Les génomes bactériens sont le siège de deux phénomènes de transferts de l'information génétique: les transferts verticaux et horizontaux. Leur coéxistence ce qui implique une délicate balance entre maintenance et instabilité des génomes. L'information génétique des bactéries est généralement portée par des réplicons circulaires : chromosomes et plasmides. Un des sérieux désavantages des réplicons circulaires est leur grande sensibilité aux réarrangements causés par recombinaison homologue. Un nombre impair de crossing-over pendant ou après la réplication de ces réplicons entraine la formation de molécules dimériques. Ces dimères correspondent à une fusion covalente des deux copies du réplicon, non-résolus les dimères ne ségrégeront pas correctement au moment de la division cellulaire. La résolution des formes multimériques des plasmides et chromosomes circulaires est médiée par un mécanisme de recombinaison spécifique de site efficace et extrêmement contrôlé : le système Xer. Les mécanismes de résolution des dimères de chromosome et de plasmide sont différents. De plus, bien que le système de résolution de dimères de chromosome soit contrôlé dans le temps et dans l'espace, chez de nombreuses bactéries il est détourné de son rôle principal par des éléments génétiques mobiles appelés IMEXs (Integrative Mobile Elements exploiting Xer). Le système Xer est alors impliqué dans les transferts verticaux et horizontaux de gènes, illustrant comment une même machine moléculaire peut intervenir dans plusieurs processus biologiques. Pour cela, des acteurs externes différents vont jouer un rôle clé dans le contrôle d'une machine Xer centrale et ainsi apporter une diversité de mécanismes, chacun dédié à une processus biologique propre. Afin de mieux comprendre les contrôles différentiels du système Xer, je me suis intéressé aux éléments génétiques mobiles. Via l'étude de la stabilité intra-chromosomique des IMEXs et de la résolution des dimères de certains grands plasmides, j'ai pu montrer que FtsK, la protéine activatrice de la résolution des dimères de chromosome, est impliquée dans la stabilisation des éléments acquis par transferts horizontaux de gènes. / The genetic information of bacteria is generally carried by circular replicons: chromosomes and plasmids. One of the serious disadvantages of circular replicons is their high sensitivity to rearrangements caused by homologous recombination. An odd number of crossing-over, during or after the replication of these replicons, results in the formation of dimeric molecules. These dimers correspond to a covalent fusion between the two copies of the replicon. If they are not resolved, the dimers will not segregate properly at the time of cell division. The resolution of multimeric forms of circular plasmids and chromosomes is mediated by an efficient and highly controlled site-specific recombination mechanism: the Xer system. The mechanisms of resolution of the chromosome and plasmid dimers are different. In addition, even if the chromosome dimer resolution system is controlled in time and space, in many bacteria it is hijacked by mobile genetic elements called IMEXs (Integrative Mobile Elements Exploiting Xer). The Xer system is then involved in the vertical and horizontal transfer of genes, illustrating how the same molecular machine can intervene in several biological processes. For this, different external actors will play a key role in controlling a central Xer machine and thus bring a variety of mechanisms, each dedicated to a specific biological process. In order to better understand the differential controls of the Xer system, I focused on mobile genetic elements. By studying the intra-chromosomal stability of IMEXs and the resolution of large plasmids dimers, I was able to show that FtsK is involved in the stabilization of the acquired mobile genetic elements.

Dans ce travail, nous proposons un nouvel appareillage, destiné à aider à la détermination du mécanisme de certaines protéines. Cet outil, qui combine un appareil de pinces magnétiques, et un microscope de fluorescence en ondes évanescentes, a été conçu pour permettre à la fois la manipulation mécanique et l'observation de l'activité d'ADN translocases à l'échelle de la molécule unique. Nous présentons d'abord ici la conception, la réalisation et l'expérimentation de ce montage. Nous montrons que, d'une part, il se compare favorablement à ses composants séparés (pinces magnétiques et microscope de fluorescence), et que d'autre part leur réunion dans un appareil unique permet d'obtenir des résultats d'un type nouveau. Nous avons orienté l'étude des ADN translocases avec cet appareil sur l'exemple de deux protéines : le moteur FtsK de Escherichia coli et l'ARN Polymérase de T7. Nous détaillons dans cette étude les questions importantes encore en suspens concernant le mécanisme et présentons les expériences que nous avons envisagées pour y répondre. Nous relatons ensuite également la difficulté que nous avons rencontrée à obtenir des substrats protéiques adaptés aux expériences que nous avons envisagées, et les solutions que nous avons mises en oeuvre pour y remédier. Enn, nous analysons les résultats obtenus dans des expériences en volume ou en pinces magnétiques seules, qui ont permis de mettre en valeur de nouveaux comportements et de préparer la réalisation de nouvelles expériences sur notre montage combiné.

Faithful chromosome segregation is essential for all living organisms. Bacterial chromosome segregation utilizes highly conserved directional SpoIIIE/FtsK translocases to move large DNA molecules between spatially separated compartments. These translocases employ an accessory DNA-interacting domain (gamma) that dictates the direction of DNA transport by recognizing specific DNA sequences. To date it remains unclear how these translocases use DNA sequence information as a trigger to expend chemical energy (ATP turnover) and thereby power mechanical work (DNA movement). In this thesis, I undertook a mechanistic study of directional DNA movement by SpoIIIE from the Gram-positive model bacterium Bacillus subtilis. Specifically, I was interested in understanding the information transfer within the protein from sequence recognition, to ATP turnover, and ultimately to chromosome translocation. How do DNA sequences trigger directional chromosome movement?

Mycobacterium tuberculosis (Mtb) is one of the most virulent and prevalent bacterial pathogens across the world. As Mtb infects millions of people a year, it remains essential to study its physiology with the goal of developing new therapeutic interventions. A critical part of the bacteria’s ability to propagate is through successful cell division. Although the process of bacterial cell division and the key proteins therein are well understood in Escherichia coli, much remains to be understood about division in mycobacteria. Genetic and cell biological approaches have recently begun to identify key divisome components in Mycobacterium smegmatis. However, questions remain regarding the role and function of one divisome protein in particular, the DNA translocase FtsK. In this dissertation, I investigated the necessity of FtsK for the growth of mycobacteria. Using an inducible knockdown of FtsK, I present evidence that complete loss of FtsK is required to inhibit growth in both Mtb and M. smegmatis, and that these orthologs share a homologous function. Additional work suggests extended loss of FtsK may be lethal to bacteria. These observations support that FtsK is an essential member of the divisome in mycobacteria, facilitating the processes of growth and division.

The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (K_d ~ 10^-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.