Bacterial genome wide epistasis: extant, emergence and molecular bases. – GeWiEp

Our experimental approach can be broken down into four phases:
1) The creation, thanks to new methods of genetic engineering, of hybrid genomes between strains.
2) The marking of these strains by genetic barcodes to follow their selective value
3) Sequencing and genetic engineering analysis of these strains
4) integration of data into models and bioinformatics analysis of genomes from databases to confirm oberservations.


We propose to use as material the strains evolved by the laboratory of Richard Lenski since 1988 as well as the pathogenic isolates of the strain ST131 which have diverged since 1987.

The objective is to measure the emergence of negative interactions between the various components of recent genomes and to try to identify the molecular bases of these interactions.

1) We have developed two alternative methods for mixing genomes and invented a new approach for selecting recombinants

2) We have succeeded in barcoding several strains including natural isolates and strains evolved at length in the laboratory. We were able to show that the barcodes were stable over time and could allow the dynamics of adaptation to be detected.

3) A phenotyping method at the transciptomic level of thousands of mutants has been developed and can be applied to the recombinants produced.

4) A database of 60,000 genomes has been constructed, the core genome phylogeny carried out and the effect of the mutations predicted.


ST131 strains and Richard Lenski strains have been better characterized in phenotypic terms.

We will now proceed to the production phase of the recombinants and analyze their selective value and their phenotypes with the techniques developed.

Methodological articles are being prepared on the methods developed;

Although the last decades witnessed many victories against infectious diseases, the spread of multidrug-resistant bacteria is now challenging these past successes. Species that were previously well controlled are now becoming health threats. For instance, Escherichia coli, which caused minor trouble 15 years ago, is now a major concern in the hospital owing to increased antibiotic resistance and extra-intestinal virulence. This pattern is partly driven by the worldwide expansion of clone ST131. It was undetected in the collections sampled in humans in the 1980s but represents now about 7% and up to 18% of strains isolated in commensal conditions in France and in extra-intestinal pathologies in the UK, respectively.

The fast emergence and evolutionary success of multi-resistant clones with some ecological specificity in the E. coli species raises both medical and fundamental interests. How can a clone or phylogroup propagate and conserve its specificity despite the presence of genetic exchanges that may transmit its genes to other species members. Experimental evolution suggests that adaptation selects rapidly for mutations scattered on the chromosome that interact epistatically with one another. We suggest that this form of Genome Wide Epistasis (GWE) may explain the diversification within E. coli species. GWE may limit genetic transfers from and to a given clone as disruption or partial transfer of a successful combination of alleles will be costly and result in recombinants with low fitness.

To test our hypothesis, we will use two unique, unprecedented and complementary strain collections that both evolved over the last 30 years. The Long-Term Experimental Evolution (LTEE) from Richard Lenski with now 70,000 generations of asexual evolution in a perfectly controlled laboratory environment is a unique analytical material to unravel the genomic consequences of adaptation. The emergence, 30 years ago, of two antibiotic resistant clades of ST131 provides an alternative well-documented system of evolution that occurred this time in the wild, in the presence of genetic exchange. Moreover, ST131 is a public health concern owing to ST131 high virulence and antibiotic resistance.

To uncover the emergence and extant of GWE, we will perform crosses between strains and study the fitness of the recombinants. Recent developments in synthetic biology tools and sequencing technologies allow now the production at high rates of recombinants and the precise measurement of their fitness in bulk through barcoding sequencing. We therefore propose to evaluate precisely the distribution of fitness effects of recombinants in the two previous systems. Their comparison and integration in a theoretical model of speciation will provide an unprecedented quantitative approach to GWE in a bacterial species. To validate this phenomenological approach, we will uncover the molecular determinants of GWE using new developments in millifluidics and microfluidics. With a method akin to quantitative trait loci analysis, our aim will be to detect regions that are restrictive to genetic exchange and to precisely measure the fitness of recombinants involving the transfer of these regions. For a subset of identified combinations of mutations, we will use a combinatorial approach coupled to microfluidic genotyping to reconstruct the precise adaptive landscapes composed of mutations or regions selected in different populations/clades.

This ambitious project tackles the fundamental question of the emergence of stable genetic specificities in a sexual bacterial species of medical relevance, and addresses it using recent technological revolutions in the fields of synthetic biology, sequencing, millifluidics and microfluidics.