Letteratura scientifica selezionata sul tema "Integrative conjugative element (ICE)"

ABSTRACTConjugative transposon Tn5253, an integrative conjugative element (ICE) ofStreptococcus pneumoniaecarrying thecatandtet(M) genes, was shown to be 64,528 bp in size and to contain 79 open reading frames, of which only 38 could be annotated. Two distinct genetic elements were found integrated into Tn5253: Tn5251(18,033 bp), of the Tn916-Tn1545family of ICEs, and Ωcat(pC194) (7,627 bp), which could not conjugate but was capable of intracellular mobility by excision, circularization, and integration by homologous recombination. The highest conjugation frequency of Tn5253was observed whenStreptococcus pyogeneswas the donor (6.7 × 10−3transconjugants/donor).

Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14 Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide–streptogramin A–pleuromutilin resistance among Firmicutes.

ABSTRACTEfflux-mediated macrolide resistance due tomef(E) andmel, carried by the mega element, is common inStreptococcus pneumoniae, for which it was originally characterized, but it is rare inStreptococcus pyogenes. InS. pyogenes, mega was previously found to be enclosed in Tn2009, a composite genetic element of the Tn916family containingtet(M) and conferring erythromycin and tetracycline resistance. In this study,S. pyogenesisolates containingmef(E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICESpy009, belonging to the ICESa2603 family. ICESpy009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated intohylA, a novel integration site for the ICESa2603 family. The modular organization of the ICE was similar to that of members of the ICESa2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICESpy009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICESpy009, in two isolates the ICE was integrated intorplL, the typical integration site of the ICESa2603 family. ICESpy009 was able to transfer macrolide resistance by conjugation to bothS. pyogenesandS. pneumoniae, showing the first evidence of the transferability of mega fromS. pyogenes.

ABSTRACTIn ICESp2905, a widespreaderm(TR)- andtet(O)-carrying genetic element ofStreptococcus pyogenes, the two resistance determinants are contained in separate fragments inserted into a scaffold of clostridial origin. ICESp2905(∼65.6 kb) was transferable not only in its regular form but also in a defective form lacking theerm(TR) fragment (ICESp2906, ∼53.0 kb). Theerm(TR) fragment was also an independent integrative and conjugative element (ICE) (ICESp2907, ∼12.6 kb). ICESp2905thus results from one ICE (ICESp2907) being integrated into another (ICESp2906).

ABSTRACT Mycoplasma genomes contain compact gene sets that approach the minimal complement necessary for life and reflect multiple evolutionary instances of genomic reduction. Lateral gene transfer may play a critical role in shaping the mobile gene pool in these organisms, yet complex mobile elements have not been reported within this genus. We describe here a large (∼23-kb) genetic element with unique features that is present in four copies in the Mycoplasma fermentans PG18 chromosome, accounting for approximately 8% of the genome. These novel elements, designated ICEF (integrative conjugal elements of M. fermentans), resemble conjugative, self-transmissible integrating elements (constins) in that circular, nonreplicative extrachromosomal forms occur in which the left and right termini of the integrated element are juxtaposed and separated by a coupling sequence derived from direct repeats flanking chromosomal copies of ICEF as a result of target site duplication. ICEF contain multiple similarly oriented open reading frames (ORFs), of which some have homology to products of known conjugation genes but others have no known counterparts. Surprisingly, unlike other constins, ICEF lack homologs of known integrases, transposases, or recombinases, suggesting that a novel enzyme may be employed for integration-excision. Skewed distribution and varied sites of chromosomal integration among M. fermentans isolates suggest a role for ICEF in promoting genomic and phenotypic variation in this species. Identification of homologs of terminal ICEF ORFs in two additional mycoplasma species indicates that ICEF is the prototype member of a family of ICE-related elements that may be widespread among pathogenic mycoplasmas infecting diverse vertebrate hosts.

Integrative and conjugative elements (ICEs) are major contributors to genome plasticity in bacteria. ICEs reside integrated in the chromosome of a host bacterium and are passively propagated during chromosome replication and cell division. When activated, ICEs excise from the chromosome and may be transferred through the ICE-encoded conjugation machinery into a recipient cell. Integration into the chromosome of the new host generates a stable transconjugant. Although integration into the chromosome of a new host is critical for the stable acquisition of ICEs, few studies have directly investigated the molecular events that occur in recipient cells during generation of a stable transconjugant. We found that integration of ICEBs1, an ICE of Bacillus subtilis, occurred several generations after initial transfer to a new host. Premature integration in new hosts led to cell death and hence decreased fitness of the ICE and transconjugants. Host lethality due to premature integration was caused by rolling circle replication that initiated in the integrated ICEBs1 and extended into the host chromosome, resulting in catastrophic genome instability. Our results demonstrate that the timing of integration of an ICE is linked to cessation of autonomous replication of the ICE, and that perturbing this linkage leads to a decrease in ICE and host fitness due to a loss of viability of transconjugants. Linking integration to cessation of autonomous replication appears to be a conserved regulatory scheme for mobile genetic elements that both replicate and integrate into the chromosome of their host.

