrações elevadas em algumas frutas e hortaliças. Este composto é o flavonoide majoritário presente em cebola (Allium cepa), mas não existem estudos que mostrem a atividade anti-QS de extratos orgânicos deste vegetal. Este trabalho avaliou o potencial antimicrobiano e anti-QS de extratos orgânicos de cebola branca e cebola roxa, assim como de alguns de seus componentes majoritários identificados, em fenótipos regulados pelo QS como a produção de violaceína em Chrormobacterium violaceum ATCC 12472, a motilidade tipo swarming e a formação de biofilmes em Pseudomonas aeruginosa PAO1 e Serratia marcescens MG1. Extratos de cebola branca e roxa foram obtidos por extração em fase sólida utilizando coluna de poliamida e seus compostos identificados e quantificados pelas técnicas de Cromatografia líquida- ionização por elétron spray-espectrometria de massas e cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodo. A atividade antimicrobiana foi avaliada pelas curvas de multiplicação de cada micro-organismo. O efeito dos compostos quercetina aglicona (inibidor do QS já relatado na literatura e encontrado no extrato de cebola roxa) e quercetina-3-β-D-glicosideo (um dos compostos majoritários encontrados em ambos extratos) sobre os micro-organismos utilizados neste estudo foi também avaliado. Foram obtidos três extratos: cebola branca em metanol (CB-MeOH), cebola branca em metanol amônia (CBMeOH/ NH4) e cebola roxa em metanol (CR-MeOH). Os compostos quercetina 3,4\'- diglicosídeio, quercetina-4-glicosídeo, quercetina-3-β-D-glicosideo e quercetina aglicona foram os predominantes nos extratos das duas variedades de cebola. Cianidina-3-O-glicosideo também foi identificada no extrato de cebola roxa. A concentração inibitória mínima (MIC) dos extratos foi igual ou superior a 125 µg/ml (p/v) de extrato seco. Não foi observada inibição significativa da produção de violaceína em C. violaceum pelos extratos orgânicos de cebola e nem pela quercetina-3-β-D-glicosideo, nas concentrações sub-inibitórias avaliadas. No entanto, a quercetina aglicona inibiu significativamente a produção de violaceína em todas as concentrações. A glicosilação da quercetina pode ter afetado sua atividade sobre a inibição da produção de violaceina, já que estudos mostram menor atividade biológica deste composto quando glicosilado. Para a motilidade tipo swarming em P. aeruginosa PAO1 houve inibição significativa pelo extrato de cebola roxa, em todas as concentrações estudadas. Os demais extratos não apresentaram inibição contra este micro-organismo. Para S. marcescens MG1, foi observada inibição da motilidade swarming somente na concentração de 125 µg/ml de CBMeOH/ NH4. As análises de comparação entre os dois tipos de quercetina revelaram que, embora para as duas bactérias testadas os dois compostos apresentaram atividade inibitória sobre a motilidade tipo swarming, a quercetina-3-β-D-glicosideo foi menos eficiente que a quercetina aglicona na concentração de 125 µg/ml. A formação de biofilmes não foi influenciada pelos extratos e, inesperadamente, não se detectou inibição da formação de biofilmes por ambos tipos de quercetina avaliados. De forma geral, os extratos orgânicos de cebola mostraram pouco efeito sobre os fenótipos controlados pelo quorum sensing e a glicosilação da quercetina provavelmente explica a baixa atividade antimicrobiana e anti-QS dos extratos. / Many bacterial genes are regulated by a communication mechanism called quorum sensing (QS). In this system, signaling molecules activate a group behavior according to cell density, allowing the control of gene expression. Studies suggest the inhibitory potential of compounds extracted from plants on the QS system, like quercetin, a flavonol present in high concentrations in some fruits and vegetables. This compound is the main flavonoid found in onion (Allium cepa); however, there are no studies showing the anti-QS activity of organic extracts of this plant. The objective of this work was to evaluate the antimicrobial and anti-QS potential of organic extracts of white and red onions, and their major components studied in QS-regulated phenotypes such as violacein production in Chromobacterium violaceum, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1.White and red onion extracts were obtained by solid phase extraction using a polyamide column and its compounds were identified and quantified by Liquid Chromatography - Electron Spray-Mass Spectrometry and high performance liquid chromatography coupled to diode array detector. O The antimicrobial activity was evaluated by growth curves of each microorganism. The effect of non-glycosylated quercetin (a QS inhibitor already reported in the literature and found in red onion extract) and