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.
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.