Dissertations / Theses on the topic 'Integrative conjugative element'

Integrative conjugative elements (ICEs) are responsible for pneumococcal genome evolution and more particularly for virulence and drug resistance acquisition. During conjugation, ICEs transfer from a donor cell containing the genetic element to a recipient cell lacking it. ICEs insert into the bacterial genome, excise from it and form a covalently closed circular intermediate which can integrate in a different genomic site and/or can integrate in a new genome upon conjugative transfer. Tn5253 is a composite ICE of Streptococcus pneumoniae conferring resistance to tetracycline and chloramphenicol. The complete nucleotide sequence showed that it is 64,528 bp in size and contains 79 ORFs, 41 of which could not be annotated. Two distinct genetic elements were found integrated in Tn5253: Tn5251 (18,033 bp), of the Tn916-Tn1545 family of CTs, and Ωcat(pC194) (7,627 bp), which could not conjugate, but was capable of intracellular mobility by homologous recombination. Ωcat(pC194) was shown to contain a copy of the staphylococcal plasmid pC194 with a 93-bp deletion in the replication origin, to be flanked by two 1,169-bp direct repeats, and to excise from Tn5253 producing a circular form of the element. The highest transfer frequency of Tn5253 was registered when Streptococcus pyogenes was the donor (6.7 x 10-3 transconjugants/donor). Upon conjugal transfer, Tn5253 was found to always integrate at a single chromosomal site. The target site (attB) was a 83-bp sequence in the rbg gene of S. pneumoniae. Strains carrying Tn5253 all contained circular forms of the ICE in which the ends were joined by a 84-bp sequence (attTn). attB and attTn differed only for an extra nucleotide in attTn. When integrated into the chromosome, Tn5253 was flanked by a copy of attB and a copy of attTn: invariably attB was adjacent to the left end and attTn to the right, suggesting a polarization in the DNA integration process. In order to study the mechanism of integration into pneumococcal chromosome, we constructed an attB mutant in which the first 63 nts were deleted and 5 nucleotide changes introduced in the remaining 20 nts. The resulting attB mutant was used as Tn5253 recipient strain in mating experiments. Conjugation frequency of Tn5253 in the attB mutant recipient was considerably lower when compared to a standard recipient (4.8 x 10-7 vs 1.7 x 10-5 transconjugants per donor cell). Five novel Tn5253 alternative insertion sites were found located in: spr1713, coding for an alpha-galactosidase, spr0540 (cell wall synthesis enzyme), spr1534 (substrate binding protein of an ABC sugar transporter), spr1983 (MFS protein) and spr0546 (nrd, putative nitroreductase). Transfer of Tn5253 from alternative attB sites occurred at lower frequencies than wild type donor ranging from 2 x 10-7 to < 3.6 x 10-8. One transconjugant harboring 3 copies of Tn5253 was able to transfer the ICE at a frequency 100-fold higher than wild type donor. Tn5253 has a strong preference for its primary insertion site, even when it is mutated, but it can also integrate at different sites. The insertion site of Tn5253 affects its transposition rate, which is decreased or abolished when integrated in one of the alternative attB.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February, 2021
Cataloged from the official PDF of thesis.
Includes bibliographical references.
Conjugative elements are mobile genetic elements that can transfer from a donor bacterium to a recipient via an element-encoded type IV secretion system. Integrative and conjugative elements (ICEs) are an abundant class of conjugative element. ICEs are typically integrated into the bacterial host chromosome, but under certain conditions, or stochastically, they can excise from the chromosome and transfer to a recipient. ICEs likely interact with their bacterial host at every stage of their life cycle, but few of these interactions have been characterized. In this work I sought to 1) identify bacterial host factors necessary for efficient transfer of the integrative and conjugative element ICEBs1 to a recipient, and 2) determine whether the ICEBs1-encoded cell wall-modifying enzyme CwlT acts on the cell wall of the donor bacterium, the recipient bacterium, or both.
I used CRISPR interference to induce a knockdown of individual essential Bacillus subtilis genes, and then screened for gene knockdowns that caused an acute defect in transfer of ICEBs1. I found that wall teichoic acids were necessary in both ICEBs1 donors and recipients for efficient conjugative transfer. I found that depletion of wall teichoic acids caused cells involved in ICEBs1 conjugation to sustain lethal envelope damage caused by active conjugation machinery. Conjugative elements must bypass the cell wall of both the donor and recipient cells in a mating pair. Conjugative elements encode cell wall hydrolases that are required for efficient transfer, which are presumed to partly degrade the cell wall of the donor bacterium during conjugation. In order to investigate the role of the ICEBs1-encoded cell wall hydrolase CwlT in conjugation, I generated cell wall-less (L-form) strains of B. subtilis which could donate or receive ICEBs1.
In the absence of either the donor or recipient cell wall, CwlT was dispensable for efficient transfer. This finding indicates that CwlT acts on both the donor and recipient cell wall in a mating pair.
by Mark Michael Harden, Jr.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Horizontal gene transfer is the acquisition of new genetic material that can confer novel phenotypes to bacteria and contribute to their evolution. Conjugation is an important mechanism of horizontal gene transfer that involves the direct transfer of DNA between two cells and is mediated by mobile genetic elements encoding type IV secretion systems. Conjugative elements prevent redundant transfer by a mechanism known as exclusion that inhibits their cognate secretion systems. Exclusion is prevalent among conjugative elements, suggesting it is advantageous and potentially essential. Yet very few exclusion mechanisms are characterized, and the advantages they provide are not well understood. My work characterizes the exclusion mechanism of an integrative and conjugative element found in a Gram-positive bacterium. In combination with several other studies, my results point to a potentially conserved mechanism and novel benefits of this phenomenon, furthering our understanding of how mobile genetic elements regulate their transfer, impact their bacterial hosts, and mediate horizontal gene transfer.
by Monika M. M. Avello.
Ph. D.

