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Investigation of the role of comA in uptake signal sequence recognition in Haemophilus influenzae

University of British Columbia

Haemophilus influenzae is a naturally competent bacterial species that preferentially takes up conspecific DNA. It recognizes this DNA by a nine base pair Uptake Signal Sequence (USS) that is over-represented in its own genome. The mechanism by which this USS is recognized is unknown, though knockout mutations in several genes have been shown to eliminate both binding and uptake of DNA. One of these genes may be involved in binding the USS. I attempted to determine if comA, one of the candidate genes, was responsible for the sequence specific binding. This gene was amplified under mutagenic PCR conditions, and then used to transform a hypercompetent strain of H. influenzae, using a novobiocin resistant PCR amplified marker sequence to identify transformants. Transformants were then screened for a reduction in transformation frequency to identify possible mutants. comA from these lines was then sequenced and compared to the known wild type sequence. None of the mutant lines differed in the sequence of comA in comparison to the known wild type, so no conclusions may be drawn concerning the role of comA. Mapping these mutations was beyond the scope of this project; however, a possible explanation for the reduced transformation frequency phenotypes is described here. Four isolates retained their phenotypes under competence inducing conditions, indicating a change in binding efficiency. None of the isolates possessed a reversion of the hypercompetent parental genotype.

Text

2010-01-05

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http://hdl.handle.net/2429/17499

Zoology

University of British Columbia

UBCV

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