Les éléments intégratifs conjugatifs (ICE) sont des îlots génomiques qui codent leur excision du chromosome, leur transfert par conjugaison et leur intégration. Ils présentent une organisation modulaire, chaque module incluant tous les gènes nécessaires pour conférer une fonction biologique. Ce travail a porté sur l'étude de la régulation ainsi que les modalités de transfert et de maintien d'ICESt1 et ICESt3, deux ICE de Streptococcus thermophilus présentant une région core étroitement apparentée et une région variable non apparentée. Les résultats obtenus ont montré que, bien qu'ICESt3 s'excise et se transfère à beaucoup plus haute fréquence qu'ICESt1, l'excision des deux éléments est activée par des stimuli identiques et est dépendante de la souche hôte. Chacun de ces ICE code des homologues de deux types de régulateurs différents, cI et ImmR, ce qui implique un mécanisme de régulation complexe et original qui pourrait être conservée chez de nombreux ICE apparentés identifiés lors de ce travail. Selon la définition initiale, les ICE se maintiendraient uniquement sous forme intégrée et ne se répliqueraient pas de façon intracellulaire. Cependant, les dommages à l'ADN induisent non seulement l'excision et le transfert d'ICESt3, mais aussi sa présence en copies multiples extrachromosomiques. Les résultats obtenus impliquent une réplication sous forme extrachromosomique, réplication codée par la région core et qui serait impliquée dans le maintien de l'élément. Une telle réplication pourrait être impliquée dans le maintien de nombreux ICE en plus de leur intégration. / Integrative and Conjugative Elements (ICEs) are genomic islands, which excise from the chromosome, self-transfer by conjugation and integrate. They harbor a modular organization: genes and sequences involved in the same biological process are grouped in the same region. This work concerns the modality of transfer and maintenance of ICESt1 and ICESt3, two ICEs of Streptococcus thermophilus that share closely related core region. ICESt1 excises much less frequently than ICESt3. Nevertheless, excision of the two elements is activated by the same stimuli (DNA damage, stationary phase and/or cell density) and depends of the host strain. Bioinformatical and transcriptional analyses highlight several differences in their organization. However, each of these two ICEs would encode two different regulators, cI and ImmR, suggesting that a complex and original pathway govern to ICESt1' and ICESt3' regulation. This regulation would be shared with numerous ICEs that we identified in the genome of various commensal or pathogenic streptococci. According to the original definition, ICE's maintenance would be exclusively due to their integration in the host chromosome, and ICEs would not be able of extracellular replication. However, in addition to the induction of ICESt3' excision and transfer, DNA damage cause replication of its extrachromosomal form. This unexpected property is encoded by the core region and would be implicated in the maintenance of the element. Comparision with data recently published on other ICEs suggest that intracellular replication could be involved in the maintenance of numerous ICEs, besides their integration.

Bovine respiratory disease (BRD) is the principal cause of morbidity and mortality among feedlot cattle. Mannheimia haemolytica is consistently implicated in this condition, but treatment options are diminishing with the rise of antimicrobial resistance and intensifying consumer pressure to reduce reliance on conventional therapies. Thus, sustainable alternatives like vaccination are required. In this study, the phenotypic and genotypic diversity of BRD pathogens were examined with the objective to identify vaccine targets using reverse vaccinology, an innovative approach to identify antigens via genomic sequence. Preliminary surveillance confirmed M. haemolytica serotype 2 isolates were predominant in healthy animals (75.5%) while serotypes 1 (70.7%) and 6 (19.5%) were common in diseased animals. Pathogens of BRD, including M. haemolytica, Pasteurella multocida and Histophilus somni were also isolated from North American BRD mortalities, and compared using pulsed-field gel electrophoresis and antimicrobial susceptibility. Concurrently, polymerase chain reaction detection of bacterial and viral agents confirmed that M. haemolytica with bovine viral diarrhea virus were the most prevalent. Whereas isolates from live cattle were found to have a relatively low level of resistance, several pathogens from the mortalities were found to contain integrative conjugative elements (ICE) conferring resistance to seven antimicrobial classes. These ICEs were transferred via conjugation to other bacterial species, emphasizing the need for alternative antimicrobial therapies. Collectively, data from these investigations informed the selection of 11 diverse M. haemolytica strains for whole genome sequencing and comparative analyses. Several bacteriophage associated genes and CRISPR-Cas regulated gene expression systems were identified and are likely contributing to virulence in M. haemolytica. Coding sequences across all genomes were screened using pan-genome analysis, identifying 291 candidates with cell-surface associated signatures. Using a cell-free translation system and enzyme-linked immunosorbent assay the candidates were screened against serum from cattle challenged with serovar 1, 2 or 6 of M. haemolytica, and ranked according to immunogenicity. The top five vaccine candidates included Ssa1, ComE, a solute binding protein, an outer membrane protein, and the periplasmic component of an ABC transporter. With further characterization, these unique antigenic candidates could be developed into a vaccine to effectively reduce the dependence on antimicrobial therapies.

