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UBC Theses and Dissertations

The TFP ATPase of haemophilus influenzae and DNA uptake Yang, Minghui


Haemophilus influenzae Rd is a Gram negative bacterium capable of taking up exogenous DNA and undergoing genetic transformation through natural competence. As in most naturally competent bacteria, H. influenzae proteins related to type IV pili are required for natural competence. The pil operon of H. influenzae is composed of four open reading frames, pilA, pilB, pilC and pilD, coding for homologues of proteins involved in the assembly and function of type IV pili. pilB is the second gene of the pil operon. It codes for the only H. influenzae homologue of Pseudomonas aeruginosa PilB and PilT, which are required for Type IV pili assembly and retraction, respectively. The deduced protein sequence of H. influenzae PilB was analyzed and nucleotide-binding motifs conserved in the VrbB/GspE family ATPases were identified. Alignment with the homologues in other naturally competent bacteria identified the most conserved regions in the C-termini of proteins in this family that are essential for ATPase activities. However, little is known with respect to the specific role of PilB in natural competence and DNA uptake. To find out H. influenzae PilB's function in DNA uptake, a pilB knockout RR1150 was constructed by an insertion at the Bell site using a Tn903 KanR cassette. To allow expression of the downstream genes pilC & pilD in this strain, an additional CRE (Competence Regulatory Element) regulatory sequence and promoter were placed in the knockout RR1150, right before pilC. Natural competence was eliminated in RR1150, indicating that pilB is essential for natural competence. DNA binding and uptake assays showed that the defect of competence in RR1150 is at the level of DNA uptake. To further investigate the specific function of pilB, the P. aeruginosa PA01 homologues pilB and pilTv/zre, introduced into the above H. influenzae p//J?-background. The two P. aeruginosa genes were strongly expressed in H. influenzae pilB and the sequences have no mutations, as indicated by real time PCR and DNA sequencing. However, DNA binding and uptake in H. influenzae pilB was not restored. The experimental data and the recently available evidence in related studies suggest that complementation failures could be attributed to either the species-specific interactions of Tfp proteins, or to the different ways by which the Tfp assembly/function systems work. Furthermore, since PilT-like ATPases were not found in any Pasteurellaceae species possessing type IV pili by BLAST search and sequence analysis, I suggest that the energetics of Pasteurellaceae Tfp is different from the PilB-PilT system of P. aeruginosa Tfp, and that an as-yet-uncharacterized protein provides the driving force required for Tfp retraction in Pasteurellaceae.

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