Final Report Summary - ICADIGE (The Integron Cassette Dynamics and the Integrase Gene Expression) Difficulties of in vitro experiments by developing a novel in vivo deep-sequencing based approach. This approach will be useful for the study of other recombinases.On our second project we have evolved the integrase using as selective pressure the attI x attI reaction. To explore a broader evolutionary landscape of the integrase protein we have recoded the intI1 gene into two alternative yet synonymous genes. We have performed directed evolution experiments on all three alleles and have recovered 8 mutations conferring enhanced recombination frequencies. We have produced mutants of our wild type and alternative genes including all mutations and have subjected them to a new round of directed evolution experiments. We have obtained five new mutations yielding recombination frequencies three orders of magnitude higher than the wild type. These frequencies mirror those for the attC x attC reaction, for which the affinity is high and a crystal structure has been obtained. In these experiments we have obtained valuable structural data on the constraints of the reaction. For instance, we have observed mutation in the residues involved in the recognition of Extra Helical Bases of the attC site, as well as an accumulation of mutations within the C-terminal region of the protein, in the multimerization domain. This suggests that there are aspects of the multimerization process, related to the flexibility of this domain, that deeply impact the attI x attI reaction. Also, many evolutionary aspects of these experiments have been remarkable. We have efficiently explored evolutionary landscapes of IntI1 that where not available in the wt code, finding mutations specific to these alleles. By performing the evolution of the three genes in parallel we have observed phenomena of convergent and parallel evolution, trade-off effects, epistatic interactions between mutations, and re-evolution of residues. This project will result in a publication in a relevant journal and paves the way to obtaining proteins that will possibly be crystallisable.Conclusions and the socio-economic impacts of the project Antimicrobial resistance (AR) is currently one of the major threats for modern medicine. A vast majority of current medical interventions, both routine and cutting-edge, ultimately rely on the efficacy of antibiotics. AR has become one of the major concerns for the eCDC and the WHO, since it kills, it hampers the control of infectious diseases, increases the costs of health care and jeopardizes health care gains to society (www.who.int/mediacentre/factsheets/fs194/en/). In a knowledge-based approach to fight AR mechanistic works such as those financed through this project serve to better understand basic aspects of antimicrobial resistance ultimately having an important impact in the fight against resistance. The characterization of the flexibility of the integrase when processing the recombination reactions sets a new approach to the genesis of cassettes and gives valuable information for the development of anti-recombination molecules.