The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

To combat widespread infections caused by Staphylococcus aureus, mupirocin was introduced at the Veterans Affairs Medical Center, Mountain Home, Tennessee. Soon after introduction, high-level mupirocin-resistance emerged. The rapid emergence was hypothesized to be due to conjugative transfer of the mupA resistance gene from S. epidermidis to S. aureus. Results have shown that transfer of high-level mupirocin-resistance from S. aureus donors commonly occurs. However, transfer from naturally-occurring S. epidermidis donors was not attainable. Staphylococcus epidermidis transconjugants, however, were capable of serving as donors. Further examination of non-transmissibility included PCR analysis of conjugative transfer genes (tra genes) in capable and non-capable donors. Results confirmed that capable donors possess full-length copies of selected transfer genes. Non-capable donors varied in the presence/absence of full-length copies of transfer genes, but none had all three genes. The genetic differences among non-capable donors suggest that non-transmissibility has arisen independently in different strains via gene deletions and recombinations.

The prokaryotic adaptive immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a defense mechanism that helps to protect the prokaryotic cell from invading mobile genetic elements. This project was performed at Uppsala University and served to answer whether the expression of Cascade, which is part of the CRISPR defense system, will have a negative effect on the cell that expresses it and to also determine whether the CRISPR defense system is effective enough to stop the spread of a conjugative plasmid. A microfluidic system was used in order to perform the experiments and images were taken with the help of fluorescent microscopy. Three different donor strains from E.coli were used. These strains had their own version of the RP4 conjugative plasmid which had the ability to infect recipient E.coli cells with said plasmid. The recipient cells had the ability to express the CRISPR system in order to defend themselves from the plasmid and CRISPR was also inducible with the help of IPTG. The different versions of the RP4 conjugative plasmid had different amounts of spacer targets that Cascade, the recognition complex in the CRISPR system, could recognize. When the recipient cells were induced and had a known target sequence of the plasmid they were able to defend themselves and keep the number of transconjugant cells low. When the recipient cells did not know the target the amount of transconjugant cells were higher. It was also noted that when the cells were induced inside the microfluidic PDMS chip they had a slower generation time. It was also noted that recipient cells had begun to die towards the end of the microfluidic experiments when the cells were induced. This raised the question as to whether the CRISPR defense system was targeting itself as well as the RP4 conjugative plasmid.

Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond. / Microbiology and Immunology

Les éléments intégratifs conjugatifs (ICE) et les éléments en dérivant jouent un rôle important dans le transfert horizontal de gènes chez les bactéries. Les ICE s'excisent par recombinaison site-spécifique sous forme circulaire, se transfèrent par conjugaison et s'intègrent dans un réplicon de la cellule réceptrice. Streptococcus agalactiae est une bactérie pathogène opportuniste responsable d'infections néonatales sévères chez l'Homme et d'infections chez les animaux (bovins, poissons, ...). Une analyse in silico antérieure de 8 génomes séquencés de Streptococcus agalactiae avait permis d'identifier plusieurs éléments intégrés dans l'extrémité 3' d'un gène codant un ARNtLys CTT dont 4 ICE putatifs. Cette étude élargie à 246 génomes a confirmé la prévalence et la diversité des éléments intégrés dans ce locus (présence d'ICE, éléments mobilisables en trans ou en cis, éléments composites, ... chez 98 % des souches). Une nouvelle famille d'éléments mobilisables putatifs s'intégrant dans l'oriT d'ICE a été caractérisée. L'étude fonctionnelle de 5 ICE a montré que 4 s'excisent du chromosome mais que seuls ICE_FSL S3-026_tRNALys et ICE_515_tRNALys se transfèrent par conjugaison au sein de l'espèce et vers S. pyogenes pour l'un des 2. Des éléments composites ont été obtenus par transfert d'ICE_515_tRNALys vers une souche possédant déjà un élément intégré dans ce locus. Un de ces éléments composites est capable de s'exciser et de se transférer par conjugaison conduisant à une mobilisation en cis de l'élément résident. En conclusion, les ICE et les éléments mobilisables (en cis ou en trans) sont très répandus chez S. agalactiae et contribuent à la plasticité génomique chez cette espèce / Integrative and Conjugative Elements (ICEs) and related elements are widespread in bacteria and play a key role in horizontal gene transfer. ICEs excise by site-specific recombination as a circular intermediate, promote their own transfer by conjugation and then integrate into a replicon of the recipient cell. Streptococcus agalactiae is an opportunistic pathogen that causes severe human invasive neonatal infections as well as infections in animals (bovine, fish...). Previous in silico analysis of eight sequenced genomes of S. agalactiae identified in each genome a different element integrated in the tRNALys CTT gene with four putative ICEs. This study, carried on 246 other genomes of S. agalactiae, confirmed the prevalence and diversity of elements integrated in this locus with 98% of the strains carrying an element (ICE, trans or cis mobilizable elements composite elements...). A novel family of putative mobilisable elements which can integrate in the oriT of ICE has been characterized. Functional analysis of 5 ICEs demonstrated that four can excise of the chromosome but that only ICE_FSLS3-026_tRNALys and ICE_515_tRNALys can transfer by conjugation inside the species or to S. pyogenes for one of them. Composite elements have been obtained after transfer of ICE_515_tRNALys to a recipient strain already carrying an element integrated in the same locus. One of this composite element is able to excise and transfer by conjugation to a new strain leading to cis-mobilization of the resident element.In conclusion, ICEs and cis and trans mobilizable elements are widespread in S. agalactiae and contribute to the genomic plasticity in this bacterial species

