UBC Theses and Dissertations
Identification and characterization of DNA receptor candidates in Haemophilus influenzae Molnar, Sebastian James
Haemophilus influenzae is a naturally transformable bacterium with a mechanism for the preferential uptake of conspecific DNA. This mechanism requires the specific recognition of uptake signal sequences (USS) on exogenous DNA by receptors on the bacterial cell surface. The USS-specific DNA receptor in H. influenzae is unknown. From the set of known and putative competence genes, candidate USS-receptor genes were identified using bioinformatic approaches. ComEl was initially identified as the best candidate receptor, since it is orthologous to proteins that bind DNA for uptake in Bacillus subtilis and other bacteria. A comEl knockout was previously found to have a 10-fold reduction in its transformability, and this retention of partial DNA uptake in ΔcomEl allowed a direct test for a change in uptake specificity. Thus, to test for a role as the USS-specific DNA-receptor, I used DNA-competition experiments to compare uptake specificity in the ΔcomEl and wild type strains. A loss of uptake specificity in ΔcomEl was not detected. Therefore, comEl does not encode the USS-specific receptor. Recently, a putative competence operon (comNOPQ) was found to have an upstream competence regulatory element (CRE), which is essential for competence gene expression, and microarray analysis showed that its genes are induced during competence development. The operon contained USS-receptor candidate genes, however, it was not known whether comNOPQ genes function in competence. To test this, I constructed a knockout of comN and measured its transformability. The transformation frequency of the ΔcomN strain was 10⁵-fold less than that of wild type. Furthermore, I found that DNA binding and uptake were completely eliminated in the ΔcomN. This suggests that comN could be a component of the DNA binding and uptake machinery or that it functions in the assembly of such apparatus. An alternative possibility is that the ΔcomN strain has polar effects on downstream genes in the comNOPQ operon, and comN does not function in the competence mechanism. Further work is needed to test these possibilities. However, I have demonstrated that the comNOPQ operon is part of the competence regulon and that it contains one or more genes that are essential for the DNA uptake mechanism in H. influenzae.
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