Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

doi: 10.1111/j.2041-1014.2011.00605.x

Figure Lengend Snippet: Plasmids and bacterial strains used in this study

Article Snippet: Antibiotics were used at the following concentrations: erythromycin, 10 μg ml −1 in P. gingivalis and 300 μg ml −1 in E. coli ; carbenicillin, 100 μg ml −1 and rifampicin 1 μg ml −1 (spontaneous mutagenicity assay) or 100 μg ml −1 (rifampicin-resistance mutation assay). table ft1 table-wrap mode="anchored" t5 caption a7 Phenotype/description Source Plasmids pCR-XL-TOPO 3.5 Ap r , Km r Invitrogen pFLL145 mutY gene cloned into pCR-XL-TOPO 3.5 This study pFLL146 ermF-ermAM cloned into S mal site of mutY in pFLL145 This study pVA2198 Sp r , ermF-ermAM ( Fletcher et al ., 1995 ) pET11a Ap r Novagen pGEV1 Ap r ’ G protein N-terminal tag, C-terminal His tag Addgene, ( Huth et al ., 1997 ) pCR-XL-TOPO-PG mutY P. gingivalis mutY cloned into pCR-XL-TOPO This study pET11a-PG mutY P. gingivalis mutY cloned into pET11a This study pGEV1-PG mutY P. gingivalis mutY cloned into pGEV1 This study Bacterial strains W83 Wild type P. gingivalis ( Abaibou et al ., 2001 ) FLL147 mutY defective mutant This study DH5α F − φ 80d lacZ ΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi-1 gyrA96 relA1 Invitrogen PR8 E. coli wild-type A-Lien Lu-Chang PR70(DE3) E. coli mutY mutant A-Lien Lu-Chang BL21 (DE3) E. coli B F − dcm ompT hsdS (r B − m B − ) gal λ (DE3) Stratagene Open in a separate window Plasmids and bacterial strains used in this study

Techniques: Clone Assay, Mutagenesis

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

doi: 10.1111/j.2041-1014.2011.00605.x

Figure Lengend Snippet: Primers and oligonucleotides used for this study

Article Snippet: Antibiotics were used at the following concentrations: erythromycin, 10 μg ml −1 in P. gingivalis and 300 μg ml −1 in E. coli ; carbenicillin, 100 μg ml −1 and rifampicin 1 μg ml −1 (spontaneous mutagenicity assay) or 100 μg ml −1 (rifampicin-resistance mutation assay). table ft1 table-wrap mode="anchored" t5 caption a7 Phenotype/description Source Plasmids pCR-XL-TOPO 3.5 Ap r , Km r Invitrogen pFLL145 mutY gene cloned into pCR-XL-TOPO 3.5 This study pFLL146 ermF-ermAM cloned into S mal site of mutY in pFLL145 This study pVA2198 Sp r , ermF-ermAM ( Fletcher et al ., 1995 ) pET11a Ap r Novagen pGEV1 Ap r ’ G protein N-terminal tag, C-terminal His tag Addgene, ( Huth et al ., 1997 ) pCR-XL-TOPO-PG mutY P. gingivalis mutY cloned into pCR-XL-TOPO This study pET11a-PG mutY P. gingivalis mutY cloned into pET11a This study pGEV1-PG mutY P. gingivalis mutY cloned into pGEV1 This study Bacterial strains W83 Wild type P. gingivalis ( Abaibou et al ., 2001 ) FLL147 mutY defective mutant This study DH5α F − φ 80d lacZ ΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi-1 gyrA96 relA1 Invitrogen PR8 E. coli wild-type A-Lien Lu-Chang PR70(DE3) E. coli mutY mutant A-Lien Lu-Chang BL21 (DE3) E. coli B F − dcm ompT hsdS (r B − m B − ) gal λ (DE3) Stratagene Open in a separate window Plasmids and bacterial strains used in this study

Techniques: Sequencing

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

doi: 10.1111/j.2041-1014.2011.00605.x

Figure Lengend Snippet: Sequence alignment comparing Porphyromonas gingivalis MutY (PG) with Escherichia coli MutY (EC). The catalytic residues in the active site are indicated by black triangles (Glu37 and Asp138). Identical residues are marked with an asterisk (*) whereas highly conserved residues are marked with a colon (:). The blue line above the sequence (PG) or below the sequence (EC) indicates the helix-hairpin-helix domain; the green line indicates the endonuclease domain; the red line indicates the iron-sulfur cluster domain. The sequence alignment indicates that the two proteins may have similar functions.