Mycoplasma agalactiae est responsable de l'agalactie contagieuse, maladie des petits ruminants difficilement contrôlée et figurant sur la liste de l’OIE. Afin d’évaluer la diversité génétique de ce pathogène, 101 isolats ont été comparés par trois techniques (VNTR, RFLP, répertoire vpma). Les résultats révèlent une grande homogénéité génétique dont la souche type PG2 est représentative. Quelques isolats font exception telle la souche 5632 que nous avons séquencée et analysée ici. La comparaison des génomes et des protéomes entre 5632 et PG2 indiquent que la plasticité de ces génomes est liée à d’importants échanges d'ADN et à la présence de nombreux éléments génétiques mobiles (10% du génome). Ces analyses révèlent également une forte dynamique au sein de répertoires de gènes codant des protéines de surfaces. Pour les mycoplasmes, bactéries minimales dépourvues de paroi, ces évènements ont certainement joués un rôle dans leur survie et leur adaptation à des hôtes complexes. / Mycoplasma agalactiae is responsible of contagious agalactia, a disease of small ruminants that is still difficult to control and is listed by the OIE. In order to evaluate the genetic diversity of this pathogen, 101 isolates were compared using three techniques (VNTR, RFLP, vpma repertoire). Results revealed a high genetic homogeneity with the PG2 type strain as representative. Some isolates however diverged such as the 5632 which was sequenced and analysed here. Whole comparative genomic and proteomic analyses of the 5632 and PG2 strains indicate that their genomic plasticity resides in important genes flux and in the presence of several mobile genetic elements (10% of the genome). These analyses also revealed that specific loci encoding repertoire of surface proteins are highly dynamic. For these minimal bacteria that lack a cell-wall, these events have most likely played a major role in their survival and adaptation to complex hosts.

