topIb, a phylogenetic hallmark gene of Thaumarchaeota encodes a functional eukaryote-like topoisomerase IB

International audienceType IB DNA topoisomerases can eliminate torsional stresses produced during replication and transcription. These enzymes are found in all eukaryotes and a short version is present in some bacteria and viruses. Among prokaryotes, the long eukaryotic version is only observed in archaea of the phylum Thau-marchaeota. However, the activities and the roles of these topoisomerases have remained an open question. Here, we demonstrate that all available thaumar-chaeal genomes contain a topoisomerase IB gene that defines a monophyletic group closely related to the eukaryotic enzymes. We show that the topIB gene is expressed in the model thaumarchaeon Ni-trososphaera viennensis and we purified the recom-binant enzyme from the uncultivated thaumarchaeon Candidatus Caldiarchaeum subterraneum. This enzyme is active in vitro at high temperature, making it the first thermophilic topoisomerase IB characterized so far. We have compared this archaeal type IB enzyme to its human mitochondrial and nuclear counterparts. The archaeal enzyme relaxes both negatively and positively supercoiled DNA like the eukaryotic enzymes. However, its pattern of DNA cleavage specificity is different and it is resistant to camptothecins (CPTs) and non-CPT Top1 inhibitors, LMP744 and lamellarin D. This newly described ther-mostable topoisomerases IB should be a promising new model for evolutionary, mechanistic and structural studies

Characterization of Integrative and Conjugative Elements (ICEs) of the ICESt3 family and factors affecting their mobility

Les éléments intégratifs conjugatifs (ICE) sont des éléments génétiques mobiles se transférant horizontalement d’une bactérie à une autre. Les ICE sont porteurs de gènes adaptatifs pouvant significativement améliorer le fitness de la bactérie hôte et permettre son adaptation à de nouvelles niches écologiques. Lors de ces travaux, des ICE apparentés à ICESt3 ont été retrouvés chez la bactérie commensale et pathogène opportuniste Streptococcus salivarius. Les analyses in silico réalisées ont démontré la diversité des ICE de cette famille, notamment au niveau de leurs modules de recombinaison, de régulation mais aussi de leurs gènes adaptatifs potentiellement mis à disposition de la communauté microbienne orale et digestive de l’Homme. La fonctionnalité de deux de ces ICE a été mise en évidence expérimentalement à travers l’évaluation de la capacité de ces éléments à se transférer intra- et inter-spécifiquement. Ces travaux ont également permis l’identification de facteurs d’hôte influençant la mobilité d’ICESt3, révélant ainsi l’importance, pour le transfert et l’acquisition de cet ICE, des molécules de surface telles que les lipoprotéines, les acides téichoïques et les exopolysaccharides. En conclusion, il a été démontré que les éléments de la famille ICESt3 participent à l’évolution du génome chez différentes espèces de streptocoques et aux échanges génétiques entre bactéries issues de l’alimentation et bactéries de la flore digestive humaine. Enfin, ces travaux ont contribué à une meilleure compréhension des mécanismes et des facteurs d’hôte influençant la mobilité de ces éléments génétiques mobilesIntegrative Conjugative Elements (ICEs) are mobile genetic elements that can be horizontally transferred from a bacterium to another, eventually regardless of the species or any other classification, allowing them to benefit from a broad host spectrum. ICEs can carry adaptive genes that can significantly improve the bacterial fitness and allow its adaptation to new ecological niches. In this work, ICEs related to ICESt3 were found in the commensal and opportunistic pathogen Streptococcus salivarius. In silico analysis highlighted the diversity of the ICESt3 family within this species, especially concerning their recombination and regulation modules, but also their adaptive genes likely available for the oral and digestive microbial community of the human host. The functionality of two ICEs found in S. salivarius was experimentally confirmed through their ability to transfer in intra- and interspecific manners. This work also allowed the identification of host factors affecting ICESt3 mobility, and revealed the importance, for the transfer and the acquisition of this ICE, of cell surface molecules such as lipoproteins, teichoic acids and exopolysaccharides. In conclusion, this thesis allowed expanding the knowledge regarding the mobile genetic elements of the ICESt3 family. This work demonstrated that these elements contribute to genome evolution of different streptococci species and gene exchanges between bacteria originated from food and the human gut flora. Finally, this study contributes to a better comprehension of the mechanisms and host factors influencing the mobility of these mobile genetic element

Caractérisation des éléments intégratifs conjugatifs de la famille ICESt3 et des facteurs influençant leur mobilité

