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Integration host factor and sequences downstream of the Pseudomonas aeruginosa algD transcription start site are
1Department of Microbiology and Immunology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157-1064.
Abstract:
Pseudomonas aeruginosa is an extremely important opportunistic pathogen in immunocompromised individuals. Strains of P. aeruginosa isolated from chronic lung infections in patients with the genetic disease cystic fibrosis have a mucoid colony morphology. This phenotype is due to overproduction of the exopolysaccharide alginate, which is believed to confer a selective advantage on P. aeruginosa in cystic fibrosis lungs. Alginate biosynthesis is controlled by a complex regulatory mechanism. Genes located in the 34-min region of the P. aeruginosa chromosome form an operon which encodes most of the biosynthetic enzymes necessary for alginate production. algD, the first gene in the operon and a critical point for the transcriptional regulation of alginate biosynthesis, is controlled by several trans, cis, and environmental factors. In this study, the involvement of the histone-like protein integration host factor (IHF) in algD expression was examined. Sequences with similarity to consensus IHF-binding sites of Escherichia coli were identified 75 bp upstream (site 1) and 90 bp downstream (site 2) of the start of algD transcription. In gel band mobility shift assays, DNA fragments containing either site bind IHF but site 2 has an approximately 90-fold higher affinity for IHF. Mutations in each of the elements were generated, and they resulted in the reduction or loss of in vitro IHF binding and a three- to fourfold decrease in algD-cat expression. This indicates that IHF binding is necessary for high-level algD transcription. The presence of a high-affinity IHF-binding site located 3' of the algD transcription start site suggested that sequences further downstream of this element are involved in algD expression. When a fragment located downstream of site 2 and upstream of the promoterless cat gene (+110 to +835) was deleted, algD-cat expression was reduced 10-fold supporting the notion that 3' enhancer elements are required for algD transcription. This is the first direct evidence of a 3' element involved in the control of a P. aeruginosa gene. It is postulated that IHF mediates the formation of a higher-order looped structure which is necessary for efficient algD transcription.
Insights
Integration host factor (IHF) is essential for high-level algD transcription in Pseudomonas aeruginosa. A downstream DNA element acts as a 3' enhancer, crucial for alginate production in cystic fibrosis lung infections.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis patients, characterized by mucoid morphology due to alginate overproduction.
- Alginate biosynthesis is regulated by a complex mechanism involving an operon, with algD being critical for transcriptional control.
- Environmental and regulatory factors influence algD expression, but the precise mechanisms remain under investigation.
Purpose of the Study:
- To investigate the role of the histone-like protein integration host factor (IHF) in regulating algD expression in Pseudomonas aeruginosa.
- To identify and characterize IHF binding sites involved in algD transcription.
- To explore the potential involvement of downstream DNA elements in algD gene expression.
Main Methods:
- Bioinformatic analysis to identify potential IHF binding sites upstream and downstream of algD.
- Gel band mobility shift assays to assess IHF binding affinity to DNA fragments.
- Site-directed mutagenesis to evaluate the impact of IHF binding sites on algD expression.
- Construction and deletion analysis of algD-cat fusion constructs to identify enhancer elements.
Main Results:
- Two potential IHF binding sites were identified: site 1 (upstream) and site 2 (downstream) of algD transcription start. Site 2 exhibited a significantly higher affinity for IHF (approx. 90-fold).
- Mutations in IHF binding sites reduced in vitro IHF binding and decreased algD-cat expression by three- to fourfold, indicating IHF is necessary for high-level transcription.
- Deletion of a downstream DNA fragment (110 to +835) resulted in a 10-fold reduction in algD-cat expression, confirming the presence of a 3' enhancer element.
Conclusions:
- Integration host factor (IHF) is a necessary factor for high-level algD transcription in Pseudomonas aeruginosa.
- A downstream DNA sequence functions as a 3' enhancer element, crucial for efficient algD expression.
- IHF likely mediates the formation of a higher-order DNA loop involving the 3' enhancer for optimal algD transcription, a novel regulatory mechanism in P. aeruginosa.