Molecular analysis of the Haemophilus influenzae type b pilin gene.

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.