Les systèmes toxine-antitoxine (TA) bactériens ont été découverts il y a une vingtaine d’année sur les plasmides à bas nombre de copie. Ils sont composés de deux gènes organisés en opéron, l’un codant pour une toxine stable et l’autre pour une antitoxine instable capable de neutraliser l’effet de la toxine. Les systèmes TA sont fortement représentés au sein de l’ensemble des génomes bactériens. Ils se localisent aussi bien sur des éléments génétiques mobiles (plasmides, phages, transposons,…) que dans les chromosomes, ce qui suggère que le transfert horizontal de gènes participe à leur dissémination. Le système TA ccd du plasmide F d’Escherichia coli (ccdF) est composé de l’antitoxine CcdA et de la toxine CcdB. Le système ccdF contribue à la stabilité du plasmide F en tuant les bactéries-filles n’ayant pas reçu de copies plasmidiques lors de la division bactérienne (tuerie post-ségrégationelle). Au cours de ce travail, nous avons caractérisé un homologue du système toxine-antitoxine ccd du plasmide F (ccdF) qui se situe dans le chromosome de la souche pathogène E. coli O157:H7 EDL933 entre les gènes folA et apaH (ccdO157). Les systèmes ccdF et ccdO157 coexistent naturellement dans les souches d’E. coli O157:H7, le système ccdF se trouvant sur le plasmide pO157 qui dérive du plasmide F. Nos résultats montrent que l’antitoxine plasmidique CcdAF neutralise l’effet de la toxine chromosomique CcdBO157, tandis que l’antitoxine chromosomique CcdAO157 ne contrecarre pas la toxicité de la toxine plasmidique CcdBF. Nous avons également montré que le système ccdF cause une tuerie post-ségrégationelle, lorsqu’il est cloné dans un plasmide instable, dans une souche possédant le système chromosomique ccdO157. Le système ccdF est donc fonctionnel en présence de son homologue chromosomique. Le système ccdO157 est absent du chromosome de la souche de laboratoire E. coli K-12 MG1655, où une région intergénique de 77 pb sépare les gènes folA et apaH. Celle-ci contient une séquence cible pour la transposition. Nous avons étudié la distribution du système ccdO157 au sein de 523 souches d’E. coli représentatives de l’ensemble des sérogroupes décrits. Nos résultats montrent que le système ccdO157 est présent au sein de souches appartenant à 47 sérogroupes différents. Nos résultats mettent en évidence la diversité de la région intergénique folA-apaH d’E. coli. Celle-ci peut contenir gènes codant pour des protéines présentant de l’homologie avec des protéines d’espèce bactériennes éloignées d’E. coli ou d’organismes eucaryotes, ainsi qu’un élément génétique mobile, l’IS621, ce qui montre que le système ccdO157 a intégré le chromosome d’E. coli via le transfert horizontal de gènes.

