Biomining is a maturing technology that uses the activity of sulfur and iron oxidising microorganisms to liberate valuable metals from ores but suffers from slow reaction kinetics. Increasing the reaction kinetics of a biomining process could produce significant bottom line improvements for mining companies worldwide and encourage the use of biomining as a green mining technology. Quorum sensing molecules have been shown to successfully modulate the behaviours of biomining bacteria in manners that may be able to improve bioreactor retention times. This study tests the potential for two different quorum sensing treatments to improve the nickel leaching ability of a biomining bacterial consortium. A novel method of delivering quorum sensing treatments to bacterial cultures is described while doubt is cast on established methods. Laboratory scale bioreactors were constructed and the leaching of nickel into solution was followed via ICP-AES to quantify improvements in bioleaching ability. Similar bioreactors were used to exhibit the inhibitory effect that a commonly used organic solvent can have on the leaching ability of bioleaching consortia. Ultimately a qualitative improvement in the bioleaching of nickel is produced using a mixture of tetradecanoyl-acylhomoserine lactone (C14-AHL) and its two derivatives, but the use of C14-AHL alone did not improve bioleaching kinetics. Use of small volumes of the solvent DMSO produced large inhibitory effects on the leaching of nickel by bioleaching consortia.

Le contrôle du biofouling sur des surfaces inertes immergées ou en atmosphère humide est une nécessité dans le secteur marin, tant pour des raisons économiques qu’environnementales. La formation de biofilm microbien, étape préalable à la formation du biofouling, est souvent intrinsèquement liée chez les bactéries au système de communication “Quorum Sensing” (QS). Chez certaines bactéries Gram négatif, le QS est basé sur la perception de petites molécules diffusibles appelées N- Acyl Homosérine Lactones (AHLs). L’une des stratégies antifouling en voie de développement de nos jours repose sur l’inhibition du QS bactérien. L’objectif de cette thèse est d’utiliser certaines bactéries marines afin d’identifier des molécules anti-QS capables de perturber la formation de biofilm. Ce travail a donc porté sur la mise en évidence de molécules AHLs impliquées dans le QS chez certaines bactéries marines isolées de la rade de Toulon, l’étude de la modulation de certains phénotypes dont la formation du biofilm, par ces molécules et, la mise en place d’un test préliminaire d’inhibition du QS. Parmi les trois bactéries isolées de la rade de Toulon (TC8, TC14 et TC15) du genre Pseudoalteromonas, connues pour produire de nombreuses molécules actives, et testées pour leur capacité à sécréter des AHLs, seule Pseudoalteromonas sp. TC15 a produit la C12-HSL. P. ulvae TC14, capable de produire un biofilm conséquent et de la violacéine, ne produit aucune AHL. Afin d’évaluer la possibilité d’utiliser une bactérie marine comme outil de criblage anti-QS, interférant avec les AHLs et les conséquences sur son biofilm, des AHLs exogènes ont été testées sur la production de violacéine, la formation de biofilm et la mobilité de TC14. Certaines AHLs ont montré qu’elles pouvaient réguler la production de violacéine et la formation de biofilm chez TC14, suggérant l’existence d’un récepteur AHLs fonctionnel. Des tests préliminaires d’inhibition du QS ont été effectués avec des molécules commerciales et des analogues synthétiques. La 3-oxo-C6-HSL commerciale, ainsi que l’esculétine et la p- benzoquinone, connues pour interférer avec le QS bactérien, ont été capables d’inhiber la production de violacéine ainsi que la formation de biofilm de TC14 à des concentrations n’affectant pas sa croissance. Cette étude suggère donc que P. ulvae TC14 pourrait être utilisée comme un outil de recherche de molécules anti-QS en conditions proches de celles trouvées dans l’environnement marin, et ce dans le but d’être ultérieurement testées sur la formation de biofilm. L’objectif à plus long terme reste de trouver un moyen de limiter la formation du biofilm en utilisant des molécules non toxiques pour l’environnement. / The biofouling control on immersed inert surfaces or in moist atmosphere is a necessity in the marine sector for both economic and environmental reasons. Microbial biofilm formation, the initial step of biofouling development, is intrinsically linked to the communication system “Quorum sensing” (QS). In some Gram negative bacteria, QS is based on the perception of small diffusible signaling molecules called Acyl Homoserine Lactones (AHLs). The inhibition of bacterial QS is part of the different antifouling strategies currently developed. This present work focused on the detection of AHLs molecules involved in this communication system in bacteria isolated from Toulon harbor and the study of modulation of some phenotypes, including biofilm formation, by these molecules as well as the development of a preliminary anti-QS assay. Three marine bacteria isolated from Toulon harbor (TC8, TC14 and TC15), belonging to the Pseudoalteromonas genus, known to synthesize many active molecules, have been tested for their ability to produce AHLs. Only Pseudoalteromonas sp. TC15 produced the C12-HSL. P. ulvae TC14 a violacein-producing and biofilm-forming bacteria, did not secrete any AHLs. Few marine bacteria are used as an anti-QS screening tool, especially by interfering with AHLs with the goal of studying the consequences on biofilm formation. In order to evaluate the possibility to use TC14 with this purpose, exogenous AHLs were tested on the violacein production, the biofilm formation and the motility of TC14. Some AHLs were able to regulate violacein production and biofilm formation suggesting the presence of a functional AHLs receptor in TC14. Preliminary QS inhibition assays were performed with commercial molecules and synthetic analogues. The commercial 3-oxo-C6-HSL as well as esculetin and p-benzoquinone, known to interfere with bacterial QS, were able to inhibit QS and biofilm formation at a non-toxic concentration. Overall, this study suggests that the marine strain P. ulvae TC14 may be used as a tool for the detection of anti-QS molecules in conditions closed to the marine environment. These molecules may subsequently be tested on the biofilm formation of marine bacteria. The long term objective is to find a way to limit biofilm formation, using non-toxic molecules for the environment.

