The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR- assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains

Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution in a model yeast genus

International audienceReconstructing genome history is complex but necessary to reveal quantitative principles governing genome evolution. Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture and gene repertoire. Here, we reconstructed the genome history of the genus Lachancea that appeared to cover a continuous evolutionary range from closely related to more diverged yeast species. Our approach integrated the generation of a high-quality genome data set; the development of AnChro, a new algorithm for reconstructing ancestral genome architecture; and a comprehensive analysis of gene repertoire evolution. We found that the ancestral genome of the genus Lachancea contained eight chromosomes and about 5173 protein-coding genes. Moreover, we characterized 24 horizontal gene transfers and 159 putative gene creation events that punctuated species diversification. We retraced all chromosomal rearrangements, including gene losses, gene duplications, chromosomal inversions and translocations at single gene resolution. Gene duplications outnumbered losses and balanced rearrangements with 1503, 929, and 423 events, respectively. Gene content variations between extant species are mainly driven by differential gene losses, while gene duplications remained globally constant in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to 14% of all gene losses by disrupting genes at their breakpoints. Finally, we found that nonsynonymous substitutions reached fixation at a coordinated pace with chromosomal inversions, translocations, and duplications, but not deletions. Overall, we provide a granular view of genome evolution within an entire eukaryotic genus, linking gene content, chromosome rearrangements , and protein divergence into a single evolutionary framework

Phenotypic impact of chromosomal rearrangements and evolution of yeast genomes

Nous avons cherché à évaluer l’impact des réarrangements chromosomiques sur l’évolution des génomes de levures selon deux approches. La première approche a consisté à retracer les réarrangements chromosomiques au cours de l’évolution des Saccharomycotina. Nous avons construit un arbre phylogénétique à partir de 66 génomes issus de bases de données publiques et reconstruit la structure des génomes ancestraux des 66 espèces. La comparaison des génomes ancestraux a permi d’inférer 5150 réarrangements chromosomiques passés. Nous avons montré que selon les clades considérés, les génomes évoluent plutôt par inversion ou par translocation et que les réarrangements chromosomiques et les mutations non-synonymes s’accumulent à un rythme coordonné au cours de l’évolution. La seconde approche a consisté à quantifier l’impact phénotypique des variations structurelles (SV) du génome en termes de taux de croissance végétative et de viabilité méiotique chez Saccharomyces cerevisiae. Nous avons développé une technique pour induire à façon des SV ciblés dans le génome de S. cerevisiae, en induisant deux coupures simultanées dans le génome de S. cerevisiae avec CRISPR/Cas9 et à guider la réparation des cassures par recombinaison homologue avec des oligonucléotides chimériques. Nous avons alors adapté cette technique pour induire en une étape un grand nombre de SV aléatoires. L’impact phénotypique des SV obtenus a été quantifié en méiose et en croissance végétative. Ces travaux montrent que même des réarrangements chromosomiques balancés n’affectant aucune phase codante génèrent une grande diversité phénotypique qui participe à l’adaptation des organismes à leur environnement.The aim of this work was to assess the impact of chromosomal rearrangements on the evolution of yeast genomes with two approaches. The first approach consisted in retracing past rearrangements during the evolution of Saccharomycotina yeast genomes. We have built a phylogenetic tree of 66 genomes gathered from public databases, then reconstructed the structure of all ancestral genomes of these species. By comparing the structure of reconstructed ancestral genomes, we have inferred 5150 past rearrangements. We showed that depending on the clades, genomes tend to evolve mostly by inversion or by translocation. In addition, we showed that chromosomal rearrangements and non-synonymous mutations tend to accumulate at a coordinated pace during evolution. The second approach aimed at quantifying the phenotypic impact of structural variations of chromosomes (SVs) in terms of vegetative growth and meiotic viability in Saccharomyces cerevisiae. We developed a technique to induce easily targeted SVs in the genome of S. cerevisiae by inducing two chromosomal breaks with CRISPR/Cas9 and providing the cells with chimerical donor oligonucleotides to repair the split chromosomes by homologous recombination. We have then adapted this technique to induce multiple random SVs in a single step. The phenotypic impact of obtained variants on vegetative growth and on spore viability was quantified. These results show that even balanced chromosomal rearrangements that do not affect coding sequence generate a wide phenotypic diversity that contributes to the adaptation of organisms to their environment

Impact phénotypique des réarrangements chromosomiques et évolution des génomes de levures