Article Snippet: Antibiotics were used at the following concentrations: erythromycin, 10 μg ml −1 in P. gingivalis and 300 μg ml −1 in E. coli ; carbenicillin, 100 μg ml −1 and rifampicin 1 μg ml −1 (spontaneous mutagenicity assay) or 100 μg ml −1 (rifampicin-resistance mutation assay). table ft1 table-wrap mode="anchored" t5 caption a7 Phenotype/description Source Plasmids pCR-XL-TOPO 3.5 Ap r , Km r Invitrogen pFLL145 mutY gene cloned into pCR-XL-TOPO 3.5 This study pFLL146 ermF-ermAM cloned into S mal site of mutY in pFLL145 This study pVA2198 Sp r , ermF-ermAM ( Fletcher et al ., 1995 ) pET11a Ap r Novagen pGEV1 Ap r ’ G protein N-terminal tag, C-terminal His tag Addgene, ( Huth et al ., 1997 ) pCR-XL-TOPO-PG mutY P. gingivalis mutY cloned into pCR-XL-TOPO This study pET11a-PG mutY P. gingivalis mutY cloned into pET11a This study pGEV1-PG mutY P. gingivalis mutY cloned into pGEV1 This study Bacterial strains W83 Wild type P. gingivalis ( Abaibou et al ., 2001 ) FLL147 mutY defective mutant This study DH5α F − φ 80d lacZ ΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi-1 gyrA96 relA1 Invitrogen PR8 E. coli wild-type A-Lien Lu-Chang PR70(DE3) E. coli mutY mutant A-Lien Lu-Chang BL21 (DE3) E. coli B F − dcm ompT hsdS (r B − m B − ) gal λ (DE3) Stratagene Open in a separate window Plasmids and bacterial strains used in this study

Techniques: Sequencing

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

doi: 10.1111/j.2041-1014.2011.00605.x

Figure Lengend Snippet: Glycosylase assays (A) Assessing MutY glycosylase activity in W83 versus FLL147 using Oligo O3 (24 mer): lane 1, positive control using purified Escherichia coli MutY; lane 2, negative control for MutY glycosylase activity containing no MutY; lane 3, W83 extract assayed for uracil-N-glycosylase activity; lane 4, FLL147 extract assayed for uracil-N-glycosylase activity; lane 5, FLL147 extract assayed for MutY glycosylase activity; lane 6, W83 extract assayed for MutY glycosylase activity (B) Assessing MutY glycosylase activity to determine if Porphyromonas gingivalis MutY is functionally interchangeable with E. coli MutY using Oligo O1 (24-mer): lane 1, positive control using purified E. coli MutY; lane 2, PR8 cell extract containing a functional E. coli MutY; lane 3, PR70(DE3)/pET11a extract lacking a functional MutY; lanes 4 and 5, PR70(DE3)/pET11a-PG mutY extract containing a functional P. gingivalis MutY. Arrows indicate cleavage product resulting from glycosylase activity. For each glycosylase reaction, the following was used: 1 pmol oligo, 2 μg protein from cell extract or 1 unit purified enzyme.

Article Snippet: Antibiotics were used at the following concentrations: erythromycin, 10 μg ml −1 in P. gingivalis and 300 μg ml −1 in E. coli ; carbenicillin, 100 μg ml −1 and rifampicin 1 μg ml −1 (spontaneous mutagenicity assay) or 100 μg ml −1 (rifampicin-resistance mutation assay). table ft1 table-wrap mode="anchored" t5 caption a7 Phenotype/description Source Plasmids pCR-XL-TOPO 3.5 Ap r , Km r Invitrogen pFLL145 mutY gene cloned into pCR-XL-TOPO 3.5 This study pFLL146 ermF-ermAM cloned into S mal site of mutY in pFLL145 This study pVA2198 Sp r , ermF-ermAM ( Fletcher et al ., 1995 ) pET11a Ap r Novagen pGEV1 Ap r ’ G protein N-terminal tag, C-terminal His tag Addgene, ( Huth et al ., 1997 ) pCR-XL-TOPO-PG mutY P. gingivalis mutY cloned into pCR-XL-TOPO This study pET11a-PG mutY P. gingivalis mutY cloned into pET11a This study pGEV1-PG mutY P. gingivalis mutY cloned into pGEV1 This study Bacterial strains W83 Wild type P. gingivalis ( Abaibou et al ., 2001 ) FLL147 mutY defective mutant This study DH5α F − φ 80d lacZ ΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi-1 gyrA96 relA1 Invitrogen PR8 E. coli wild-type A-Lien Lu-Chang PR70(DE3) E. coli mutY mutant A-Lien Lu-Chang BL21 (DE3) E. coli B F − dcm ompT hsdS (r B − m B − ) gal λ (DE3) Stratagene Open in a separate window Plasmids and bacterial strains used in this study

Techniques: Activity Assay, Positive Control, Purification, Negative Control, Functional Assay