The genome sequences from two strains of the acidophilic, autotrophic, chemolithotrophic proteobacterium A. ferrooxidans are available from genome databases. Bioinformatic sequence comparison revealed the existence in one strain of a putative integrative conjugative element (ICE), containing an entire set of clustered tRNA genes. ICE is missing in the other strain, suggesting that this element as well as the tRNA genes cluster is dispensable for the bacterium. Bioinformatic predictions suggest that the tRNA genes cluster might mainly contribute to the translation of ICE encoded genes.

ABSTRACTThe genetic structures involved in the dissemination ofblaCMY-2carried byProteus mirabilisisolates recovered from different gull species in the South of France were characterized and compared to clinical isolates.blaCMY-2was identified inP. mirabilisisolates from 27/93 yellow-legged gulls and from 37/65 slender-billed gulls. It was carried by a conjugative SXT/R391-like integrative and conjugative element (ICE) in all avian strains and in 3/7 human strains. Two clinical isolates had the same genetic background as six avian isolates.

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<br>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

Les ICE (Éléments intégratifs conjugatifs) et les IME (Éléments intégratifs mobilisables) sont des éléments mobiles bactériens qui jouent un rôle clé dans les transferts horizontaux. Ils ont la capacité de s'intégrer et de se transférer par conjugaison d'une bactérie à une autre. Ces éléments sont très répandus dans les génomes bactériens mais sont encore mal connus. Leur identification automatique est un défi et ils ne sont en général pas annotés dans les génomes. Jusqu'à présent, seules deux approches bioinformatiques permettent la détection des ICE et la détection des IME, mais leur fiabilité reste très variable, en particulier chez les Firmicutes. De plus, aucune de ces approches ne permet de détecter avec précision les éléments composites constitués d’ICE et d’IME emboîtés ou en accrétions, qui sont fréquemment observés dans des génomes bactériens. Nous avons développé une stratégie et un outil nommé ICEscreen permettant d’identifier les ICE et IME dans les génomes des Firmicutes, y compris les éléments emboîtés ou en accrétions. Notre outil, commence par la détection de quatre protéines signatures (SP) indispensables au fonctionnement de ces éléments puis effectue la détection et le typage des éléments à partir de la colocalisation des SP et de la caractérisation de leur contenu. Notre outil utilise un algorithme dédié permettant de résoudre la structure des éléments qu'ils soient composites ou non. Pour réaliser ces étapes nous avons construit une banque de protéines signatures d’ICE et d’IME de référence à partir d’une liste de gènes connus pour être impliqués dans la dynamique de ces éléments chez les streptocoques ainsi que de profils HMM publics ou construits pour cette étude. Pour valider les résultats d’ICEscreen nous avons construit un jeu de données, FirmiData, constitué de 40 génomes de Firmicutes pour lesquels les ICE et IME ont été annotés semi-manuellement et expertisés. Nous avons ensuite comparé les résultats de ICEscreen avec ceux de deux outils de référence : CONJscan et ICEfinder. ICEscreen détecte la quasi-totalité des éléments de la référence (96 %) ce qui en fait un outil plus performant que CONJscan (58 %) et surtout ICEfinder (53 %) sur notre jeu de données. ICEscreen est ainsi un outil d’aide à l’annotation et à la découverte d’ICE et d’IME dans les génomes de Firmicutes, ce qui peut aider à mieux caractériser leur contribution aux transferts horizontaux de gènes, notamment lors de la transmission de la résistance aux antibiotiques, auxquels ils sont fréquemment associés<br>ICEs (Integrative Conjugative Elements) and IMEs (Integrative Mobilizable Elements) are bacterial mobile elements that play a key role in horizontal transfers. They have the capacity to integrate and transfer by conjugation from one bacterium to another. These elements are widespread in bacterial genomes but are still poorly understood. Their automatic identification is a challenge and they are generally not annotated in genomes. So far, only two bioinformatic approaches allow the detection of ICEs and IMEs, but their reliability remains highly variable, particularly among Firmicutes. Moreover, neither of these approaches can accurately detect composite elements consisting of nested or accreted ICEs and IMEs, which are frequently observed in bacterial genomes. We have developed a strategy and a tool called ICEscreen to identify ICEs and IMEs in the genomes of Firmicutes, including nested or accreted elements. Our tool starts with the detection of four signature proteins (SPs) that are essential to the functioning of these elements and then carries out the detection and typing of the elements based on the colocalization of the SPs and the characterisation of their content. Our tool uses a dedicated algorithm to solve the structure of the elements whether they are composite or not. To perform these steps, we have built a bank of ICEs and IMEs signature proteins from a list of genes known to be involved in the dynamics of these elements in streptococci and also public HMM profiles and HMM profiles constructed especially for this study. To validate the ICEscreen results, we built a dataset, FirmiData, consisting of 40 genomes of Firmicutes for which the ICEs and IMEs were annotated semi-manually and curated. We then compared the results of ICEscreen with those of two reference tools: CONJscan and ICEfinder. ICEscreen detects almost all the elements of the reference (96%) making it a more powerful tool than CONJscan (58%) and especially ICEfinder (53%) on our dataset. ICEscreen is thus a tool for the annotation and discovery of ICE and IME in the genomes of Firmicutes, which can help to better characterize their contribution to horizontal gene transfers, particularly during the transmission of antibiotic resistance, with which they are frequently associated