The aim of this work was to assess the impact of chromosomal rearrangements on the evolution of yeast genomes with two approaches. The first approach consisted in retracing past rearrangements during the evolution of Saccharomycotina yeast genomes. We have built a phylogenetic tree of 66 genomes gathered from public databases, then reconstructed the structure of all ancestral genomes of these species. By comparing the structure of reconstructed ancestral genomes, we have inferred 5150 past rearrangements. We showed that depending on the clades, genomes tend to evolve mostly by inversion or by translocation. In addition, we showed that chromosomal rearrangements and non-synonymous mutations tend to accumulate at a coordinated pace during evolution. The second approach aimed at quantifying the phenotypic impact of structural variations of chromosomes (SVs) in terms of vegetative growth and meiotic viability in Saccharomyces cerevisiae. We developed a technique to induce easily targeted SVs in the genome of S. cerevisiae by inducing two chromosomal breaks with CRISPR/Cas9 and providing the cells with chimerical donor oligonucleotides to repair the split chromosomes by homologous recombination. We have then adapted this technique to induce multiple random SVs in a single step. The phenotypic impact of obtained variants on vegetative growth and on spore viability was quantified. These results show that even balanced chromosomal rearrangements that do not affect coding sequence generate a wide phenotypic diversity that contributes to the adaptation of organisms to their environment.Nous avons cherché à évaluer l’impact des réarrangements chromosomiques sur l’évolution des génomes de levures selon deux approches. La première approche a consisté à retracer les réarrangements chromosomiques au cours de l’évolution des Saccharomycotina. Nous avons construit un arbre phylogénétique à partir de 66 génomes issus de bases de données publiques et reconstruit la structure des génomes ancestraux des 66 espèces. La comparaison des génomes ancestraux a permi d’inférer 5150 réarrangements chromosomiques passés. Nous avons montré que selon les clades considérés, les génomes évoluent plutôt par inversion ou par translocation et que les réarrangements chromosomiques et les mutations non-synonymes s’accumulent à un rythme coordonné au cours de l’évolution. La seconde approche a consisté à quantifier l’impact phénotypique des variations structurelles (SV) du génome en termes de taux de croissance végétative et de viabilité méiotique chez Saccharomyces cerevisiae. Nous avons développé une technique pour induire à façon des SV ciblés dans le génome de S. cerevisiae, en induisant deux coupures simultanées dans le génome de S. cerevisiae avec CRISPR/Cas9 et à guider la réparation des cassures par recombinaison homologue avec des oligonucléotides chimériques. Nous avons alors adapté cette technique pour induire en une étape un grand nombre de SV aléatoires. L’impact phénotypique des SV obtenus a été quantifié en méiose et en croissance végétative. Ces travaux montrent que même des réarrangements chromosomiques balancés n’affectant aucune phase codante génèrent une grande diversité phénotypique qui participe à l’adaptation des organismes à leur environnement

Reshuffling yeast chromosomes with CRISPR/ Cas9

International audienceGenome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions

The evolution of the temporal program of genome replication

International audienceGenome replication is highly regulated in time and space, but the rules governing the remodeling of these programs during evolution remain largely unknown. We generated genome-wide replication timing profiles for ten Lachancea yeasts, covering a continuous evolutionary range from closely related to more divergent species. We show that replication programs primarily evolve through a highly dynamic evolutionary renewal of the cohort of active replication origins. We found that gained origins appear with low activity yet become more efficient and fire earlier as they evolutionarily age. By contrast, origins that are lost comprise the complete range of firing strength. Additionally, they preferentially occur in close vicinity to strong origins. Interestingly, despite high evolutionary turnover, active replication origins remain regularly spaced along chromosomes in all species, suggesting that origin distribution is optimized to limit large inter-origin intervals. We propose a model on the evolutionary birth, death, and conservation of active replication origins

The evolution of the temporal program of genome replication

AbstractComparative analyses of temporal programs of genome replication revealed either a nearly complete conservation between closely related species or a comprehensive reprogramming between distantly related species. Therefore, many important questions on the evolutionary remodeling of replication timing programs remain unanswered. To address this issue, we generated genome-wide replication timing profiles for ten yeast species from the genus Lachancea, covering a continuous evolutionary range from closely related to more divergent species. The comparative analysis of these profiles revealed that the replication program linearly evolves with increasing evolutionary divergence between these species. We found that the evolution of the timing program mainly results from a high evolutionary turnover rate of the cohort of active replication origins. We detected about one thousand evolutionary events of losses of active replication origins and gains of newborn origins since the species diverged from their last common ancestor about 80 million years ago. We show that the relocation of active replication origins is independent from synteny breakpoints, suggesting that chromosome rearrangements did not drive the evolution of the replication programs. Rather, origin gains and losses are linked both in space, along chromosomes, and in time, along the same branches of the phylogenetic tree. New origins continuously arise with on average low to medium firing efficiencies and increase in efficiency and earliness as they evolutionarily age. Yet, a subset of newborn origins emerges with high firing efficiency and origin losses occur concomitantly to their emergence and preferentially in their direct chromosomal vicinity. These key findings on the evolutionary birth, death and conservation of active replication origins provide the first description of how the temporal program of genome replication has evolved in eukaryotes.</jats:p

A Reversible markerless translocation.

A. PAM sequences are symbolized by the small black bars within the CAN1 and ADE2 genes in dark blue and orange. RT- and PM-donors stand for Reciprocal Translocation and Point Mutation donor DNAs used to repair the DSBs. The point mutation responsible for the introduction of the STOP codon in the PAM sequence is indicated by an asterisk. Strain names are written in diagonal. B. Left: plots indicating the number of transformants for 108 transformed cells obtained in 3 independent experiments. Panels (1) and (2) illustrate the efficiency of transformation with the Cas9 plasmid and cutting efficiency of the gRNAs, respectively. Panels (3) and (4) show DSB repair efficiency at the ADE2 and CAN1 loci by both the Reciprocal Translocation (RT) and Point Mutation (PM) donor DNAs (Methods). Right: Histograms of the numbers of white [ADE2], pink [ade2], canavanine resistant [CANR] and sensitive [CANS] colonies obtained with the RT (3) and PM donors (4), 100% and 96% of [ade2] transformants are also mutated for the CAN1 gene, respectively. C. PFGE karyotypes of two strains carrying the ADE2-CAN1 translocation (YAF190, YAF192) and two strains with the reverse translocations restoring the original chromosomes V and XV (YAF194 and YAF199 originating from YAF190 and YAF192, respectively). Chimerical chromosomes are denoted VtXV and XVtV. Original chromosome XV and V from the reference strain BY4741 are indicated in orange and blue respectively. Chromosome size is indicated in kb.</p