Almost all bacteria contain genes that may lead to their growth stasis and death.Normally, these toxins are believed to be neutralized with their cognate antitoxinsfrom a toxin-antitoxin (TA) operon. These modules are also abundant in pathogenic bacteria suggesting a role for them both in normal bacterial physiology and pathogenicity. Their functions have been subject to intense debates. Due to the cell killing capability of the toxin and the gene silencing capability of the antitoxin, they have been utilized for basic research, biotechnology and medical applications. However, further advancements of these applications have been impeded by our limited knowledge of the biology of TAs. Among these TA systems is the Ibs/Sib (A-E) family. Here, we discuss our efforts in characterizing these systems, with a focus on the IbsC/SibC member. Studying them has shown to not be straightforward due to the complexity of their underlying mechanisms and the current approaches being laborious and lacking sensitivity to be applied to these low abundant molecules. We have developed fluorescence-based platforms to take advantage of sensitive and high throughput and resolution techniques such as Fluorescence Assisted Cell Sorting (FACS) to study these molecules instead of relying on traditional culturing methods. While developing these platforms, we gained insights about the biology and regulation of these molecules. To expand this knowledge, we actively pursued investigating the regulation of these molecules at the transcriptional and post-transcriptional levels, both in their native context and in artificial systems. The rest of this thesis summarizes our efforts in solving one of the biggest pieces of the Ibs/Sib puzzle, namely their physiological expressions. With the strategies we have optimized for specific detection of these low abundance molecules, and the knowledge of their biology and regulation presented, we are now at an exciting phase to interrupt the long pause in the study of functions by these molecules and advancement of TA-based applications. / Thesis / Doctor of Philosophy (PhD) / Almost all bacteria contain genes that may lead to their growth stasis or death. Normally, these toxins are believed to be neutralized with their cognate antitoxins. In spite of the efforts to understand these toxin-antitoxin (TA) systems, their physiological roles are subject to intense debate. These systems are hard to study mainly because 1) they are only activated under specific conditions and 2) they are low in abundance. Current approaches are not high throughput and sensitive enough. In this thesis, we developed DNA constructs, bacterial strains and methodologies to facilitate the study of these molecules, particularly the Ibs-Sib family. We next employed these tools to gain a fundamental knowledge of their expression under different conditions, which revealed surprising information about the function of these molecules. We believe that future studies can greatly benefit from the tools offered here to tremendously enhance our understanding of these systems and lead to useful applications.

Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.

Type II toxin-antitoxin (TA) systems are composed of a stable toxin and its cognate unstable antitoxin that impedes the toxin through direct interaction. The human oral pathogen Streptococcus mutans uses a quorum-sensing peptide (CSP) as a stress-inducible pheromone to synchronize gene expression in response to specific stressors. The objectives of this study were to investigate the role of S. mutans MazEF TA in cell survival and characterize the functionality of CSP-inducible chromosomal type II TAs. Our results suggest that MazEF represents a stress-response element. Interestingly, S. mutans negatively regulates its MazEF system under high-cell-density environment that is characteristic of oral biofilms. S. mutans also encodes a novel chromosomal type II TA involved in biofilm formation and development of dormant persister cells. The results from this study suggest a complex interplay between quorum-sensing (signal), type II TA activation (response), and persister formation (phenotype) as a reaction to environmental perturbations.

Type II toxin-antitoxin (TA) systems are composed of a stable toxin and its cognate unstable antitoxin that impedes the toxin through direct interaction. The human oral pathogen Streptococcus mutans uses a quorum-sensing peptide (CSP) as a stress-inducible pheromone to synchronize gene expression in response to specific stressors. The objectives of this study were to investigate the role of S. mutans MazEF TA in cell survival and characterize the functionality of CSP-inducible chromosomal type II TAs. Our results suggest that MazEF represents a stress-response element. Interestingly, S. mutans negatively regulates its MazEF system under high-cell-density environment that is characteristic of oral biofilms. S. mutans also encodes a novel chromosomal type II TA involved in biofilm formation and development of dormant persister cells. The results from this study suggest a complex interplay between quorum-sensing (signal), type II TA activation (response), and persister formation (phenotype) as a reaction to environmental perturbations.

