We study the mechanisms responsible of the bacterial genome variability, with a special interest for those involved in exogenous gene acquisition – the horizontal gene transfer. Our model system is the integron, a natural genetic engineering system involved in the development and dissemination of antibiotic resistance genes among Gram-negative species. We are also investigating other factors playing a role in the plasticity of the Vibrio species genomes, which are all constituted of two circular chromosomes with distinct dynamic characteristics. Our last but not least topic of interest, we are working on the evolutionary constrains which are exerted at the gene level by the host codon usage.
Extension of the evolutionary potential of any gene
Evolutionary processes largely rely on the production of diversity. Genetic robustness, by allowing the accumulation of neutral diversity within a population, has been associated with increase in evolutionary potential (evolvability). We have already shown that one can use a well-known source of robustness, the redundancy of the genetic code, to alter the evolvability of any protein. The topology of the code allows synonymous codons to sample different mutational neighborhoods. Using this property, we developed an algorithm to design synonymous sequences with maximally divergent evolutionary potentials relative to the input sequences (see publication). A web version is accessible here for academic use. At the population level, each of these sequences expands the scope of the evolutionary landscape that can be explored by the encoded protein, and ultimately increase the odds of uncovering adaptive mutants. We applied this principle to evolve new antibiotic resistance phenotype variants. Fundamentally, our results provide an example of how neutral diversity may favor evolvability. Moreover, in light of the rapid development in nucleic acid synthesis, the use of rationally designed synonymous genes offers a profitable enhancement to any directed evolution procedure. We are currently applying this principle to other genes of interest, in order to access to novel phenotypes, so far undescribed.
We are studying different aspects of this gene capture system: their distribution, their contribution to the adaptive capacity of their host and their recombination processes.
This natural genetic system is composed of two basic elements: a gene coding an integrase of the site-specific tyrosine recombinase family and a primary recombination site, attI. The integrase activity allows the insertion of open reading frames, in the form of a circular cassette, at the recombination site. All these cassettes are composed of a single gene associated to a recombination site, the attC site, indispensable for the integrase recognition and recombination with attI.
We have shown that the resistance integrons derived from sedentary super-integrons carried by environmental species, such as the different Vibrio.
Recently, the structural characteristics of the attC sites led us to propose a new model for the recombination in integrons, which only involved the attC bottom strand folded in a stem-and loop, based on its symmetrical structure, and a canonical double-strand (ds) attI site. Recognition and recombination by the IntI integrase of such a structure with a canonical ds-attI site would lead to a Holliday junction (HJ) intermediate which may be resolved by a replication step.
We have sustained this model with in vivo experiments, but also through the resolution of the 3D structure of integron integrase tetramer bound to single stranded substrates (collaboration with D. Gopaul).
We are currently studying the pathways allowing the formation of the attC secondary structure in vivo and the resolution of the unconventional Holliday junction.
We also showed that in most integrons, be they chromosomal or mobile, the integrase expression was controlled by the SOS response regulator LexA. This control allow to subdue the cassette capture and array reorganization to the episodes of stress met by bacteria during their life, such as the antibiotics treatments. We also showed that horizontal gene transfer carried on by both conjugation and natural transformation were potent inducers of the SOS response, and that they also triggered the integrase expression and cassette recombination.
Plasticity of the Vibrio species genomes
This third project is to investigate other factors involved in genome plasticity of the complex genome of Vibrio species. The Vibrio group includes a large number of pathogenic species whose hosts range from human to aquatic animals. The few species so far characterized have been found to carry two circular chromosomes showing a high variability. The selective advantage conferred by such an organization is unknown.
To increase our knowledge, we sequenced the genome of V. splendidus LGP 32 in collaboration with C. Bouchier, a strain which is only remotely related to the Vibrio species sequenced so far. We are currently sequencing (collaboration with the French genoscope) another vibrio, V. nigripulchritudo, a shrimp patohgen, which has the largest genome among characterized Vibrio (>6.5 Mb). Togeteher with comparative analyses with the other sequenced Vibrio genome, we have undertaken different in vivo and in silico genome subtraction approaches to identify the hot spot of variability. We expect better understanding of the rules governing the overall organization and the gene partition between the two chromosomes in Vibrio.
We have now undertaken the experimental study of the 2 chromosome organisation, by developing tools that allow to precisely remodel the geome architecture. We have now built V. cholerae strains with either a single chromosome, two chromosomes of even size, and two chromosomes controlled by the same oriC1. We are currently studyng these strains to understand their different physiological properties.