Projet ANR-08-MIEN-0016 – IIRE : Integrase d'integron recombinaison et expression
Programme : Maladies Infectieuses et environnement (MIE) 2008
Année de financement : 2008
Coordinateur du projet : INSTITUT PASTEUR (INSTITUT PASTEUR)
Partenaires : Institut Pasteur; Institut Pasteur; Université de Limoges
Montant : 449999.0 euros
Date de début : 2009
Durée : 36 mois
Résumé : Integrons are a fast reaction device for the Gram negative bacteria to overcome the use of novel antibiotics. They are a means of capture and dissemination of genes conferring resistance to antibiotics. It is therefore essential to identify and develop strategies to stop, or at least limit, the cassettes recruitment and exchange in integrons. Our present knowledge shows that the recombination process in these elements is unique, and could offer an opportunity to develop specific antagonist molecules, targeting the cassette integration reaction, the cassette excision, or both.  To reach these goals we need to precisely characterize the different recombination reactions , namely the integration step (attI x attC), the Holliday junction intermediate resolution, and moreover, determine when and where they occur. Multiple events including the SOS response, and the use of antibiotics themselves, impact on the expression and regulation of the integrase, these will also be addressed.We propose, in this context, that the recombination reaction for the insertion step requires a different set of specific paradigms by the enzyme in order to recognize both the hairpin attC and double stranded attI. We will determine the role of base substitution on the recombination frequency. We will determine the role of DNA phasing and propensity of the sequences to generate hairpins by insertions and deletions in the attC. We will test the availability of single strand DNA by using conjugation systems to deliver specific strands. We would like to investigate the steps beyond the first strand exchange of single strand DNA, namely the mechanism/cellular components required for the resolution of the Holliday junction intermediate (HJ). We will test the effect of components of the RuVABC system and DNA polymerases on recombination frequencies. We will determine the crystal structure of the IntI1-HJ (attIxattC). We will monitor binding to the HJ by gel retardation assays using 5' end fluophore labeled DNA. Furthermore, we believe that the programmed integration event is dependent on a set of cellular processes that result from an ideal series of conditions and participants, in particular to see when in an animal model the integrase expression is triggered, and if antibiotics intake is triggering resistance cassette relocation through an SOS response induction. We will monitor the expression of the promoter for the integrase, and will monitor the rearrangement of cassettes in the integron under antibiotic selective pressure.
We would therefore like to address these issues using a multidisciplinary approach including genetics, biochemical and structural methods in order to overcome limitations in individual techniques when it comes to the study of such complex events. We hope to pinpoint the specific features of the double stranded DNA recognition and contrast it with that of the single DNA attC. We will reveal the crucial role of the nucleotides at the core of the site, the implication of their sequences, structure, interaction with the enzyme, and their role in DNA-DNA micro-homology pairing for strand exchange. We will obtain the three dimensional structure of the integrase IntI1-Holliday junction (HJ)(attIxattC) complex. We will determine the role of cellular components in the resolution of the HJ. Additionally, we will shed light on the mechanism of single strand DNA production in the bacterial cell and the role this state of DNA in the physiology of the cell, specifically pertaining to integron integrase activity.
We will obtain information on the control of the integron integrase expression with respect to treatment with antibiotics, the triggering of the recombination process for adaptation by rearrangements, and dispersion of cassettes.