Le Quorum sensing (QS) est un système de communication bactérienne capable de réguler divers processus cellulaires qui dépendent de la densité de la population microbienne. Chez les bactéries à Gram négatif, cela se produit par production de molécules de signalisation auto-inductrices (AI), les acyl homosérine lactones (AHL). La libération d´AHL à l'extérieur de la cellule est détectée par la population bactérienne provoquant en réponse la régulation de l'expression de certains gènes (régulon QS).Notre laboratoire a étudié et identifié un système QS fonctionnel dans la souche Acidithiobacillus ferrooxidans ATCC 23270T. En outre, nous avons montré que des analogues synthétiques d´AHL modulent l´adhésion d´At. ferrooxidansT sur un substrat minéral, tels des coupons de soufre.Dans ce projet de recherche, nous nous proposons d'identifier les gènes qui sont régulés par le QS chez At. ferrooxidansT, en particulier ceux impliqués dans la biogénèse du biofilm. Notre hypothèse est que des analogues synthétiques d’AHL induisent le système QS. Ainsi, nous nous proposons de moduler l´adhésion sur substrat minéral grâce à l'utilisation de ces molécules. L'utilisation de ces AHL permettra de caractériser le régulon QS dans cette souche bactérienne.L'identification d'analogues synthétiques d´AHL qui favorisent l'adhésion à des coupons de soufre nous a permis d'étudier le transcriptome d´At. ferrooxidansT dans des conditions où le régulon QS est stimulé. Puces à ADN d´At. ferrooxidansT avec/sans ces analogues synthétique d´AHLs nous a permis de caractériser le régulon QS et les gènes impliqués dans la biogénèse du biofilm dans les conditions utilisées. / Quorum sensing (QS) is a bacterial communication system capable of controlling several cellular processes dependent on the density of the microbial population. In Gram-negative bacteria, it occurs mainly through the production by bacteria of small diffusible signaling molecules, termed autoinducers (AI), of the acyl homoserine lactones type (AHLs). The release of AHLs outside the cell is detected by the bacterial population generating the regulation of the expression of several genes (regulon QS).Our laboratory has studied and identified a functional QS system in the Acidithiobacillus ferrooxidans ATCC 23270T type strain. Besides, by using synthetic analogs of AHLs, we have shown that AHL-type QS molecule analogs modulate adhesion of At. ferrooxidansT to minerals, such as sulfur coupons. In this research, we propose to identify the genes that are regulated by QS in At. ferrooxidansT, particularly those that are associated with biofilm formation. For this, we propose to modulate the adhesion of At. ferrooxidansT to mineral substrate through the use of a synthetic AHL analog. Our working hypothesis postulates that AHLs molecules induce the QS system, and that their use will allow the characterization of the QS regulon of this bacterial strain by transcriptomic analysis.The identification of synthetic AHLs improving adherence of At. ferrooxidansT on sulfur coupons allowed us to study the transcriptome of this organism in conditions in which QS regulon is stimulated. DNA microarrays of At. ferrooxidansT with/without one of these AHLs synthetic analogues allowed us to identify the QS regulon and to determine genes involved in biofilm formation.

