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IN VITRO AND IN VIVO ACTIVITY OF DINB: ROLE IN MUCOID CONVERSION OF PSEUDOMONAS AERUGINOSA

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abstract
The opportunistic pathogen Pseudomonas aeruginosa undergoes a phenotypic change called mucoid conversion in the lungs of chronically infected cystic fibrosis (CF) patients. This phenotypic change leads to the production of the exopolysaccharide alginate, which gives the organism a selective advantage in the CF lung, as alginate protects against reactive oxygen species, antibiotics and host immune cells. The majority of the mucoid variants that emerge from CF patients have mutations in mucA, which encodes an anti-sigma factor and this mutation allows for the biosynthesis of alginate. The mechanisms leading to the mutagenesis of mucA have yet to be discovered, and are the main focus of this work. We first characterized the protein DinB of P. aeruginosa, which we hypothesized would be involved in the response to DNA damage. We have shown that DinB is a DNA polymerase with no exonuclease activity, and that it is able to utilize both magnesium and manganese as metal ion cofactors for catalysis. The Km for dNTP is over 600-fold lower when manganese is present compared with magnesium, indicating a role for the cofactor in changing the affinity of the enzyme for dNTP. Given the E. coli DinB’s preference for creating -1 frameshifts in poly-guanine runs, we also assayed the activity of P. aeruginosa DinB on the poly-guanine run of a mucA-derived template. When there was a G:G mismatch as the terminal base pair of the template/primer, DinB was able to extend the primer but caused -1 frameshifts. We have determined that this effect is most likely due to DNA slippage of the template and primer strands during replication, based on DinB’s lack of frameshifting on a template without the homopolymeric run. In the context of a chronic CF respiratory infection, where a high frequency of hypermutable mutants emerge, this ability of DinB to extend a mismatch at this site in mucA with a -1 frameshift could be a partial explanation for mucoid conversion in vivo. Having examined the activity of DinB, we have also shown that dinB is directly repressed by LexA as part of an SOS-type response, and that DinB is up-regulated during response to DNA damage by mitomycin C and hydrogen peroxide. Using a Lac+ reversion assay, plasmid-expressed DinB increased the frequency of -1 frameshift mutations in a poly-guanine run. Given the similarity of this sequence with the mutation hotspot in mucA, we next considered DinB’s role in mucoid conversion. After developing a selection system for detecting mucoid variants, we determined the effect of both hydrogen peroxide treatment as well as the role of DNA repair. Following sub-lethal hydrogen peroxide treatment, mucoid conversion frequency increased in both the wild-type and isogenic lexA- mutant. When a strain has lost mismatch repair function by deletion of mutS, the mucoid conversion frequency was drastically increased. Both with the single deletion dinB- as well as the double deletion mutS-dinB-, mucoid conversion is essentially abolished. This indicates a crucial role for both DinB and MutS in mucoid conversion. Given this data, we conclude that there is a DinB-dependent pathway for mucoid conversion, and that impairment of MMR serves to exacerbate this phenomenon.
subject
Pseudomonas aeruginosa
mucoid conversion
DinB
Y family DNA polymerase
mismatch repair
cystic fibrosis
contributor
Rockel, Andrea (author)
Fred W. Perrino, PhD (committee chair)
Daniel J. Wozniak, PhD (committee member)
Karin D. Scarpinato, PhD (committee member)
David A. Ornelles, PhD (committee member)
Sean D. Reid, PhD (committee member)
date
2009-03-19T14:24:49Z (accessioned)
2010-06-18T18:59:03Z (accessioned)
2009-03-19T14:24:49Z (available)
2010-06-18T18:59:03Z (available)
2009-03-19T14:24:49Z (issued)
degree
Microbiology & Immunology (discipline)
identifier
http://hdl.handle.net/10339/14820 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work for access only to the Wake Forest University system for one year from the date below. After one year, release the entire work for access worldwide. (accessRights)
title
IN VITRO AND IN VIVO ACTIVITY OF DINB: ROLE IN MUCOID CONVERSION OF PSEUDOMONAS AERUGINOSA
type
Dissertation

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