Epoxide-Mediated CifR Repression of cif Gene Expression Utilizes Two Binding Sites in Pseudomonas aeruginosa
| Autor: | George A. O'Toole, Dean R. Madden, Emily L. Dolben, Deborah A. Hogan, Christopher D. Bahl, Allia K. Lindsay, Jessica D. St. Laurent, Alicia E. Ballok |
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| Rok vydání: | 2012 |
| Předmět: |
DNA
Bacterial Cystic Fibrosis Virulence Factors Operon Repressor ATP-binding cassette transporter Biology Microbiology Bacterial Proteins Gene expression Humans Pseudomonas Infections Cloning Molecular Binding site Promoter Regions Genetic Molecular Biology Gene Binding Sites Promoter Articles Gene Expression Regulation Bacterial Apical membrane Molecular biology Mutagenesis Mutation Pseudomonas aeruginosa Epoxy Compounds DNA Intergenic Protein Binding |
| Zdroj: | Journal of Bacteriology. 194:5315-5324 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.00984-12 |
| Popis: | Pseudomonas aeruginosa secretes an epoxide hydrolase virulence factor that reduces the apical membrane expression of ABC transporters such as the cystic fibrosis transmembrane conductance regulator (CFTR). This virulence factor, named CFTR inhibitory factor (Cif), is regulated by a TetR-family, epoxide-responsive repressor known as CifR via direct binding and repression. We identified two sites of CifR binding in the intergenic space between cifR and morB , the first gene in the operon containing the cif gene. We have mapped these binding sites and found they are 27 bp in length, and they overlap the −10 and +1 sites of both the cifR and morB regulatory region and the start of transcription, respectively. In addition, we found that CifR binds to each repression site with differing affinity. Mutagenesis of these binding sites resulted in a loss of DNA binding in vitro , and mutation of one of these sites in vivo resulted in an increase in transcription of both the cif and cifR genes. We characterized cif and cifR gene expression in sputum and found that, whereas cif gene expression varied relative to an in vitro coculture control, cifR gene expression was consistently higher. Analysis of a longitudinal sample of CF isolates from nine patients revealed that Cif protein was expressed over time, although variably, and these changes could not be linked to mutations in the cifR gene or the promoters of these genes. Finally, we tested CifR responsiveness to other epoxides and showed that CifR can respond to multiple epoxides to various degrees. |
| Databáze: | OpenAIRE |
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