Les éléments génétiques mobiles contribuent grandement à la diversité et à l’évolution des génomes bactériens par le biais du transfert horizontal. Parmi eux, les éléments intégratifs conjugatifs (ICE) codent leur propre excision, leur transfert par conjugaison et leur intégration. En revanche, les éléments intégratifs et mobilisables (IME) ne sont autonomes que pour leur excision et intégration et ne codent seulement que certaines des protéines/fonctions (oriT) dont ils ont besoin pour leur transfert conjugatif. Par conséquent, les IME ont besoin d’un élément conjugatif « helper » pour se transférer. Malgré leur impact sur le flux des gènes et l’évolution des génomes, la prévalence des ICE reste peu étudiée et seulement très peu d’IME avaient été identifiés au début de cette étude. De plus, bien que plusieurs méthodes de détection des ilots génomiques existent, aucune d’elles n’est dédiée aux ICE ou aux IME. Ce qui ne facilite pas l’analyse exhaustive de ces éléments. Le genre Streptococcus appartient au phylum des firmicutes. La quasi-totalité des streptocoques sont des bactéries commensales ou pathogènes de l’homme et d’autres animaux. Aussi, 2 espèces de streptocoques sont utilisées en tant que ferments lactiques lors la production de laits fermentés et divers fromages. Globalement, le genre streptocoques représente un groupe d’intérêt pour l’homme, l’étude du flux de gènes au sein de ces organismes et l’impact qu’il peut avoir sur leur mode vie est primordiale. Au cours de cette thèse, nous avons recherché les ICE et les IME dans 124 souches de streptocoques appartenant à 27 espèces en utilisant une base de données de référence comportant des protéines dites « signatures » d’IME et d’ICE (de leurs modules de conjugaison/mobilisation et d’integration/excision). Cette analyse exhaustive a permis l’identification et la délimitation de 131 ICE ou ICE légèrement dégénérés et 144 IME. Tous ces éléments ont été délimités, ce qui nous a permis de déterminer leur spécificité d’intégration dans les génomes. Au total, 17 spécificités d’intégration ont été identifiées pour les ICE dont 8 encore jamais décrites (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG and ybaB/EbfC) et 18 spécificités pour les IME dont seulement 5 étaient connues chez les firmicutes. Les modules d’intégration des ICE codent soit une intégrase à tyrosine pouvant avoir une faible spécificité (1 famille d’intégrase) ou une forte spécificité (13 spécificités différentes), soit des intégrases à sérine seule ou en triplet (4 spécificités différentes), soit une transposase à DDE. Les IME codent soit des intégrases à tyrosine (10 spécificités différentes) soit des intégrases à serine seule (8 spécificités différentes). Les ICE ont été groupés en 7 familles distinctes selon les protéines codées par leur module de conjugaison. Les IME présentaient une très forte diversité au sein de leur module de mobilisation, empêchant ainsi leur regroupement en famille selon les gènes portés par ce module. Les analyses phylogénétiques des protéines signature codées par tous les ICE et les IME ont montré des échanges de modules d’intégration entre les ICE et les IME et de nombreux échanges entre les modules de mobilisation des IME. L’ensemble de ces résultats révèle la forte prévalence et l’extrême diversité des ICE et des IME au sein des génomes de streptocoques. Une meilleure connaissance et compréhension de ces éléments nous a incité à construire un outil informatique semi-automatisé de détection des ICE et des IME de Streptocoques ainsi que leurs sites d’insertion / Mobile genetic elements largely contribute to the evolution and diversity of bacterial genomes through horizontal gene transfer. Among them, the integrative and conjugative elements (ICEs) encode their own excision, conjugative transfer and integration. On the other hand, integrative mobilizable elements (IMEs) are autonomous for excision and integration but encode only some of the proteins needed for their conjugative transfer. IMEs therefore need a “helper” conjugative element to transfer. Despite their impact on gene flow and genome dynamics, the prevalence of ICEs remains largely underscored and very few IMEs were identified at the beginning of this study. Furthermore, although several in silico methods exist to detect genomic islands, none are dedicated to ICEs or IMEs, thus complicating exhaustive examination of these mobile elements. The Streptococcus genus belongs to the firmicutes’ phylum. Almost all streptococci are commensal bacteria or pathogenes to men and animals. Two species of Streptococcus are also used in the dairy industry as lactic ferments in order to produce fermented milk and different types of cheese. Studying the gene flux of the Steptococci genus and the impact it can have on the lifestyle of these organisms is essential, as it has a lot of interest for human health and activities. In this work, we searched for ICEs and IMEs in 124 strains of streptococci belonging to 27 species using a reference database of ICE and IME signature proteins (from their conjugation, mobilization and integration/excision modules). This exhaustive analysis led to the identification and delimitation of 131 ICEs or slightly decayed ICEs and 144 IMEs. All these elements were delimited, which allowed us to identify their integration specificities in the genomes. In total, 17 ICE integration specificities were identified. Among them, 8 had never been described before (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG and ybaB/EbfC). 18 specificities were also identified for IMEs, among which only 5 were known for the firmicutes. ICEs encode high or low-specificity tyrosine integrases (13 different specificities), single serine intégrases (1 specificity), triplet of serine integrases (3 different specificities), or DDE transposases while IMEs encode either tyrosine integrases (10 different specificities) or single serine integrases (8 different specificities). ICE were grouped in 7 distinct families according to the proteins encoded by their conjugation module whereas the mobilization modules of IMEs were highly diverse, preventing them from grouping into families according to their mobilization modules. The phylogenetic analysis of the signature proteins encoded by all ICEs and IMEs showed integration module exchanges between ICEs and IMEs and several mobilization module exchanges between IMEs. The overall results reveal a strong prevalence and extreme diversity of these elements among Streptococci genomes. Better understanding and knowledge of ICEs and IMEs prompted us to build a semi-automated command-line tool to identify streptococcal ICEs and IMEs as well as to determine their insertion site