Les bactéries du genre xanthomonas sont responsables de nombreuses maladies des plantes, telles que la nervation noire des brassicacées causée par x. campestris pv. campestris (xcc). Lors des phases précoces du processus infectieux, ces bactéries doivent identifier des sites favorables à leur pénétration dans les tissus et les atteindre afin de s'internaliser dans les tissus végétaux et s’y multiplier. Le chimiotactisme est le mécanisme par lequel les bactéries détectent des signaux et se dirigent vers des attractants ou s’éloignent de signaux répulsifs. L’objectif de ce travail est de comprendre les rôles du chimiotactisme et de la mobilité flagellaire dans la fitness des xanthomonas. Nous avons montré que la mobilité flagellaire n’est pas une caractéristique partagée par tous les xanthomonas mais qu’environ 5% des souches perdent cette capacité sans altération majeure de leur fitness in planta. Un senseur du chimiotactisme, dénommé hsb1, probablement acquis par transfert horizontal, présente un groupe d’allèles spécifique à x. campestris. Une mutation de hsb1 dans la souche xcc atcc 33913 entraine une diminution de l’internalisation de cette souche dans les tissus de plantes hôtes combinée à une augmentation de l’internalisation dans les tissus des plantes non-hôtes. Hsb1 perçoit un signal émis par les blessures des feuilles de chou. Un glucosinolate, la sinigrine, et un acide aminé, la l-phénylalanine, sont détectés in vitro par ce senseur, mais ne sont pas métabolisés. Des travaux complémentaires seront nécessaires pour identifier le signal détecté par ce senseur et envisager la conception de méthodes de lutte basées sur la confusion d’informations. / Xanthomonads are responsible for plant diseases such as black rot of Brassicaceae caused by X. campestris pv. campestris (Xcc). During the early stages of the infection, pathogenic bacteria such as Xcc must detect favorable sites and ingress into host plant tissues to colonize and multiply in the apoplast or the xylem vessels. Chemotaxis is the mechanism used by bacteria to detect attractants and repellents and adapt in consequence its direction. The aim of this work is to understand the roles of chemotaxis and flagellar motility in the fitness of xanthomonads. We showed that flagellar motility is not a general feature of xanthomonads. About 5 % of tested strains lost this ability without major impact on their fitness in planta. A chemotaxis sensor, named Hsb1, probably acquired by horizontal transfer shows a group of alleles that are specific of X. campestris. In Xcc ATCC 33913, a mutation in hsb1 resulted in a decreased penetration of this strain in the host plant tissues combined with an increase penetration in the non-host plant tissues. Hsb1 sense a signal from wounds of cabbage leaves. In vitro, a glucosinolate, the sinigrin, and an amino acid, the L-phenylalanine are detected by Hsb1 but are not metabolized. Further work is needed to identify the signal detected by the sensor and to design control methods based on confusion.

The bacterial flagellum is a fascinating corkscrew-shaped macromolecular rotary machine used primarily to propel bacterial cells through their environment via the conversion of chemical potential energy into rotational power and thrust. Flagella are the principal targets of complex chemotaxis systems, which allow microbes to navigate their habitats to locate favorable conditions and avoid harmful ones by continuous sampling of environmental compounds and cues. Flagella serve as surface and temperature sensors, mediators of host cell adherence by bacterial pathogens and symbionts alike, and important virulence factors for disease-causing microbes. They play several essential roles in accelerating the foundational stages of biofilm formation, during which bacteria build highly intricate microbial communities with increased resistance to predation and environmental assaults. Flagellum-mediated chemotaxis has broad and impactful implications in fields of bioremediation, targeted drug delivery, bacterial-mediated cancer therapy and diagnostics, and cross-kingdom horizontal gene transfer. While the core structural and functional components of flagella have been well characterized in the closely related enteric bacteria, Escherichia coli and Salmonella typhimurium, major departures from this paradigm have been identified in other diverse species that merit further investigation. Many bacteria employ additional reinforcement modules to surround and stabilize their more powerful flagellar motors and provide increased contact points in the inner membrane, the peptidoglycan sacculus, and, in Gram-negative bacteria, the outer membrane. Additionally, the soil-dwelling bacterium Sinorhizobium meliloti exhibits marked distinctions in the regulation, structure, and function of its navigation systems. S. meliloti is a nitrogen-fixing symbiont of the agronomically valuable leguminous plant, Medicago sativa Lucerne, and uses its coupled chemotaxis and flagellar motility systems to search for host plant roots to colonize. Following root colonization, the bacterium converts to a nitrogen-fixing factory for the plant and the combined influences of this symbiosis can quadruple the yields of the host. This dissertation is aimed at delivering a thorough representative overview of the processes facilitating bacterial flagellum-mediated chemotaxis and motility. Chapter 1 describes the interplay between chemotaxis and flagellar motility pathways as well as the structure, function, and regulation of these systems in several model bacteria. Particular emphasis is placed on the comparison of flagellar systems from the soil-dwelling legume symbiont, Sinorhizobium meliloti with other model systems, and a brief introduction is provided for its primary counterpart, the agronomically valuable legume, Medicago sativa, more commonly referred to as alfalfa. Chapter 2 embodies the first report of a flagellar system to require two copies of a protein known as FliL for its function. FliL is found in all bacterial flagellar systems reported to date but is only essential for some to drive motility. The more conserved copy of the protein has retained the title of FliL and several experiments to assay the proficiency of flagellar motor function revealed that in the absence of FliL swimming is essentially abolished as is the presence of flagella on the cell body. Flagellar motor