Les génomes bactériens évoluent principalement grâce au transfert horizontal de gènes. La conjugaison bactérienne en est un des mécanismes majeurs. Elle est notamment médiée par les éléments intégratifs et conjugatifs (ICE). En plus de leur transfert, les ICE codent d'autres fonctions conférant à leur hôte un avantage adaptatif, comme par exemple des résistances aux antibiotiques dont la diffusion est un enjeu majeur en santé publique. Il est donc nécessaire de comprendre comment les ICE se transfèrent si l'on veut limiter leur dissémination. Le transfert d'un ICE d'une cellule donatrice vers une cellule réceptrice implique son excision du chromosome, son transfert puis son intégration dans les génomes des deux cellules partenaires. Les données de la littérature révèlent que l'initiation de ce transfert est médiée par un complexe nucléoprotéique appelé relaxosome, dont la protéine clé est la relaxase, une transestérase codée par l'élément. Le rôle de la relaxase est d'effectuer une coupure simple brin sur l'ADN de l'ICE au niveau d'un site conservé, appelé nic. Ce clivage libère une extrémité 3'OH libre, servant d'amorce pour initier la réplication en cercle roulant. Le complexe ADN-relaxase est alors dirigé vers le pore de conjugaison. Au cours de ma thèse j'ai étudié un ICE modèle, ICESt3 de Streptococcus thermophilus qui appartient à la superfamille ICESt3/Tn916/ICEBs1, très répandue chez les Firmicutes. Ces ICE possèdent une relaxase non canonique, appartenant à la famille MOBT, apparentée aux initiateurs de réplication à cercle roulant de la famille Rep_trans. L'objectif de ma thèse était d'élucider le fonctionnement de la relaxase RelSt3 afin de décrypter les mécanismes moléculaires d'initiation du transfert conjugatif médié par une relaxase MOBT. Mes recherches ont conduit à l'identification du site de liaison de RelSt3 sur l'origine de transfert (oriT) d'ICESt3. Ce site, appelé bind, a pour originalité d'être distant du site nic, ce qui n'est pas le cas des autres familles de relaxases. RelSt3 présente un domaine HTH à son extrémité N-terminal. J'ai montré que ce domaine est requis pour la fixation de RelSt3 sur le site bind, et important pour son activité catalytique. Des tests de conjugaison ont démontré que ce domaine HTH est crucial pour le transfert conjugatif d'ICESt3. Des prédictions structurales de ce domaine en complexe avec l'ADN ont conduit à l'identification de l'interface d'interaction avec le site bind, confirmée par mutagénèse dirigée. J'ai également démontré que RelSt3 présente une activité de coupure-religature et qu'elle se fixe de façon covalente sur l'extrémité 5' du brin clivé, démontrant ainsi que cette enzyme participe aux étapes initiale et terminale de la conjugaison. Dans la littérature, il a été démontré que les relaxases interagissent fréquemment avec d'autres protéines accessoires, codées par l'ICE ou la bactérie hôte pour former le relaxosome. Le deuxième objectif de ma thèse était d'identifier des partenaires de RelSt3. L'analogie avec ICEBs1 chez Bacillus subtilis a permis d'identifier deux protéines candidates OrfL et OrfM codées par ICESt3, ainsi qu'une hélicase cellulaire, probablement impliquée dans la réplication en cercle roulant, nommée PcrA. Une caractérisation de ces protéines candidates a été effectuée en utilisant des approches biochimiques et biophysiques. Le réseau d'interaction entre l'ensemble de ces protéines a été dressé en utilisant des approches in vitro, ainsi que l'approche double hybride in vivo. Ces données nous permettent d'avoir un premier aperçu des constituants du relaxasome d'ICESt3. J'ai par ailleurs montré que OrfL et OrfM stimulent l'activité catalytique de RelSt3 in vitro, et qu'elles sont toutes les deux essentielles à la conjugaison d'ICESt3.Ce travail nous apporte une meilleure compréhension des mécanismes moléculaires en jeu lors de la conjugaison d'un ICE pilotée par une relaxase de la famille MOBT<br>Bacterial genomes evolve mainly through horizontal gene transfer. Bacterial conjugation is one of the major mechanisms for these transfers. Conjugation is mediated by integrative and conjugative elements (ICE). In addition to their transfer function, ICEs encode other functions that may provide an adaptive advantage to their