In the present thesis, I studied the activation of an SOS-like response in Streptococcus pneumoniae encoded by the Streptococcus pyogenes prophage φ1207.3. This system leads to the temporary activation of an hypermutable phenotype, which resulted in increased survival and increased mutation rate upon exposure to mitomycin C or UV-C light. Then, a different type of stress response, the Envelope Stress Response (ESR), was exploited as a strategy for sensitization of Escherichia coli to several antibiotics, by disbalancing five pathways, namely σE, Cpx, Rcs, Bae, and Psp. Disbalancing the Psp pathway increased E. coli susceptibility to some beta-lactam antibiotics. Prophage φ1207.3, carrying a two-genes macrolide efflux system, was originally described as an Integrative and Conjugative Element (ICE). In this thesis, φ1207.3 was transferred to the standard pneumococcal laboratory strain Rx1, for which the whole genome sequence was obtained. It was demonstrated that φ1207.3 is a functional phage of the Siphoviridae family, able to form mature phage particles. It was shown that φ1207.3 does not enter the lytic cycle, even upon induction with mitomycin C. Since φ1207.3 transfers through a mechanism requiring cell-to-cell contact resembling conjugation, the cellular localization of φ1207.3 was investigated. It was demonstrated that the number of φ1207.3 phage particles on the bacterial cells exceeds the number of phages in the culture supernatant by 3 orders of magnitude. φ1207.3 transfer to a variety of streptococcal species was obtained by setting up a mating protocol for the transfer of large mobile genetic elements. Tn5253 is a composite ICE of Streptococcus pneumoniae carrying two elements: i) the ICE Tn5251, carrying the tet(M) tetracycline resistance gene, and ii) the Ωcat(pC194) not-conjugative element, harbouring the cat chloramphenicol resistance gene and able of intracellular transposition. The Tn5253 chromosomal integration site (attB) was investigated in S. pneumoniae with different backgrounds and in other streptococcal and enterococcal species. Finally, during the sequencing of two Mycobacterium chimaera strains, it was reported the presence of an ICE carrying putative genes involved in the catabolic degradation of polycyclic aromatic hydrocarbons, important environmental pollutants.