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Bacterial infections are an increasing cause for concern as resistance spreads to the majority of our front line antibiotics. To counter antibiotic resistance, new treatment regimens and drug targets are being investigated, including directly targeting bacterial virulence (pathogen-induced harm to the host), and therapies which target resistance mechanisms. The outcome of successful treatment with these compounds is not always killing or halting growth of bacteria, therefore selection for resistance to these types of therapeutics is complex. This complexity is increased by the secretion of many virulence factors, meaning their effects are shared with neighbouring individuals. In addition virulence factors show high phenotypic plasticity due to regulation by processes like quorum sensing (QS), which further complicates treatments targeting virulence, or the regulatory processes themselves. Using the example of quorum sensing inhibitors this study shows the importance of understanding the function and ecology of targeted virulence factors, to predict the selection for resistance to anti-virulence drugs. Later chapters elaborate on this to show how quorum sensing control affects selection on secreted virulence factors. The use of anti-virulence drugs as adjuvants is discussed, with a study showing that the interaction between QS inhibition and translation inhibitors is dependent on the environment. The selection for resistance to combinations of antibiotics and adjuvants is investigated using co-amoxiclav as an example, showing that treatment with high doses of adjuvant are robust to the evolution of resistance.

The phenylacetic acid degradation pathway of Burkholderia cenocepacia is necessary for full pathogenicity of B. cenocepacia in nematode; however, the reasons of such requirements are unknown. Unlike wild type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (ΔpaaABCDE) released phenylacetic acid extracellularly in conditions that allow infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the ΔpaaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild type, due to phenylacetic acid consumption. Detection of acyl-homoserine lactones was reduced in spent medium from ΔpaaABCDE compared to that of the wild type strain. Phenotypes repressed in ΔpaaABCDE, protease activity and pathogenicity against C. elegans, increased with the addition of exogenous N-octanoyl-L-homoserine lactone. Thus, it was demonstrated that the attenuated phenotype of B. cenocepacia ΔpaaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signaling. / October 2014

This study provided evidence of cell-density dependent signalling interactions between the autotrophic ammonia-oxidising bacterium Nitrosomonas europaea and a variety of terrestrial heterotrophic bacteria. The autoinducer signal molecule N-(3-hexanoyl)-L-homoserine lactone (HHL) extended the lag phase of N. europaea recovering from starvation while N-(3-oxooctanoyl)-L-homoserine lactone (OOHL) reduced the lag phase. However, the autoinducers N-(3-oxohexanoyl)-L-homoserine lactone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), N-(3-oxobutanoyl)-L-homoserine lactone(OBHL) and N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) did not exert an effect. Spent cell-free medium from early stationary phase cultures of Agrobacterium tumefaciens, Pseudomonas fluorescens and Micrococcus luteus reduced the lag phase of N. europaea recovering from starvation. Supplementing solid medium with spent cell-free medium from the heterotrophs A. tumefaciens, P. fluorescens and M. luteus reduced the incubation period required for the development of colonies of the ammonia-oxidiser. For example, the presence of spent cell-free medium from A. tumefaciens reduced the required incubation period from 20 weeks to 2 weeks. Spent cell-free medium from the heterotrophs Comomonas testosteroni, Erwinia carotovora and Rhizobium leguminosarum had no effect on the growth of N. europaea. A. tumefaciens produces the N-acyl autoinducer OOHL which reduces the lag phase of N. europaea. A mutant unable to produce this autoinducer was generated and spent cell-free medium from this organism had no effect on the recovery of N. europaea from starvation or the incubation period required for the development of colonies of N. europaea on solid medium. Enrichment cultures of ammonia-oxidising bacteria were established from soil and the heterotrophs present in the final stages of enrichment were isolated and identified. Spent cell-free medium from these organisms also reduced the lag phase of N. europaea recovering from starvation and reduced the incubation period required for the development of colonies of N. europaea on solid medium.

Helicobacter pylori is a human pathogen that colonizes the stomach and increases the risk of diseases such as ulcers and gastric cancer. The distribution of H. pylori within the stomach is associated with different disease outcomes, with more dispersed colonization correlated with gastric cancer. The focus of this research is to study the chemotactic responses that promote dispersal of H. pylori within the stomach. We have shown previously that H. pylori perceive the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report that H. pylori chemorepulsion from endogenous AI-2 influences the proportion and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS) or are defective for chemotaxis (∆cheA) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wildtype biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified and characterized two novel periplasmic binding proteins, AibA and AibB, that independently both bind AI-2 and are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenously administered AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal and may play an important role in H. pylori colonization of the stomach by promoting dispersal away from areas of high cell density, thereby modulating the disease spectrum of the host. This dissertation contains previously unpublished co-authored material.