DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis

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 the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. 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 to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G 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-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.

FIG. 1.

Inactivation of P. gingivalis uvrB by allelic mutagenesis. PCR amplification was used to determine the inactivation of the P. gingivalis uvrB gene in seven erythromycin-resistant P. gingivalis colonies. (A) Lanes 1 to 7, primers P1 and P2 (Table 2) were used to amplify the 5.1-kb uvrB::ermF-ermAM cassette; lane 8, pFLL143; lane 9, P. gingivalis wild-type W83 (Table 2). (B) Lanes 1 to 7, amplification of ermF-ermAM cassette from seven erythromycin-resistant colonies, using the P3 and P4 primers; lane 8, amplification of ermF-ermAM cassette from pFLL143; lane 9, pVA2198; lane 10, P. gingivalis W83.

FIG. 2.

RT-PCR analysis of P. gingivalis W83 and P. gingivalis FLL144. DNase-treated total RNAs extracted from P. gingivalis strains W83 and FLL144 were subjected to RT-PCR. Lanes 1 and 3, primers P5 and P6 (Table 2) were used to amplify the 1.2-kb uvrB gene from P. gingivalis W83 and FLL144, respectively; lanes 2 and 4, primers P7 and P8 (Table 2) were used to amplify the 0.9-kb vimA gene from P. gingivalis W83 and FLL144, respectively; lanes 5 to 8, no-RT negative controls for uvrB and vimA from P. gingivalis W83 and FLL144. All lanes contained 10 μl of amplified mixture.

FIG. 3.

Proteolytic activity of P. gingivalis FLL144. P. gingivalis strains were grown to late log phase (OD₆₀₀ of 1.2) in 50 ml of BHI broth supplemented with hemin and vitamin K. (A) A whole-cell culture was analyzed for Rgp (BAPNA) activity. (B) A whole-cell culture was analyzed for Kgp (ALNA) activity. The results shown are representative of three independent experiments performed in triplicate.

FIG. 4.

UV sensitivity of P. gingivalis is increased by inactivation of uvrB. P. gingivalis strains W83 and FLL144 were grown to mid-log phase (OD₆₀₀ of 0.6), spread on BHI plates, and then subjected to irradiation at increasing doses (0 μJ, 500 μJ, and 1,000 μJ) of UV in a Stratalinker 2400 apparatus (Stratagene, La Jolla, CA). After 7 to 10 days of incubation, surviving colonies were counted and compared to those on untreated plates. The results are representative of three independent experiments. Error bars represent standard errors of the means.

FIG. 5.

Sensitivity of P. gingivalis strains W83 and FLL144 to hydrogen peroxide. P. gingivalis was grown to early log phase (OD₆₀₀ of 0.2) in BHI broth, 0.25 mM (A) or 0.5 mM (B) H₂O₂ was then added to the cell cultures, and the cultures were further incubated for 24 h. Cell cultures without H₂O₂ were used as controls. The results shown are representative of three independent experiments.

FIG. 6.

8-oxo-G and uracil removal activities of P. gingivalis strain W83 and FLL144 cell extracts. [γ-³²P]ATP-5′-end-labeled oligonucleotide (O1) was incubated with P. gingivalis extracts for 1 h (Fpg) or 1 min (Ung), electrophoresed, and visualized. (A) Lane −, negative control containing O1; lane +, positive control containing O1 and Fpg enzyme; lane 1, O1 and treated P. gingivalis W83 extract; lane 2, O1 and treated P. gingivalis FLL144 extract; lane 3, O1 and P. gingivalis W83 extract; and lane 4, O1 and P. gingivalis FLL144 extract. (B) [γ-³²P]ATP-5′-end-labeled oligonucleotides (O3 and O4) were incubated with P. gingivalis extracts for 20 min, electrophoresed, and visualized. Lane −, negative control containing O4 and Fpg enzyme; lane +, positive control containing O3 and Fpg enzyme; lane 1, O4 and treated P. gingivalis W83 extract; lane 2, O3 and treated P. gingivalis W83 extract; lane 3, O4 and treated P. gingivalis FLL144; and lane 4, O3 and treated P. gingivalis FLL144 extract. (C) Lane +, positive control containing O2 and Ung enzymes; lane 1, O2 and P. gingivalis W83 extract; and lane 2, O2 and P. gingivalis FLL144 extract.