Recombinational and transcriptional regulation within the class 1 integron
Cagle, Caran Astor
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The integron is a site-specific recombination system that uses IntI integrase to mediate the insertion and excision of gene cassettes among bacterial genomes, notably the dissemination of antibiotic resistance genes. Integrons carry three promoters, a well conserved integrase promoter (Pint), and seven variants of a pair of promoters (Pc and P2) responsible for gene cassette transcription. I investigated integron regulation at the levels of recombination and transcription. Qualitative PCR and protein pull-down assays done here did not identify growth-phase- dependent, or accessory protein(s) involvement in recombination regulation. However, others work revealed growth phase involvement as recombination products increased in the transition from log to stationary phase, and crystallography revealed site-directed recognition through specific substrate conformations. Competitive electro-mobility binding assays done in this body of work suggested additional levels of recombination regulation through differing IntI1 folding conformations resulting in the preference of one recognition site over another. Bi-directional transcriptional fusion data of Pint, and Pc and P2 established the weak expression of Pint and confirmed Pc and P2 strength; in contrast to prior speculation, deletion of 3-bp within the P2 spacer region as it exists in four other versions, resulted in dramatically reduced but not completely inhibited expression of P2. The transcription regulator predictor program PRODORIC 8.9 predicted FIS, LexA, and IHF sites. In vitro binding assays confirmed the direct interaction of all three proteins with the promoter region. Transcriptional fusion data showed FIS repressed Pint and the cassette promoters; however, LexA did not repress Pint or P2 in the native IntI state (i.e. without the 3-bp deletion resulting in formation of a LexA site). IHF activated Pint and the cassette promoters, and did not affect P2 with 3-bp deletion. Finally, H-NS, a transcription regulator with no sequence specific recognition sites, directly repressed Pint and P2 promoters with 3-bp deletion, but activated the cassette promoters (Pc and P2) in vivo. Integrase mediated recombination is an intricate multi-faceted network of regulatory components entailing growth phase and conformations of the substrate DNA and IntI. Furthermore, the integrase and cassettes are regulated transcriptionally through equally complicated nucleo-protein complexes involving FIS, LexA, IHF, and H-NS.