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

doi: 10.1111/j.2041-1014.2011.00605.x

Figure Lengend Snippet: Complementation of Escherichia coli mutY mutant with Porphyromonas gingivalis mutY

Article Snippet: Antibiotics were used at the following concentrations: erythromycin, 10 μg ml −1 in P. gingivalis and 300 μg ml −1 in E. coli ; carbenicillin, 100 μg ml −1 and rifampicin 1 μg ml −1 (spontaneous mutagenicity assay) or 100 μg ml −1 (rifampicin-resistance mutation assay). table ft1 table-wrap mode="anchored" t5 caption a7 Phenotype/description Source Plasmids pCR-XL-TOPO 3.5 Ap r , Km r Invitrogen pFLL145 mutY gene cloned into pCR-XL-TOPO 3.5 This study pFLL146 ermF-ermAM cloned into S mal site of mutY in pFLL145 This study pVA2198 Sp r , ermF-ermAM ( Fletcher et al ., 1995 ) pET11a Ap r Novagen pGEV1 Ap r ’ G protein N-terminal tag, C-terminal His tag Addgene, ( Huth et al ., 1997 ) pCR-XL-TOPO-PG mutY P. gingivalis mutY cloned into pCR-XL-TOPO This study pET11a-PG mutY P. gingivalis mutY cloned into pET11a This study pGEV1-PG mutY P. gingivalis mutY cloned into pGEV1 This study Bacterial strains W83 Wild type P. gingivalis ( Abaibou et al ., 2001 ) FLL147 mutY defective mutant This study DH5α F − φ 80d lacZ ΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi-1 gyrA96 relA1 Invitrogen PR8 E. coli wild-type A-Lien Lu-Chang PR70(DE3) E. coli mutY mutant A-Lien Lu-Chang BL21 (DE3) E. coli B F − dcm ompT hsdS (r B − m B − ) gal λ (DE3) Stratagene Open in a separate window Plasmids and bacterial strains used in this study

Techniques: Mutagenesis

Journal: Molecular oral microbiology

Article Title: Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

doi: 10.1111/j.2041-1014.2011.00605.x

Figure Lengend Snippet: Porphyromonas gingivalis MutY binds to an oligo containing an 8-oxoG:A. Lanes 1–3 represent the kit controls using EBNA (Epstein–Barr nuclear antigen) as the protein and EBNA oligo with or without biotin. In lanes 4–7, an oligo containing an 8-oxoG:A mispair with or without biotin was used. Lane 5 contains Escherichia coli MutY. Lanes 6 and 7 contain purified P. gingivalis MutY. For lanes 4–7, the following was used: 20 fmol oligo-biotin, 4 pmol unlabelled oligo, and 2 μg protein.

Article Snippet: Antibiotics were used at the following concentrations: erythromycin, 10 μg ml −1 in P. gingivalis and 300 μg ml −1 in E. coli ; carbenicillin, 100 μg ml −1 and rifampicin 1 μg ml −1 (spontaneous mutagenicity assay) or 100 μg ml −1 (rifampicin-resistance mutation assay). table ft1 table-wrap mode="anchored" t5 caption a7 Phenotype/description Source Plasmids pCR-XL-TOPO 3.5 Ap r , Km r Invitrogen pFLL145 mutY gene cloned into pCR-XL-TOPO 3.5 This study pFLL146 ermF-ermAM cloned into S mal site of mutY in pFLL145 This study pVA2198 Sp r , ermF-ermAM ( Fletcher et al ., 1995 ) pET11a Ap r Novagen pGEV1 Ap r ’ G protein N-terminal tag, C-terminal His tag Addgene, ( Huth et al ., 1997 ) pCR-XL-TOPO-PG mutY P. gingivalis mutY cloned into pCR-XL-TOPO This study pET11a-PG mutY P. gingivalis mutY cloned into pET11a This study pGEV1-PG mutY P. gingivalis mutY cloned into pGEV1 This study Bacterial strains W83 Wild type P. gingivalis ( Abaibou et al ., 2001 ) FLL147 mutY defective mutant This study DH5α F − φ 80d lacZ ΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 (r K − m K + ) phoA supE44 thi-1 gyrA96 relA1 Invitrogen PR8 E. coli wild-type A-Lien Lu-Chang PR70(DE3) E. coli mutY mutant A-Lien Lu-Chang BL21 (DE3) E. coli B F − dcm ompT hsdS (r B − m B − ) gal λ (DE3) Stratagene Open in a separate window Plasmids and bacterial strains used in this study