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

O reconhecimento da resistência antimicrobiana como um fenômeno emergente em saúde pública, tem constituído um problema em nível mundial. O abuso na utilização de antibióticos na medicina humana e veterinária, e na agricultura, tem originado incremento na diversidade de micro-organismos resistentes, refletindo em falha terapêutica. Os mecanismos de resistência a antibióticos em micro-organismos são mediados principalmente por genes adquiridos de DNA exógeno. A dinâmica da transferência horizontal é realizada por meio de elementos genéticos móveis que carregam genes de resistência. A ampla distribuição deste tipo de estruturas, como o elemento SXT, isolado inicialmente em V. cholerae, tem contribuído para a disseminação de complexos específicos clonais em determinadas áreas geográficas. Este estudo pioneiro no Brasil pesquisou a presença de elementos SXT, em espécies bacterianas do grupo das gama proteobactérias em espécies ambientais, determinou suas características estruturais e funcionais, incluindo genes de resistência a antibióticos, bem como a sensibilidade aos antibióticos dentre os isolados bacterianos que os abrigam. O resultado foi a classificação de 43 elementos SXT obtidos no Brasil, através da comparação com aqueles descritos na literatura. Dentre os elementos SXT obtidos, quatro são albergados por Morganella morganii, fato inédito na literatura. O conhecimento da evolução bacteriana constitui importante ferramenta para estabelecer estratégias eficazes de controle e tratamento de infecções, sem aumentar a pressão seletiva sobre os micro-organismos, bem como instrumento preciso e de grande importância para subsidiar estudos epidemiológicos.
Recognition of antimicrobial resistance as an emerging phenomenon in public health has been a problem worldwide. The abuse in the use of antibiotics in human and veterinary medicine, and agriculture, has caused an increase in the diversity of resistant microorganisms, reflecting in treatment failure. The mechanisms of antibiotic resistance in microorganisms are primarily mediated by genes acquired from exogenous DNA. The dynamics of the horizontal transfer is performed by mobile genetic elements which carry resistance genes. The wide distribution of these structures, such as the SXT element originally isolated from V. cholerae, has contributed to the spread of specific clonal complexes in certain geographical areas. This pioneering study in Brazil researched the presence of SXT elements in the group of bacterial species in environmental gamma-proteobacteria species, determined their structural and functional characteristics, including genes for resistance to antibiotics and the antibiotic susceptibility among bacterial isolates that harbor them. The result was the classification of 43 SXT elements found in Brazil, by comparison with those found in the literature. Among the SXT elements found, four are sheltered by Morganella morganii, unprecedented in the literature. Knowledge of bacterial evolution is an important to establish effective strategies to control and treat infections without increasing the selective pressure on microorganisms, as well as a precise instrument and very important tool to support epidemiological studies.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Mobile genetic elements facilitate movement of genes, including those conferring antibiotic resistance and other traits, between bacteria. Integrative and conjugative elements (ICEs), also known as conjugative transposons, are a large family of mobile genetic elements that can transfer between neighboring cells. ICEs are found integrated in the chromosome of their host bacterium, where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICE DNA will excise and form a circular plasmid-like intermediate. It was previously thought that ICEs were incapable of autonomous replication. However, my research, along with the work of others, shows that ICEs can replicate autonomously, and that many ICEs utilize a rolling circle replication mechanism. Plasmids and phages that use rolling circle replication encode a single strand origin (sso) that enhances priming of DNA synthesis. We identified a functional single strand origin, sso1, in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. Sso activity was important for autonomous, rolling circle replication of ICEBs1 in host cells, and for stable acquisition of the element in new host cells. I also showed that the broad-host range ICE Tn916 replicates autonomously by a rolling circle mechanism. Replication of Tn916 was dependent on the relaxase encoded by Tn916 orf20. The origin of transfer of Tn916, oriT(916), also functioned as an origin of replication. I found that the relaxase (Orf20) and the two putative helicase processivity factors (Orf22 and Orf23) encoded by Tn916 likely interact in a complex to facilitate replication. Lastly, I identified a functional single strand origin of replication (sso) in Tn916 that I predict primes second strand synthesis during rolling circle replication. The importance of autonomous replication by rolling circle in the ICE lifecycle and horizontal gene transfer processes is discussed.
by Laurel D. Wright.
Ph. D.

