Abstract

The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike other organisms, a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxoG) in P. gingivalis was suggested. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1 kb fragment containing the uvrB gene was PCR-amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on Brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black-pigmentation and beta-hemolysis when compared to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity or sensitivity to hydrogen peroxide when compared to the parent strain. However, in contrast to the wild-type, P. gingivalis FLL144 was significantly more sensitive to UV irradiation. The enzymatic removal of 8-oxoG from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxoG lesion. Analysis of these proteins indicates that a conserved hypothetical protein, PG1037, was of particular interest. This protein is encoded as a part of an operon which is flanked by two genes, namely, PG1036 (uvrA) and PG1038 (prcA). The uvrA-pg1037-prcA operon in P. gingivalis is upregulated in the presence of H2O2. A PCR-based linear transformation method was successfully used to inactivate the uvrA and prcA genes by allelic exchange mutagenesis. Similar to the wild-type when plated on Brucella blood agar, the isogenic mutants were blackpigmented and beta-hemolytic. The mutants showed different generation time and levels of proteolytic activities compared to the wild-type strains. The uvrA- and prcA-defective mutants were more sensitive to H2O2 and were significantly more sensitive to UV irradiation than the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. In protein-protein interaction studies we identified a protein complex associated with the removal of the 8-oxoG lesion. Collectively, these findings suggest that the uvrA-pg1037-prcA operon plays an important role in peroxide resistance in P. gingivalis and that a complex may be required to remove the 8-oxoG lesion. Also, the repair of oxidative stress-induced DNA damage involving 8-oxoG occurs by a yet to be described mechanism.

LLU Discipline

Microbiology and Molecular Genetics

Department

Basic Sciences

School

School of Medicine

First Advisor

Fletcher, Hansel

Second Advisor

Johnson, Mark

Third Advisor

Perry, Christopher

Fourth Advisor

Soto, Ubaldo

Fifth Advisor

Sowers, Lawrence

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

January 2011

Date (Title Page)

6-1-2011

Language

English

Library of Congress/MESH Subject Headings

Porphyromonas gingivalis;

Subject - Local

Periodontal pocket, Oxidative stress, Proteolytic activity

Type

Dissertation

Page Count

185 p.

Digital Format

Application/PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses & Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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