Characterization of a site-specific gene-transfer mechanism in bacteria

Sammanfattning: Integrons capture and rearrange single genes borne on mobile units called cassettes using site-specific recombination. The integrons increase the variation of antibiotic resistance among bacterial plasmids and may also reorganize clustered genes in bacterial genomes.The integrons consist of a conserved sequence coding for a recombinase of the lambda phage integrase family, and adjacent cassettes in tandem. Palindromic recombination sites, attC, occur 3' of all cassettes but the conserved sequence is joined to the cassettes by another type of site, attI. All sites contain a core with the reactive sequence GTT.Constructed deletions and point mutations were used to characterize attC and attI. Evidence was found that attC contains two symmetrical and co-operating subsites. The subsite containing the core was 1000-fold more reactive than that containing an inverse core. Mutations in the inverse core reduced recombination at the remote core strongly which suggests that the low-reactive end in attC has significance for the assembly of a synaptic nucleoprotein complex. The low-reactive end of attC could furthermore be used to fuse the site to sequences in the process of being recruited to new cassettes.Most integrations of cassettes occurred at attI. Recombination between two attI sites responded to upstream deletions progressing closer than 9-14 bp from the core. Recombination with an attC-site required another area of the conserved sequence 32 bp from the core of attI. The cross-over point was localized between G and TT in the core.Nucleotide sequencing of integrons documented new cassettes harboring ORFS, the dihydrofolate reductase gene dfr2a and a new spectinomycin resistance gene aadA4.The integron of class 2 in Tn7 was sequenced and functionality was demonstrated after mutagenesis of an internal stop codon in the integrase gene. Also the third class of integron was shown to be functional. Limited cross-specificity between the three integron classes was observed.The integrases of integron classes 1 and 3 were expressed as polyhistidine conjugates and purified by metal ion affinity chromatography. The purified recombinases catalyzed site-specific recombination in cell-free reactions stimulated by the architectural proteins HU and IHF.