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Genetics and Molecular Biology |
Microbial Pathogenesis Unit, Department of Microbiology and Immunology, University of Adelaide, Adelaide, South Australia 50051
Author for correspondence: Paul A. Manning. Tel: +1 781 839 4000. Fax: +1 781 839 4500. e-mail: paul.manning{at}astrazeneca.com
Until the discovery of the Vibriocholerae repeat (VCR), the gene capture and expression systems termed integrons had been typically associated with antibiotic-resistance gene cassettes with usually less than five genes in an array. A method is described for the cloning of the ends of large cassette arrays. Conserved restriction sites within VCRs facilitated the mapping by Southern hybridization and cloning of the 5’ end of the VCR array, and using appropriate fragments it was possible to develop a physical map of the region of the V. cholerae chromosome. Sequence determination of the predicted beginning of this region revealed intI4, a member of the integron family of integrases. Comparison of these sequences from El Tor, Classical and serotype O134 V. cholerae strains identified the 3’ end of the attI site, thereby defining the class 4 integron in one of the V. cholerae chromosomes, and providing the first evidence for integron-like site-specific recombination within V. cholerae. Conduction assays demonstrated IntI1-mediated recombination between VCRs. Restriction mapping places the sequences of intI4 and 26 VCR gene cassettes in arrays within a 120 kb region of the V. cholerae O1 strain 569B genome. This region contains an estimated 150 VCR gene cassettes, dwarfing previously described arrays. Southern analysis of genomic DNA from strains of Vibrio anguillarum, Vibrio mimicus and a number of V. cholerae serotypes revealed fragments that hybridized with VCR-specific probes but showed a high degree of restriction fragment length polymorphism. These data facilitate the identification of part of a new class 5 integron from V. mimicus.
Keywords: gene capture, site-specific recombination, repetitive sequences, evolution
Abbreviations: 59-be, 59-base element; 5’-CS, 5’ conserved sequence; VCCI, Vibrio cholerae chromosomal integron; VCR, Vibrio cholerae repeat
The GenBank accession numbers for the sequences determined in this work are AF025662, AF055586, X64097, AF179593 and AF179596.
a Present address: AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA.
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