title: Protective role of the PG1036-PG1037-PG1038 operon in oxidative stress in Porphyromonas gingivalis W83.
abstract: As an anaerobe, Porphyromonas gingivalis is significantly affected by the harsh inflammatory environment of the periodontal pocket during initial colonization and active periodontal disease. We reported previously that the repair of oxidative stress-induced DNA damage involving 8-oxo-7,8-dihydroguanine (8-oxoG) may occur by an undescribed mechanism in P. gingivalis. DNA affinity fractionation identified PG1037, a conserved hypothetical protein, among other proteins, that were bound to the 8-oxoG lesion. PG1037 is part of the uvrA-PG1037-pcrA operon in P. gingivalis which is known to be upregulated under H2O2 induced stress. A PCR-based linear transformation method was used to inactivate the uvrA and pcrA genes by allelic exchange mutagenesis. Several attempts to inactivate PG1037 were unsuccessful. Similar to the wild-type when plated on Brucella blood agar, the uvrA and pcrA-defective mutants were black-pigmented and beta-hemolytic. These isogenic mutants also had reduced gingipain activities and were more sensitive to H2O2 and UV irradiation compared to the parent strain. Additionally, glycosylase assays revealed that 8-oxoG repair activities were similar in both wild-type and mutant P. gingivalis strains. Several proteins, some of which are known to have oxidoreducatse activity, were shown to interact with PG1037. The purified recombinant PG1037 protein could protect DNA from H2O2-induced damage. Collectively, these findings suggest that the uvrA-PG1037-pcrA operon may play an important role in hydrogen peroxide stress-induced resistance in P. gingivalis.
authors.affiliation: Division of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, California, United States of America.
authors.forename: Leroy G
authors.initials: LG
authors.lastname: Henry
authors.forename: Wilson
authors.initials: W
authors.lastname: Aruni
authors.forename: Lawrence
authors.initials: L
authors.lastname: Sandberg
authors.forename: Hansel M
authors.initials: HM
authors.lastname: Fletcher
mesh_terms: D018829:Adhesins, Bacterial / Q000378:metabolism; D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D045647:DNA Glycosylases / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D000080867:Gingipain Cysteine Endopeptidases; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D007249:Inflammation; D013058:Mass Spectrometry; D016296:Mutagenesis; D009154:Mutation; D009876:Operon; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000080867:Gingipain Cysteine Endopeptidases; D011994:Recombinant Proteins; D006151:Guanosine; C046215:8-hydroxyguanosine; D006861:Hydrogen Peroxide; D045647:DNA Glycosylases; D003546:Cysteine Endopeptidases