Bacterial integrative & conjugative elements (ICEs) are chromosomally-integrated DNA islands that excise to form circular molecules capable of horizontal self-transmission via conjugation (cell-to-cell contact). Symbiosis ICEs, such as ICEMlSymR7A of Mesorhizobium loti, are a group of ICEs that carry genes enabling rhizobial bacteria to engage in N2-fixing symbioses with leguminous plants. Transfer of symbiosis ICEs can convert non-symbiotic rhizobia into legume symbionts in a single evolutionary step. In this thesis, a novel form of “tripartite” ICE (ICE3) is reported that exists as three entirely separated regions of DNA residing in the chromosomes of genetically diverse N2-fixing Mesorhizobium spp. These ICE3 regions did not excise independently, rather through multiple recombinations with the host chromosome they formed a single contiguous region of DNA prior to excision and conjugative transfer. Upon integration into a recipient chromosome, the ICE3 recombined the recipient chromosome to disassemble into the tripartite form. These recombination reactions were catalysed by three Integrase proteins IntG, IntM, and IntS, acting on three associated integrase attachment sites. The “excisive” recombination reactions (i.e. assembly and excision) were stimulated by three recombination directionality factors RdfG, RdfM, and RdfS. Expression of ICE3 transfer and conjugation genes were found to be induced by quorum-sensing. Quorum-sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a “master controller” of ICE3 assembly and excision. A model for ICE3 recombination and transfer is presented in this thesis. The conservation of gene content between symbiosis ICE and ICE3 indicated that these elements share a common evolutionary history. However, the persistence of ICE3 structure in diverse mesorhizobia is perplexing due to its seemingly unnecessary complexity. Bioinformatic comparisons of ICE and ICE3 indicated that the tripartite configuration itself may provide selective benefits to the element, including enhanced host range, host stability and resistance to destabilization by tandem insertion of competing integrative elements. In congruency with ICEMlSymR7A, ICE3 acquisition can convey upon recipients the ability to form N2-fixing symbiosis with the host-legume of the ICE3 donor. Interestingly, the effectiveness of N2-fixation may be impaired. The consequences of the emergence of sub-optimal N2-fixing strains following ICE3 transfer in agriculture is discussed. If ICE3 transfer poses a barrier for future inoculation success, the elucidation of the mechanism of ICE3 assembly, excision, and transfer will allow for the development of strategies for management.

Rhizobia are soil-dwelling bacteria capable of forming symbiotic associations with legumes, where they reduce atmospheric dinitrogen (N2) into ammonia (NH3), providing the host plant with a bioavailable nitrogen source. For rhizobia in the genus Mesorhizobium, genes essential to the establishment (nod) and maintenance of nitrogen fixing-legume symbioses (nif and fix) are chromosomally-encoded on mobile segments of DNA known as symbiosis integrative and conjugative elements (ICEs). Symbiosis ICEs are capable of excising from the host chromosome and transferring to a recipient ICE-free cell via bacterial conjugation. In the recipient cell, the invading ICE integrates into the recipient chromosome at conserved sites known as attachment sites. As well as encoding genes for excision, conjugation and integration, symbiosis ICEs often encode genes for vitamin biosynthesis, such as those for the synthesis of nicotinate, biotin and thiamine. Symbiosis ICEs can be monopartite or tripartite in nature and it appears that these two conformations of elements may be evolutionarily related, with the more complex tripartite ICE hypothesised to have evolved from two recombination reactions between three independent monopartite ancestral ICEs that co-existed in an ancestral bacterial cell. Testing this hypothesis for tripartite ICE evolution requires conjugation experiments between two strains that harbour different symbiosis ICEs, which has not previously been experimentally attempted. Symbiosis ICE transfer studies have been conducted between an ICE donor strain and an ICE-devoid vitamin auxotrophic recipient strain, with ICE transfer exconjugants selected on the basis of vitamin prototrophy. However, this method of selecting exconjugants is ineffective for conjugation experiments between two ICE-harbouring strains, as both usually harbour the operons for nicotinate, biotin and thiamine biosynthesis. Genetically-marking two structurally similar, yet phenotypically distinct monopartite ICEs with a selectable marker, such as an antibiotic resistance gene would allow for screening exconjugants solely on the presence of the selectable marker. Two such ICEs are ICEMcSym1192 from the genome of the Cicer arietinum (chickpea) nodulating microsymbiont Mesorhizobium ciceri CC1192 and ICEMlSymR7A from the genome of the Lotus-nodulating M. loti R7A. Therefore, the aims of this thesis were to first genetically mark the symbiosis ICEs ICEMcSym1192 and ICEMlSymR7A, with antibiotic resistance genes that could facilitate selection of exconjugants in ICE transfer studies. Second, to investigate the free-living growth and symbiotic phenotype of marked symbiosis ICEs. Finally, to test the in vitro mobility of these genetically marked elements. Using a site-directed mutagenesis approach, M. ciceri CC1192 ICEMcSym1192 was successfully marked within an intergenic region of the symbiosis ICE, with a gene encoding resistance to neomycin/kanamycin (nptII), yielding ICEMcSym1192::nptII. The free-living phenotype of two M. ciceri ICEMcSym1192::nptII derivatives, MCC91 and MCC92, was assessed alongside wild-type CC1192, in a bacterial growth experiment to compare the mean generation times of all three strains cultured in both rich (TY) and minimal (AMS with either glucose or succinate as the sole carbon source) media. Mean generation times were not significantly different between these strains (p > 0.05) in all media tested. Additionally, the symbiotic phenotype of the ICEMcSym1192::nptII derivatives did not differ significantly (p > 0.05) to wild-type CC1192, as measured by mean nodule number, nodule weight and shoot dry weight of inoculated C. arietinum. Using a similar site-directed approach, M. loti R7A ICEMlSymR7A was also marked within an intragenic region, however, with Ω-aadA encoding resistance to spectinomycin/streptomycin, yielding ICEMlSymR7A::Ω-aadA. Two ICEMlSymR7A::Ω-aadA derivatives, MCC93 and MCC94, were completed towards the end of this Honours project, therefore, the free-living and symbiotic phenotype could not be achieved within the scope of this project. The transfer of ICEMcSym1192::nptII in MCC91 and MCC92, into the ICE-devoid recipient strain, R7ANS, was tested with conjugation mixtures plated onto three different sets of media to select for R7ANS (ICEMcSym1192::nptII) exconjugants. Integration of ICEMcSym1192::nptII in R7ANS should allow exconjugants to be selected solely on the presence of the antibiotic marker conferring neomycin resistance (encoded on ICEMcSym1192::nptII) and tetracycline resistance (encoded on R7ANS plasmid, pFAJ1700). The three selection conditions included rich or minimal media, with reduced tetracycline concentrations and the addition of the vitamin thiamine. While, putative exconjugants were extracted from two selection conditions, PCR screening confirmed these were not R7ANS exconjugants but instead were most likely spontaneous CC1192 tetracycline resistant isolates. The inability to isolate R7ANS (ICEMcSym1192::nptII) exconjugants could be due to a number of reasons, including the use of HEPES-buffered, rather than phosphate-buffered, minimal media, the combination of neomycin and tetracycline in the selection media attenuating the rate of ICE excision and/or transfer or the insertion of the nptII cassette in ICEMcSym1192::nptII affecting ICE mobility. Nevertheless, the marked strains generated in this thesis and the assessment performed on them to date, provides a solid foundation for subsequent experiments to further characterise the mobility of these derivatives. The discovery that Mesorhizobium symbiosis ICEs can exist in both monopartite and tripartite configurations raises many questions about ICE stability and persistence. The genetically-marked ICEMcSym1192::nptII and ICEMlSymR7A::Ω-aadA produced in this thesis represent an important set of experimental tools necessary to further investigate the evolution of these symbiotic elements.