Integrative Conjugative Elements (ICEs) are mobile genetic elements that can be horizontally transferred from a bacterium to another, eventually regardless of the species or any other classification, allowing them to benefit from a broad host spectrum. ICEs can carry adaptive genes that can significantly improve the bacterial fitness and allow its adaptation to new ecological niches. In this work, ICEs related to ICESt3 were found in the commensal and opportunistic pathogen Streptococcus salivarius. In silico analysis highlighted the diversity of the ICESt3 family within this species, especially concerning their recombination and regulation modules, but also their adaptive genes likely available for the oral and digestive microbial community of the human host. The functionality of two ICEs found in S. salivarius was experimentally confirmed through their ability to transfer in intra- and interspecific manners. This work also allowed the identification of host factors affecting ICESt3 mobility, and revealed the importance, for the transfer and the acquisition of this ICE, of cell surface molecules such as lipoproteins, teichoic acids and exopolysaccharides. In conclusion, this thesis allowed expanding the knowledge regarding the mobile genetic elements of the ICESt3 family. This work demonstrated that these elements contribute to genome evolution of different streptococci species and gene exchanges between bacteria originated from food and the human gut flora. Finally, this study contributes to a better comprehension of the mechanisms and host factors influencing the mobility of these mobile genetic elementsLes éléments intégratifs conjugatifs (ICE) sont des éléments génétiques mobiles se transférant horizontalement d’une bactérie à une autre. Les ICE sont porteurs de gènes adaptatifs pouvant significativement améliorer le fitness de la bactérie hôte et permettre son adaptation à de nouvelles niches écologiques. Lors de ces travaux, des ICE apparentés à ICESt3 ont été retrouvés chez la bactérie commensale et pathogène opportuniste Streptococcus salivarius. Les analyses in silico réalisées ont démontré la diversité des ICE de cette famille, notamment au niveau de leurs modules de recombinaison, de régulation mais aussi de leurs gènes adaptatifs potentiellement mis à disposition de la communauté microbienne orale et digestive de l’Homme. La fonctionnalité de deux de ces ICE a été mise en évidence expérimentalement à travers l’évaluation de la capacité de ces éléments à se transférer intra- et inter-spécifiquement. Ces travaux ont également permis l’identification de facteurs d’hôte influençant la mobilité d’ICESt3, révélant ainsi l’importance, pour le transfert et l’acquisition de cet ICE, des molécules de surface telles que les lipoprotéines, les acides téichoïques et les exopolysaccharides. En conclusion, il a été démontré que les éléments de la famille ICESt3 participent à l’évolution du génome chez différentes espèces de streptocoques et aux échanges génétiques entre bactéries issues de l’alimentation et bactéries de la flore digestive humaine. Enfin, ces travaux ont contribué à une meilleure compréhension des mécanismes et des facteurs d’hôte influençant la mobilité de ces éléments génétiques mobile

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and their intra- and interspecies transfers

International audienc

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and their intra- and interspecies transfers

International audienc

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and their intra- and interspecies transfers

International audienc

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by wholegenomesequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3= end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appearparticularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcusfaecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase. IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restrictionmodification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius. Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating thatdiffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and their intra- and interspecies transfers

International audienc

Diversity of Integrative and Conjugative Elements of Streptococcus salivarius and Their Intra- and Interspecies Transfer

Integrative and conjugative elements (ICEs) are widespread chromosomal mobile genetic elements which can transfer autonomously by conjugation in bacteria. Thirteen ICEs with a conjugation module closely related to that of ICESt3 of Streptococcus thermophilus were characterized in Streptococcus salivarius by wholegenomesequencing. Sequence comparison highlighted ICE evolution by shuffling of 3 different integration/excision modules (for integration in the 3= end of the fda, rpsI, or rpmG gene) with the conjugation module of the ICESt3 subfamily. Sequence analyses also pointed out a recombination occurring at oriT (likely mediated by the relaxase) as a mechanism of ICE evolution. Despite a similar organization in two operons including three conserved genes, the regulation modules show a high diversity (about 50% amino acid sequence divergence for the encoded regulators and presence of unrelated additional genes) with a probable impact on the regulation of ICE activity. Concerning the accessory genes, ICEs of the ICESt3 subfamily appearparticularly rich in restriction-modification systems and orphan methyltransferase genes. Other cargo genes that could confer a selective advantage to the cell hosting the ICE were identified, in particular, genes for bacteriocin synthesis and cadmium resistance. The functionality of 2 ICEs of S. salivarius was investigated. Autonomous conjugative transfer to other S. salivarius strains, to S. thermophilus, and to Enterococcusfaecalis was observed. The analysis of the ICE-fda border sequence in these transconjugants allowed the localization of the DNA cutting site of the ICE integrase. IMPORTANCE The ICESt3 subfamily of ICEs appears to be widespread in streptococci and targets diverse chromosomal integration sites. These ICEs carry diverse cargo genes that can confer a selective advantage to the host strain. The maintenance of these mobile genetic elements likely relies in part on self-encoded restrictionmodification systems. In this study, intra- and interspecies transfer was demonstrated for 2 ICEs of S. salivarius. Closely related ICEs were also detected in silico in other Streptococcus species (S. pneumoniae and S. parasanguinis), thus indicating thatdiffusion of ICESt3-related elements probably plays a significant role in horizontal gene transfer (HGT) occurring in the oral cavity but also in the digestive tract, where S. salivarius is present

Impact of cell surface molecules on conjugative transfer of the integrative and conjugative element ICESt3 of Streptococcus thermophilus

International audienceIntegrative Conjugative Elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules including cell surface proteins, wall teichoic acids, lipoteichoic acids and exopolysaccharides on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild type donor towards mutated recipient cells increased 5- to 400-fold compared to WT when recipients cells were affected in lipoproteins, teichoic acids or exopolysaccharides. These mutants displayed an increased biofilm-forming ability compared to WT suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation.By contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport. IMPORTANCE: ICEs contribute to horizontal gene transfer of adaptive traits (for example virulence, antibiotic resistance or biofilm formation) and play a considerable role in bacterial genome evolution thus underlying the need of a better understanding of their conjugative mechanism of transfer. While most studies are focusing on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model of study, we demonstrated the impact of lipoproteins, teichoic acids and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfers mediated by ICEs