Mutagenesis and Homology Modeling of the Tn21 Integron Integrase IntI1

摘要

Horizontal DNA transfer between bacteria is widespread and a major cause of antibioticnresistance. For logistic reasons, single or combined genes are shuttled between vectors such as plasmidsnand bacterial chromosomes. Special elements termed integrons operate in such shuttling and are thereforenvital for horizontal gene transfer. Shorter elements carrying genes, cassettes, are integrated in the integrons,nor excised from them, by virtue of a recombination site, attC, positioned in the 3′ end of each unit. It isna remarkable and possibly restricting elementary feature of attC that it must be single-stranded while thenpartner target site, attI, may be double-stranded. The integron integrases belong to the tyrosine recombinasenfamily, and this work reports mutations of the integrase IntI1 from transposon Tn21, chosen within anwell-conserved region characteristic of the integron integrases. The mutated proteins were tested for bindingnto a bottom strand of an attC substrate, by using an electrophoresis mobility shift assay. To aid in interpretingnthe results, a homology model was constructed on the basis of the crystal structure of integron integrasenVchIntIA from Vibrio cholerae bound to its cognate attC substrate VCRbs. The local stability and hydrogennbonding network of key domains of the modeled structure were further examined using molecular dynamicsnsimulations. The homology model allowed us to interpret the roles of several amino acid residues, fournof which were clearly binding assay responsive upon mutagenesis. Notably, we also observed featuresnindicating that IntI1 may be more prone to base-specific contacts with VCRbs than VchIntIA.