Genes are able to replicate horizontally and vertically- a given gene may be more successful on horizontally mobile elements than others. This includes genes that exhibit a post-segregational killing (PSK) phenotype. PSK is generated by expression of a toxin and antitoxin from a mobile element, such that if a bacterium loses the element the toxin becomes active in the cell and the cell dies. All PSKs described to date involve a toxin and an antitoxin function, though within a given group of toxin and antitoxin gene pairs only some are likely to exhibit this phenotype. Here, I investigate what differentiates genes that induce PSK from biochemically similar genes that do not. One group of genes of which some are known to induce PSK is toxinantitoxin (TA) systems, composed of a stable toxin and an unstable antitoxin. I analyzed computational data on the distribution of type I TA systems (RNA antitoxin), which appear to be less mobile than type II TA systems (protein toxin). Data on validated TAs suggests a correlation between distribution, mobility and the PSK phenotype. Differences in phylogeny could be due to differences in tendency to exhibit PSK in different environments. This connection between distribution and PSK was explored by experimentally testing a computationally described operon, plasmid_Toxin-ptaRNA1, that exhibited structural and distributional similarities to a mobile type I TA system. Despite this, expression of the predicted toxin ORFs did not reduce growth (as measured by saturation density) in E. coli, and the operon did not induce PSK. The conditions of PSK were further tested with the toxin (barnase) and antitoxin (barstar), which are not known to have the phenotype. A number of heterologous expression systems were developed with these genes in E. coli to test their ability to exhibit PSK in a manner akin to both type II TA systems, with a cytoplasmic toxin, and bacteriocins,which have a secreted toxin. I used equations of logarithmic decay to model the necessary expression of the proteins in the cell and their rate of decay after plasmid loss to enable PSK. My results suggest there is likely to be an evolutionary trend toward TA systems with high expression levels of very unstable antitoxins. Secreted barnase was also tested experimentally for its ability to induce PSK similar to bacteriocins, which exhibit a PSK-like phenotype in monoculture by driving maintenance of the immunity encoding plasmid. Barnase did not induce PSK, possibly due to its inability to cause antibiosis in our test system. Structural similarities and biochemical similarities are not sufficient to determine whether a given system will act as a PSK because numerous contextual factors have an effect on whether the genes are addictive. A given set of genes may have the phenotype in one species but not another, under one set of environmental conditions but not another, or on one replicon but not another. This is consistent with the competition hypothesis, which states that genes will be selected for on mobile elements due to their ability to increase horizontal reproductive success, depending on the environmental conditions.

Chromosomal toxin-antitoxin (TA) modules have been proposed to function as regulators of cell growth in response to environmental perturbations. The objective of this study was to characterize the MazEF TA system of the human pathogen Streptococcus mutans. Our data showed that the mazEF genes form a bicistronic operon. MazF toxin had a toxic effect on cells and this effect can be neutralized by coexpression of its cognate antitoxin MazE. Furthermore, we demonstrated that MazE and MazF proteins interact with each other in vivo, confirming the nature of this TA as a type II addiction system. We also demonstrated that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a protein complex. Our results suggest that the MazEF TA might represent a cell growth modulator facilitating the persistence of S. mutans in the oral cavity.

Chromosomal toxin-antitoxin (TA) modules have been proposed to function as regulators of cell growth in response to environmental perturbations. The objective of this study was to characterize the MazEF TA system of the human pathogen Streptococcus mutans. Our data showed that the mazEF genes form a bicistronic operon. MazF toxin had a toxic effect on cells and this effect can be neutralized by coexpression of its cognate antitoxin MazE. Furthermore, we demonstrated that MazE and MazF proteins interact with each other in vivo, confirming the nature of this TA as a type II addiction system. We also demonstrated that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a protein complex. Our results suggest that the MazEF TA might represent a cell growth modulator facilitating the persistence of S. mutans in the oral cavity.