quercetin-3-β-D-glycoside (one of the major compounds found in both extracts) on the microorganisms used in this study was also evaluated. Three extracts were obtained: white onion in methanol (CB-MeOH), white onion in methanol ammonia (CB-MeOH / NH4) and red onion in methanol (CR-MeOH). Our results showed that quercetin 3,4\'- diglycoside, quercetin-4-glycoside, quercetin-3-β-D-glycoside and non-glycosylated quercetin were predominant in the extracts of the two onion varieties. Cyanidin-3-O-glycoside has also been identified in the purple onion extract. The minimum inhibitory concentration (MIC) of extracts was equal or greater than 125 µg / ml (w / v) of dry extract. There was no significant inhibition of violacein production in C. violaceum by organic onion extracts or by quercetin-3-β- D-glycoside at the sub-inhibitory concentrations evaluated. However, non-glycosylated quercetin showed a significant inhibition of violacein production in all tested concentrations. The glycosylation of Quercetin could have altered its inhibition activity towards violacein production, and in fact, some studies have shown less biological activity of some phenolic compounds when they have been glycosylated. For swarming motility in P. aeruginosa PAO1 there was significant inhibition by red onion extract, in all studied concentrations. The other extracts did not present inhibition against this microorganism. For S. marcescens MG1, inhibition of swarming motility was observed only at the concentration of 125 µg / ml of CB-MeOH / NH4. Comparative analyses between the two types of quercetin showed that, although for the two bacteria tested the two compounds showed inhibitory activity on swarming motility, quercetin-3-β-D-glycoside was less efficient than non-glycosylated quercetin in the concentration of 125 µg / ml. Biofilm formation was not influenced by the extracts and unexpectedly, both types of quercetin evaluated did not show inhibition towards biofilm formation. In general, organic onion extracts showed little effect on quorum sensing controlled phenotypes and glycosylation of quercetin probably explains the low antimicrobial and anti-QS activity of the extracts.

Muitos genes bacterianos são regulados pelo mecanismo de comunicação denominado quorum sensing (QS). Neste sistema, moléculas sinalizadoras ativam um comportamento de grupo, conforme a densidade celular, permitindo o controle da expressão gênica. Estudos sugerem o potencial de compostos extraídos de plantas sobre o QS, a exemplo da quercetina, um flavonol presente em concentrações elevadas em algumas frutas e hortaliças. Este composto é o flavonoide majoritário presente em cebola (Allium cepa), mas não existem estudos que mostrem a atividade anti-QS de extratos orgânicos deste vegetal. Este trabalho avaliou o potencial antimicrobiano e anti-QS de extratos orgânicos de cebola branca e cebola roxa, assim como de alguns de seus componentes majoritários identificados, em fenótipos regulados pelo QS como a produção de violaceína em Chrormobacterium violaceum ATCC 12472, a motilidade tipo swarming e a formação de biofilmes em Pseudomonas aeruginosa PAO1 e Serratia marcescens MG1. Extratos de cebola branca e roxa foram obtidos por extração em fase sólida utilizando coluna de poliamida e seus compostos identificados e quantificados pelas técnicas de Cromatografia líquida- ionização por elétron spray-espectrometria de massas e cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodo. A atividade antimicrobiana foi avaliada pelas curvas de multiplicação de cada micro-organismo. O efeito dos compostos quercetina aglicona (inibidor do QS já relatado na literatura e encontrado no extrato de cebola roxa) e quercetina-3-β-D-glicosideo (um dos compostos majoritários encontrados em ambos extratos) sobre os micro-organismos utilizados neste estudo foi também avaliado. Foram obtidos três extratos: cebola branca em metanol (CB-MeOH), cebola branca em metanol amônia (CBMeOH/ NH4) e cebola roxa em metanol (CR-MeOH). Os compostos quercetina 3,4\'- diglicosídeio, quercetina-4-glicosídeo, quercetina-3-β-D-glicosideo e quercetina aglicona foram os predominantes nos extratos das duas variedades de cebola. Cianidina-3-O-glicosideo também foi identificada no extrato de cebola roxa. A concentração inibitória mínima (MIC) dos extratos foi igual ou superior a 125 µg/ml (p/v) de extrato seco. Não foi observada inibição significativa da produção de violaceína em C. violaceum pelos extratos orgânicos de cebola e nem pela quercetina-3-β-D-glicosideo, nas concentrações sub-inibitórias avaliadas. No entanto, a quercetina aglicona inibiu significativamente a produção de violaceína em todas as concentrações. A glicosilação da quercetina pode ter afetado