The genome of Klebsiella pneumoniae HS11286 carries two integrative and conjugative elements (ICEs), ICEKpnHS-1 and ICEKpnHS-2; the former belongs to the ICEKp1 family and the latter was unexplored. This study was designed to further characterise the two ICEs, and investigate their prevalence in local clinical K. pneumoniae isolates. Sequence analysis showed that ICEKpnHS-2 belonged to the subfamily of PAPI elements with XerC-like integrases and PCR screening revealed that ICEKpnHS-1 associated elements were more (30%) prevalent in local isolates compared to ICEKpnHS-2 (12.5%). Contributions of these elements to cell physiology, virulence and antibiotic resistance were investigated. Deletion of ICEKpnHS-1 reduced siderophore secretion and growth, particularly in iron restricted conditions. The ΔICEKpnHS-1 strain also showed reduced resistance to various antibiotics, especially those known to be affected by the ATP binding cassette (ABC) and major facilitator superfamily (MFS) exporters. Assays in Galleria mellonella provided preliminary evidence that deletion of ICEKpnHS-2 reduced the virulence of K. pneumoniae HS11286. Type IV secretion systems (T4SS) are major component modules of ICEs responsible for conjugative transfer of nucleoprotein complexes. Observed reduction of conjugative transfer of one of the ICEs following deletion of the other led us to study interactions between the two distinct T4SSs encoded on the ICEs in HS11286. A marker plasmid containing oriT of ICEKpnHS-1 (P-oriT1) was constructed and deletion of ICEKpnHS-2 was shown to produce a six–fold reduction in trans-conjugants. Deletion of the mobB (ICEKpnHS-1) orthologue in ICEKpnHS-2 (mob-2) and traI (relaxase of ICEKpnHS-2) both produced similar defects in transconjugants frequencies that could be complemented in-trans. The ATPase Mob-1 was bioinformatically shown to lack a Walker B and it was speculated that Mob-2 might compensate for this. Accordingly, point mutations in Walker motifs of mob-2 provided evidence supporting this complementary function in energy transduction contributing significantly to the crosstalk between the two non-homologous ICEs. Data on frequency of transfer of the native chromosomal ICEKpnHS-1 and its effects on the phenotypes of an E. coli were also shown. However, ICEKpnHS-2 self-conjugation was not detected.

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

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

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.