Heterogeneity of the GFP fitness landscape and data-driven protein design.

Studies of protein fitness landscapes reveal biophysical constraints guiding protein evolution and empower prediction of functional proteins. However, generalisation of these findings is limited due to scarceness of systematic data on fitness landscapes of proteins with a defined evolutionary relationship. We characterized the fitness peaks of four orthologous fluorescent proteins with a broad range of sequence divergence. While two of the four studied fitness peaks were sharp, the other two were considerably flatter, being almost entirely free of epistatic interactions. Mutationally robust proteins, characterized by a flat fitness peak, were not optimal templates for machine-learning-driven protein design - instead, predictions were more accurate for fragile proteins with epistatic landscapes. Our work paves insights for practical application of fitness landscape heterogeneity in protein engineering.

A Versatile Protocol to Generate Translocations in Yeast Genomes Using CRISPR/Cas9.

Genomic engineering methods represent powerful tools to examine chromosomal modifications and to subsequently study their impacts on cellular phenotypes. However, quantifying the fitness impact of translocations, independently from base substitutions or the insertion of genetic markers, remains a challenge. Here we report a rapid and straightforward protocol for engineering either targeted reciprocal translocations at the base pair level of resolution between two chromosomes or multiple simultaneous rearrangements in the yeast genome, without inserting any marker sequence in the chromosomes. Our CRISPR/Cas9-based method consists of inducing either (1) two double-strand breaks (DSBs) in two different chromosomes with two distinct guide RNAs (gRNAs) while providing specifically designed homologous donor DNA forcing the trans-repair of chromosomal extremities to generate a targeted reciprocal translocation or (2) multiple DSBs with a single gRNA targeting dispersed repeated sequences and leaving endogenous uncut copies of the repeat to be used as donor DNA, thereby generating multiple translocations, often associated with large segmental duplications (Fleiss, et al. PLoS Genet 15:e1008332, 2019).

Reshuffling yeast chromosomes with CRISPR/Cas9.

Genome 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.

A brief review of bioluminescent systems (2019).

Despite being widely used in reporter technologies, bioluminescent systems are largely understudied. Of at least forty different bioluminescent systems thought to exist in nature, molecular components of only seven light-emitting reactions are known, and the full biochemical pathway leading to light emission is only understood for two of them. Here, we provide a succinct overview of currently known bioluminescent systems highlighting available tools for research and discussing future applications.

The evolution of the temporal program of genome replication.

Genome 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 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.

Reconstructing 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.