sua atividade sobre a inibição da produção de violaceina, já que estudos mostram menor atividade biológica deste composto quando glicosilado. Para a motilidade tipo swarming em P. aeruginosa PAO1 houve inibição significativa pelo extrato de cebola roxa, em todas as concentrações estudadas. Os demais extratos não apresentaram inibição contra este micro-organismo. Para S. marcescens MG1, foi observada inibição da motilidade swarming somente na concentração de 125 µg/ml de CBMeOH/ NH4. As análises de comparação entre os dois tipos de quercetina revelaram que, embora para as duas bactérias testadas os dois compostos apresentaram atividade inibitória sobre a motilidade tipo swarming, a quercetina-3-β-D-glicosideo foi menos eficiente que a quercetina aglicona na concentração de 125 µg/ml. A formação de biofilmes não foi influenciada pelos extratos e, inesperadamente, não se detectou inibição da formação de biofilmes por ambos tipos de quercetina avaliados. De forma geral, os extratos orgânicos de cebola mostraram pouco efeito sobre os fenótipos controlados pelo quorum sensing e a glicosilação da quercetina provavelmente explica a baixa atividade antimicrobiana e anti-QS dos extratos. / Many bacterial genes are regulated by a communication mechanism called quorum sensing (QS). In this system, signaling molecules activate a group behavior according to cell density, allowing the control of gene expression. Studies suggest the inhibitory potential of compounds extracted from plants on the QS system, like quercetin, a flavonol present in high concentrations in some fruits and vegetables. This compound is the main flavonoid found in onion (Allium cepa); however, there are no studies showing the anti-QS activity of organic extracts of this plant. The objective of this work was to evaluate the antimicrobial and anti-QS potential of organic extracts of white and red onions, and their major components studied in QS-regulated phenotypes such as violacein production in Chromobacterium violaceum, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1.White and red onion extracts were obtained by solid phase extraction using a polyamide column and its compounds were identified and quantified by Liquid Chromatography - Electron Spray-Mass Spectrometry and high performance liquid chromatography coupled to diode array detector. O The antimicrobial activity was evaluated by growth curves of each microorganism. The effect of non-glycosylated quercetin (a QS inhibitor already reported in the literature and found in red onion extract) and quercetin-3-β-D-glycoside (one of the major compounds found in both extracts) on the microorganisms used in this study was also evaluated. Three extracts were obtained: white onion in methanol (CB-MeOH), white onion in methanol ammonia (CB-MeOH / NH4) and red onion in methanol (CR-MeOH). Our results showed that quercetin 3,4\'- diglycoside, quercetin-4-glycoside, quercetin-3-β-D-glycoside and non-glycosylated quercetin were predominant in the extracts of the two onion varieties. Cyanidin-3-O-glycoside has also been identified in the purple onion extract. The minimum inhibitory concentration (MIC) of extracts was equal or greater than 125 µg / ml (w / v) of dry extract. There was no significant inhibition of violacein production in C. violaceum by organic onion extracts or by quercetin-3-β- D-glycoside at the sub-inhibitory concentrations evaluated. However, non-glycosylated quercetin showed a significant inhibition of violacein production in all tested concentrations. The glycosylation of Quercetin could have altered its inhibition activity towards violacein production, and in fact, some studies have shown less biological activity of some phenolic compounds when they have been glycosylated. For swarming motility in P. aeruginosa PAO1 there was significant inhibition by red onion extract, in all studied concentrations. The other extracts did not present inhibition against this microorganism. For S. marcescens MG1, inhibition of swarming motility was observed only at the concentration of 125 µg / ml of CB-MeOH / NH4. Comparative analyses between the two types of quercetin showed that, although for the two bacteria tested the two compounds showed inhibitory activity on swarming motility, quercetin-3-β-D-glycoside was less efficient than non-glycosylated quercetin in the concentration of 125 µg / ml. Biofilm formation was not influenced by the extracts and unexpectedly, both types of quercetin evaluated did not show inhibition towards biofilm formation. In general, organic onion extracts showed little effect on quorum sensing controlled phenotypes and glycosylation of quercetin probably explains the low antimicrobial and anti-QS activity of the extracts.

