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DNA uptake specificity of Haemophilus influenzae

University of British Columbia

DNA binding and uptake by the naturally transformable Gram-negative bacterium Haemophilus influenzae Rd has been studied for over twenty years. It is well characterized that H. influenzae cells preferentially bind and take up DNA from their own species. This preferential uptake is dependent on the ability of cells to recognize a 9-base pair uptake signal sequence (USS), 5 ' - AAGTGCGGT, in the DNA molecule. Genomic analysis has shown that there are 1465 copies of the 9-base pair uptake sites. Further analysis revealed an extended consensus region of 29 base pairs which includes the core region and two down stream 6-base pair A/T-rich regions, each spaced about one helix turn apart. To determine properties of the DNA molecule that, in addition to the presence of the USS, are necessary for uptake by H. influenzae I designed oligonucleotides with variations in the regions flanking the core USS. Oligonucleotides were varied in both the length and base composition of 3' and 5' flanking sequences. I showed that nucleotides 5' to the USS are required for high levels of binding and uptake of DNA. Also, I showed that both length and base composition of the 3' flanking region greatly affect binding and uptake. If sequence 3' to the USS is G/C rich, uptake proceeds at a very low level. However, if DNA lacks a 3' sequence, both binding and uptake are abolished. Based on these findings I propose a speculative model of how cells bind and take up DNA in a sequence specific manner. I attempt with this model to supplement other proposed models which do not address the initial steps of binding and uptake by H. influenzae. A second goal of m y research was to isolate the receptor that allows competent cell to preferentially bing and take up sequence specific DNA. The method I used was UV laser crosslinking. Conditions used in crosslinking experiments were varied including the time of incubation of DNA and cells, the presence and absence of competing DNAs and attachment of bulky groups to the DNA to prevent uptake. Following many attempts to isolate the receptor I concluded that under the conditions used, it was impossible to isolate the receptor using laser crosslinking.

Thesis/Dissertation

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2009-07-13

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http://hdl.handle.net/2429/10775

Zoology

University of British Columbia

UBCV

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