All bacteria initiate translation using formylated methionine, yet directly after translation, the formyl-group is removed. This sequence of addition and removal appears futile, yet every sequenced bacterial genome encodes the enzymes for formylation and deformylation, suggesting this process is essential. Puzzlingly, the process is absent from both Archaea and Eukaryotes, and moreover, bacterial mutants lacking both the formylase and deformylase activities are viable, albeit with a diminished growth rate. We created an Escherichia coli strain devoid of formylase and deformylase activity. This strain was then allowed to evolve over 1500 generations whereupon it reached wild-type growth rate, demonstrating that formylation can be completely dispensed with. This raises an additional question: if the formylation cycle is unnecessary, how did it emerge and why has it persisted? Our results show that the formylation-deformylation cycle could have evolved as a toxin-antitoxin pair (TA) with post-segregational killing (PSK) activity. TAs ‘addict’ cells to the plasmids that carry them by inducing PSK. We measured the stability of formylase-deformylase encoding plasmids and their ability to elicit PSK in our evolved E. coli strain. We report several lines of evidence consistent with the formylation-cycle having evolved from a plasmid-borne PSK element: 1) in the absence of deformylation, formyl-methionine on proteins is cytotoxic in bacteria 2) deformylation relieves the cytotoxicity of formyl-methionine, 3) the loss of a plasmid containing formylase and deformylase genes from evolved cells results in cessation of growth – a standard PSK phenotype. In addition, we introduced the E. coli formylase and deformylase genes into yeast and demonstrate that Met-tRNA formylation is not lethal, even in the absence of deformylation. This suggests PSK would be ineffectual in yeast, accounting for the absence of formylation from eukaryotic cytoplasmic translation. We also report the presence of formylase and deformylase genes in the two representative members of the archaeal Methanocopusculum genus. Moreover, we demonstrate that these genes have been acquired by a recent horizontal gene transfer from bacteria. Our results indicate that formylmethionine use in bacteria evolved, not through a direct functional benefit to cells, but through competition between infectious genetic elements.

Les systèmes Toxine Antitoxine (TA) sont présents chez la plupart des génomes bactériens. Nous rapportons dans cette étude la présence de tels systèmes chez la bactérie pathogène Helicobacter pylori. Ce nouveau système TA de type I, de la même manière que les autres systèmes de type I décrits, est composé d’une toxine peptidique membranaire (AapA) dont la traduction est inhibée par un ARNnc (IsoA) suivant une interaction côté 5’ non traduit de l’ARNm. La structuration particuliere de l’ARNm a permit l’identification d’orthologue de ce système dans les chromosomes des genres Helicobacter et Campylobacter mais aussi sur leur patrimoine génétique mobiles, tels les plasmides. Ceci impliquant leur potentielle acquisition dans le génome par transfert génétique horizontal. La deuxième partie de l’étude se focalise sur la résolution par RMN du liquide de la structure atomique de la toxine AapA1 dans un environnement pseudo-membranaire. Une approche par mutation révèle les determinants structuraux de la toxicité, pointant la présence d’une empreinte de charge dans la partie helicoidale transmenbranaire de la toxine présente aussi chez d’autres toxines de Type I. La dernière partie de l’étude se centre sur l’expression et la purification de la toxine afin d’en étudier la structure en environnement membranaire complexe par RMN du solide. Les résultats prometteurs ouvrent la voie à une caractérisation de la toxine par l’expérience de PISEMA. / Toxin-antitoxin (TA) systems are present in almost all bacterial genomes. Here we report that the genome of the major human gastric pathogen, Helicobacter pylori, is hosting several copies of a new family of type I TA systems. Similarly to other type I TA systems, the toxin (AapA) is a small membrane protein whose expression is controlled by a small antisense RNA (IsoA, antitoxin) that binds to the 5’ untranslated region (UTR) of the mRNA. In addition we used the strong conservation of the mRNA folding to identify homologs of this TA system not only in other Helicobacter and Campylobacter chromosomes but also on plasmids, indicating that this new TA system might have been spread over different genomes via horizontal gene transfer.The second part of the study take account of the AapA toxin itself. We acutely determine the structure of AapA1 by liquid state NMR in membrane mimicking environment. We then probe first structural insight on atomic structural determinants of its toxicity following a mutation studies.These results reveal a particular charge pattern on the transmembrane α helix domain of AapA toxin similary to other Type I toxin. A third part of the studies is based on expression and purification of the toxin in order to determine the structure in complex membrane environment by solid state NMR. The promosisng result open the way to characterize the toxin by PISEMA experiment.