title: Regulation of the integrase and cassette promoters of the class 1 integron by nucleoid-associated proteins.
abstract: The integrase IntI1 catalyses recombination of antibiotic-resistance gene cassettes in the integron, a widely found bacterial mobile element active in spreading antibiotic multi-resistance. We have previously shown that resistance cassette recombination rate and specificity depend on the amount of intracellular integrase. Here, we used in vivo and in vitro methods to examine convergent expression of the integrase promoter (P(int)) and of the cassette promoters (P(c) and P(2)) in the prototypical plasmid-borne class 1 integron, In2. Highly conserved P(int) has near consensus -10 and -35 hexamers for σ(70) RNA polymerase, but there are 11 naturally occurring arrangements of P(c) alone or combinations of the P(c)+P(2) cassette promoters (note that P(2) occurs with a 14 or 17 bp spacer). Using a bi-directional reporter vector, we found that P(int) is a strong promoter in vivo, but its expression is reduced by converging transcription from P(c) and P(2). In addition to cis-acting convergence control of integrase expression, the regulator site prediction program, prodoric 8.9, identified sites for global regulators FIS, LexA, IHF and H-NS in and near the integron promoters. In strains mutated in each global regulator, we found that: (1) FIS repressed integrase and cassette expression; (2) LexA repressed P(int) and P(2) with the 14 bp spacer version of P(2) and FIS was necessary for maximum LexA repression; (3) IHF activated P(int) when it faced the strong 17 bp spacer P(2) but did not elevate its expression versus LexA-repressed P(2) with the 14 bp spacer; and (4) H-NS repressed both P(int) and the 14 bp P(2) but activated the 17 bp P(2) cassette promoters. Mobility shift assays showed that FIS and IHF interact directly with the promoter regions and DNase I footprinting confirmed extensive protection by FIS of wild-type In2 integron promoter sequence. Thus, nucleoid-associated proteins, known to act directly in site-specific recombination, also control integron gene expression directly and possibly indirectly.
authors.affiliation: Department of Microbiology, The University of Georgia, Athens, GA 30602-2605, USA.
authors.forename: Caran A
authors.initials: CA
authors.lastname: Cagle
authors.affiliation: Department of Microbiology, The University of Georgia, Athens, GA 30602-2605, USA.
authors.forename: Julia E S
authors.initials: JES
authors.lastname: Shearer
authors.affiliation: Department of Microbiology, The University of Georgia, Athens, GA 30602-2605, USA.
authors.forename: Anne O
authors.initials: AO
authors.lastname: Summers
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D032023:Integrons; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; C529458:FimG protein, E coli; D036781:Fimbriae Proteins; D019426:Integrases