Le biocontrôle est défini comme un ensemble de méthodes de protection des végétaux par l’utilisation de mécanismes naturels. Son principe repose sur la gestion des équilibres des populations d’agresseurs plutôt que sur leur éradication. La protection des cultures de la pomme de terre Solanum tuberosum contre les bactéries pectinolytiques (Dickeya et Pectobacterium) a été précédemment proposée comme une application du biocontrôle. Il s’agit ici de perturber (quencher) la communication quorum-sensing (QS) utilisée par l’agent pathogène pour coordonner son attaque et sa virulence. Afin d’optimiser cette méthode de lutte par quorum-quenching (QQ) et d’en contrôler l’efficacité, nous avons étudié la voiecatabolique des -lactones d’un agent de biocontrôle, la bactérie Rhodococcus erythropolis. Cette voie est impliquée dans la dégradation des signaux N-acyl-homoserine lactones du pathogène. Nous avons d’abord étudié le rôle du répresseur QsdR ainsi que la régulation transcriptionnelle de l’opéron qsd impliqué dans la dégradation des signaux. La compréhension de cette régulation a permis de générer des biosenseurs capables de monitorer les activités QS du pathogène et QQ du protecteur. Sous microscopie confocale à balayage laser, ces outils ont apporté des preuves visuelles du rôle et du lien entre ces deux activités dans les tissus du tubercule. Enfin, la faible spécificité du répresseur QsdR pour ses ligands, apermis de proposer la -caprolactone, un analogue structural des signaux de QS, comme inducteur de l’opéron qsd. Dans l’ensemble, ces travaux permettent d’approfondir nos connaissances sur le rôle et le fonctionnement du QQ chez R. erythropolis. Ils permettent aussi d’envisager le contrôle de la maladie via un agent dont l’activité de QQ pourra être biostimulée par des lactones peu coûteuses lors de la formulation puis de l’épandage aux champs. / Biocontrol is defined as a set of plant protection methods through the use of natural mechanisms. Its principle involves the control of populations of aggressors rather than their eradication. The protection of the potato Solanum tuberosum against soft-rot bacteria (Dickeya and Pectobacterium) has been previously proposed as an application of biocontrol. This involves disturbing the quorum-sensing (QS) communication used by the pathogen to coordinate its attack and virulence. In order to optimize this quorum-quenching (QQ) biocontrol method and to control its effectiveness, we have studied the catabolic pathway of -lactones of a biocontrol agent, the Rhodococcus erythropolis bacterium. This pathway is involved in the degradation of the pathogen N-acyl-homoserine lactones signals. We firststudied the role of the QsdR repressor as well as the transcriptional regulation of the qsd operon involved in signal degradation. The understanding of this regulation has made it possible to generate biosensors capable of monitoring the QS of the pathogen and QQ of the protector. Under confocal laser scanning microscopy, these tools provided visual evidence of the role and link between these two activities in the tuber tissues. Finally, the low specificity of the QsdR repressor for its ligands made it possible to propose the -caprolactone, a structural analog of QS signals, as an inducer of the qsd operon. Overall, this work provides insight into the role and function of QQ in R. erythropolis. It also allows to envisage the control of the disease using a biocontrol agent whose QQ activity can be biostimulated by inexpensive lactones during formulation then spreading in the field.

Yes / Bacteria sense their own population size, tune the expression of responding genes, and behave accordingly to environmental stimuli by secreting signaling molecules. This phenomenon is termed as quorum sensing (QS). By exogenously manipulating the signal transduction bacterial population behaviors could be controlled, which may be done through quorum quenching (QQ). QS related regulatory networks have been proven their involvement in regulating many virulence determinants in pathogenic bacteria in the course of infections. Interfering with QS signaling system could be a novel strategy against bacterial infections and therefore requires more understanding of their fundamental mechanisms. Here we review the development of studies specifically on the inhibition of production of N-acyl-homoserine lactone (AHL), a common proteobacterial QS signal. The opportunistic pathogen, Pseudomonas aeruginosa, equips the alkylquinolone (AQ)-mediated QS which also plays crucial roles in its pathogenicity. The studies in QQ targeting on AQ are also discussed. / University of Malaya High Impact Research Grants (UMC/625/1/HIR/MOHE/CHAN/01, A-000001-50001,and UMC/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Yes / Lysinibacillus fusiformis strain RB21 is a quorum-quenching bacterium that is able to degrade quorum-sensing signaling molecules. Here, we present the first complete genome sequence of L. fusiformis strain RB21. The finished genome is 4.8 Mbp in size, and the quorum-quenching gene was identified. / University of Malaya for High Impact Research (UM-MOHE HIR) grant UM C/625/1/HIR/MOHE/CHAN/01, no. A000001-50001 and grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027