host, such as resistance to antibiotics whose dissemination is a major public health issue. It is therefore necessary to understand how ICEs are transferred in order to limit their dissemination.The transfer of an ICE from a donor cell to a recipient cell requires its excision from the chromosome, its transfer from one cell to the other and then its integration into the genomes of the two partner cells. According to the literature, the initiation of ICE transfer is mediated by a nucleoprotein complex called relaxosome, whose key protein is the relaxase, a transesterase encoded by the element. The role of the relaxase is to perform a single-stranded cleavage on the DNA of the ICE at a conserved site, called nic. This cleavage releases a free 3'OH end, used as a primer to initiate rolling circle replication. The DNA-relaxase complex is then driven to the conjugation pore.During my PhD thesis, I studied ICESt3 from Streptococcus thermophilus which belongs to the ICESt3/Tn916/ICEBs1 superfamily, widespread among Firmicutes. These ICEs encode a non-canonical relaxase belonging to the MOBT family, which is related to the rolling circle replication initiators of the Rep_trans family. The general objective of my thesis was to elucidate the function of the RelSt3 relaxase in order to decipher the molecular mechanisms of initiation of conjugative transfer mediated by a MOBT relaxase.My work led to the identification of the RelSt3 binding site on ICESt3 origin of transfer (oriT). This site, called bind, is peculiar in that it is distant from the nic site, which is not the case for other relaxase families. RelSt3 possesses an HTH domain at its N-terminus. I have shown that this domain is required for the binding of RelSt3 to its bind site, and that it is important for its catalytic activity. Conjugation assays demonstrated that this HTH domain is crucial for the conjugative transfer of ICESt3. Structural predictions of the HTH domain in complex with DNA led to the identification of the interaction interface with the bind site, confirmed by mutagenesis. I also demonstrated that RelSt3 exhibits a nicking-closing activity and that it covalently binds to the 5' end of the cleaved strand, demonstrating that this enzyme participates in both initial and final steps of conjugation.In the literature, it has been shown that relaxases interact frequently with other accessory proteins, encoded by the ICE or by the host bacteria, participating in relaxosome formation. The second objective of my thesis was to identify RelSt3 partners. Comparisons with available data on ICEBs1 from Bacillus subtilis allowed to identify two candidate proteins, OrfL and OrfM, that may belong to the relaxosome of ICESt3, as well as a cellular helicase, PcrA , probably involved in the rolling circle replication. A characterization of these proteins was performed using biochemical and biophysical approaches. The interaction network between all of these proteins was established using in vitro approaches, as well as with the in vivo two-hybrid approach. These data provide a first insight into the components of the ICESt3 relaxasome. I also showed that OrfL and OrfM stimulate the catalytic activity of RelSt3 in vitro, and that they are both essential for ICESt3 conjugation.This work lead to a better understanding of the molecular mechanisms required during the conjugation of an ICE driven by a MOBT family relaxase

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<br>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

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<br>Cataloged from the official PDF of thesis.<br>Includes bibliographical references.<br>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.<br>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.<br>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.<br>by Mark Michael Harden, Jr.<br>Ph. D.<br>Ph.D. Massachusetts Institute of Technology, Department of Biology

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2018.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>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.<br>by Monika M. M. Avello.<br>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<br>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