Related Experiment Videos
Molecular analysis of the Haemophilus influenzae type b pilin gene
1Department of Molecular Biology and Microbiology, Tufts University Health Sciences Campus, Boston, Massachusetts 02111.
Abstract:
A Haemophilus influenzae DNA library was prepared in the vector lambda EMBL3, and recombinant phage were screened for the pilin gene (pil) using a synthetic oligonucleotide. Southern blot analysis of the positive clones revealed a 2.5kb PstI/PvuI fragment that hybridized with the oligonucleotide probe. This fragment was subcloned into pBR322 and sequenced. The nucleotide sequence disclosed an open reading frame of 653 bases. The deduced amino acid sequence corresponded with the known amino acid sequence of the purified pilin protein. Primer extension analysis using total RNA from piliated H. influenzae cells delineated a start site for the gene, -10 and -35 promoter regions, and a ribosome-binding site. No transcripts were seen with the RNA derived from a non-piliated strain. Southern blots of DNA from a number of H. influenzae strains revealed homology with the pil structural gene. DNA from a non-piliated strain of H. influenzae also hybridized with the pil probe. Transcriptional and translational studies were performed in Escherichia coli with plasmids containing: (i) the pil gene on the 2.5 kb PstI/PvuI fragment, (ii) the pil gene fused to the phoA gene, and (iii) the pil gene present on a 12.2 kb insert containing extensive H. influenzae DNA flanking the pil gene. The results suggest that the H. influenzae pil gene is expressed in Escherichia coli, but from a promoter other than the one used in H. influenzae.
Insights
Researchers identified and sequenced the Haemophilus influenzae pilin (pil) gene. Expression studies in E. coli suggest the gene is transcribed from a different promoter than in H. influenzae.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Haemophilus influenzae possesses pili, which are protein appendages crucial for bacterial adhesion and colonization.
- Understanding the genetic basis of pilin production is essential for developing strategies against H. influenzae infections.
Purpose of the Study:
- To isolate, clone, and characterize the pilin (pil) gene from Haemophilus influenzae.
- To investigate the transcriptional regulation and expression of the H. influenzae pil gene.
Main Methods:
- Construction of a Haemophilus influenzae DNA library in lambda EMBL3.
- Screening for the pilin gene using a synthetic oligonucleotide probe.
- Southern blot analysis, DNA subcloning into pBR322, nucleotide sequencing, and primer extension analysis.
- Transcriptional and translational studies in Escherichia coli using various plasmid constructs.
Main Results:
- A 2.5kb PstI/PvuI fragment containing the pil gene was isolated and sequenced, revealing a 653-base open reading frame.
- The deduced amino acid sequence matched the known pilin protein sequence.
- Promoter regions (-10 and -35) and a ribosome-binding site were identified.
- Homology with the pil structural gene was found in various H. influenzae strains.
- The H. influenzae pil gene was expressed in E. coli, indicating functional transcription and translation, albeit from a different promoter.
Conclusions:
- The pilin gene from Haemophilus influenzae has been successfully cloned and sequenced.
- The genetic elements for pilin gene expression, including promoter and ribosome-binding sites, were identified.
- The H. influenzae pil gene can be expressed in Escherichia coli, suggesting potential for heterologous expression studies.