Techniques: Purification

Journal: BMC Oral Health

Article Title: A molecular survey of S. mutans and P. gingivalis oral microbial burden in human saliva using Relative Endpoint Polymerase Chain Reaction (RE-PCR) within the population of a Nevada dental school revealed disparities among minorities

doi: 10.1186/1472-6831-12-34

Figure Lengend Snippet: RE-PCR using DNA from P. gingivalis ( PG) standards and saliva samples. A) DNA standards obtained from PG samples containing 5.0 x 10 3 - 10 6 CFU/mL established minimum threshold (C T ) and saturation (C S ) cycles; (high concentration) 5/0 x 10 6 CFU/mL C T = C15, C S = C35; (low) 5.0 x 13 6 CFU/mL , C T = C30, C S = 55. B) RE-PCR at C30 (at low concentration C T = C30, below high concentration C S = C35) revealed strong, positive correlations (R 2 = 0.8507) between signal band intensity (SBI) and CFU/mL. C) RE-PCR using DNA extractions from all saliva samples revealed (n = 10/56 had elevated PG levels. Plotting the PG-positive sample SBI (*) with the DNA standards revealed samples with moderate to very high concentrations; Very high (n = 4), high risk (n = 4), moderate (n = 2). No significant differences in gender (not shown) between PG-positive and overall sample demographics were noted, however 90% (n = 9/10) of the PG-positive samples came from Minority patients, which was significantly different than in the overall sample (64.9%) ( X 2 = 17.921, d.f. = 1; p < 0.0001; M = minority, W = white). In addition, the ages of PG-positive patients were not found to significantly different than those of the study sample ( p = 0.05).

Article Snippet: The oral bacteria cell lines Streptococcus mutans ( S. mutans or SM) 25175 (NCTC-10449) and Porphyromonas gingivalis ( P. gingivalis or PG) BAA-1702 (FDC-381) were also obtained from ATCC.

Techniques: Concentration Assay

Journal: Microbiology Spectrum

Article Title: Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome

doi: 10.1128/spectrum.00599-24

Figure Lengend Snippet: P. gingivalis factors inhibit SARS-CoV-2 pseudovirus infection. ACE2-overexpressing 293T cells were infected with a single-round HIV-1 lentiviral vector pseudotyped with SARS-CoV-2 (CoV-2) spike (Wuhan strain) or VSV-G. The viruses contain luciferase reporter to allow for the measurement of infection in RLU. (a) Inhibition of infection by various concentrations (% vol/vol) of supernatants of P. gingivalis W83 WT ( Pg WT) grown in brain-heart infusion (BHI), tryptic soy broth (TSB), and 10% FCS DMEM medium and medium control (Control) is shown. (b) The inhibitory effect of supernatant of P. gingivalis grown in 10% FCS DMEM on CoV-2 Omicron BA.4/5 pseudotyped lentivirus was measured. (c) Comparison of CoV-2 pseudovirus versus VSV control inhibition by P. gingivalis supernatant (grown in DMEM/10%FCS) is shown and inhibition of infection (%Change of RLU) is indicated. The mean luciferase activity ± SD ( N = 3) of infected cells was determined. Difference between infection with treatment and control as well as between infection with CoV-2 and VSV pseudovirus was considered significant (* P < 0.05) using Student’s t -test.

Article Snippet: P. gingivalis ( Pg WT) W83 BAA-308 was purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Plasmid Preparation, Luciferase, Inhibition, Control, Comparison, Activity Assay

Journal: Microbiology Spectrum

Article Title: Inhibition of SARS-CoV-2 infection by Porphyromonas gingivalis and the oral microbiome

doi: 10.1128/spectrum.00599-24

Figure Lengend Snippet: Effects P. gingivalis gingipains in SARS-CoV-2 pseudovirus infection. (a) For SARS-CoV-2 pseudovirus inhibition of ACE2 + 293T infection, various concentrations (%vol/vol) of supernatants from wildtype P. gingivalis ( Pg WT) and triple gingipain knockout P. gingivalis mutant ( Pg W83 Δ kgp Δ rgpA Δ rgpB ), and 10% FCS DMEM only control (DMEM ctrl) were used. (b) Inhibition of ACE2 + 293T infection at higher bacterial supernatant concentrations (7.5% and 15%) is shown. (c) Cytotoxicity of ACE2 + 293T (measured by WST) at high concentrations of Pg WT supernatant but not triple gingipain knockout is shown. Infection was measured by luciferase activity (RLU) and the mean RLU ± SD ( N = 3) of infected cells with or without P. gingivalis supernatant was determined. Difference between infection with treatment and control was considered significant (* P < 0.05) using Student’s t -test.

Article Snippet: P. gingivalis ( Pg WT) W83 BAA-308 was purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Infection, Inhibition, Knock-Out, Mutagenesis, Control, Luciferase, Activity Assay