activity and swimming proficiency of mutants lacking the FliL-paralog MotF was nearly as abysmal as those without FliL but flagellation was essentially normal indicating distinct roles for the two proteins. FliL is implicated in initial stator recruitment to the motor while MotF was found to serve as a power or speed modulator. A model to accommodate and explain the roles of these proteins in the flagellar motor of S. meliloti is provided. Chapter 3 links a never-before characterized flagellar protein, currently named Orf23, to a role in promoting maximum swimming velocity and perhaps stator alignment with the rotor in a peptidoglycan-dependent manner. The loss of LdtR, a transcriptional regulator of peptidoglycan-modification genes, caused defects in swimming motility that are restored only by removal of Orf23 or by replacing a nonpolar glycine with a polar serine in the periphery of stator units. Bioinformatics analyses, immunoblotting, and membrane topology reporter assays revealed that Orf23 is likely embedded in the inner membrane and that the remainder of the protein extends into the periplasm. Building on findings from Chapter 2, Orf23 is anticipated to influence stator positioning through interactions with MotF, FliL, and/or stator units directly. The chapter is concluded with the description of future experiments aimed to more thoroughly characterize Orf23. Altogether, this work increases the depth and breadth of knowledge regarding the composition and function of the speed-variable bacterial flagellar motor. We have identified several components required for stator incorporation and function, as well as an accessory component that improves stator performance. A wise society will draw inspiration from these fascinating and powerful machines to inform new technologies to achieve modern goals including targeted drug delivery, bioremediation, and perhaps one day our own exploration. / Doctor of Philosophy / Bacteria are small autonomous single-celled organisms capable of existing and thriving in highly diverse environments. Motility is achieved by these organisms in various ways, but the most common approach is to produce one or more corkscrew-shaped propeller systems known as flagella that are constructed upon and anchored within the wall of the bacterial cell. Rotation of these propellers relies on power converters known as stators to transform the flow of ions down self-produced gradients into useful rotational energy. This process can be likened to the way that the stored energy of water behind a dam can be harnessed and used to power hydroelectric generators. While the core components of flagellar motors are well conserved and understood among distantly related bacteria, billions of years of evolution and refinement of additional structures have allowed bacteria to accommodate swimming in diverse habitats with e.g. low nutrient availability or high viscosity. Here we describe the discovery and characterization of additional components in the flagellar motor system of the soil-dwelling bacterium Sinorhizobium meliloti to navigate soil environments. We report the first identification of a flagellar motor that requires two copies of a pervasive flagellar motor protein known as FliL and have named the more distinct version of the protein MotF. We found that FliL is required for the power converter components to install into the motor and that MotF is necessary to activate them. Next, we identify another motor component, Orf23, that is dispensible for motility but appears to be required to achieve maximum swimming velocity and may serve to shift the motor into a "higher gear". We find that disruption of a regulator of cell wall modification systems leads to defects in motility that are only restored when Orf23 is removed or when the power converter is modified. Ideas are proposed for how FliL, MotF, and Orf23 are integrated into the motor and may contribute to stator function. An advanced understanding of the mechanisms governing flagellar motor structure and function will provide avenues for the improvement of bacteria-based agricultural improvements, development of optimized bacteria-mediated drug delivery systems, bioremediation techniques, and more.

Serratia sp. ATCC 39006 (S39006) is known for producing carbapenem and prodiginine antibiotics; 1-carbapen-2-em-3-carboxylic acid (car) and prodigiosin. It displays different motility mechanisms, such as swimming and swarming aided by flagellar rotation and biosurfactant production. In addition, S39006 produces gas vesicles to float in aqueous environments and enable colonization of air-liquid interfaces. Gas vesicles are thought to be constructed solely from proteins expressed from a gene cluster composed of two contiguous operons, gvpA1-gvpY and gvrA-gvrC. Prior to this study, three cognate regulators, GvrA, GvrB, and GvrC, encoded by the right hand operon were known to be essential for gas vesicle synthesis. Post-transcriptional regulators such as RsmA-rsmB were also known to be involved in the inverse regulation of gas vesicles and flagella based motility. Furthermore, gas vesicle formation, antibiotic production, and motility in S39006 were affected by cell population densities and de-repressed at high cellular densities through a quorum sensing (QS) system. The aim of this research study was to identify novel regulatory inputs to gas vesicle production. Mutants were generated by random transposon mutagenesis followed by extensive screening, then sequencing and bioinformatic identification of the corresponding mutant genes. After screening, 31 mutants and seven novel regulatory genes impacting on cell buoyancy were identified. Phenotypic and genetic analysis revealed that the mutations were pleiotropic and involved in cell morphology, ion transport and central metabolism. Two new pleiotropic regulators were characterized in detail. Mutations in an ion transporter gene (trkH) and a putative transcriptional regulator gene (floR) showed opposite phenotypic impacts on flotation, flagella-based motility and prodigiosin, whereas production of the carbapenem antibiotic was affected in the transcription regulator mutant. Gene expression assays with reporter fusions, phenotypic assays in single and double mutants, and proteomics suggested that these regulatory genes couple different physiological inputs to QS and RsmA-dependent and RsmA-independent pathways.