A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence
Porphyromonas gingivalis is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of P. gin...
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Frontiers Media S.A.
2024-10-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1476171/full |
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| author | Kimihiro Abe Kimihiro Abe Hiroko Yahara Ryoma Nakao Takehiro Yamaguchi Yukihiro Akeda |
| author_facet | Kimihiro Abe Kimihiro Abe Hiroko Yahara Ryoma Nakao Takehiro Yamaguchi Yukihiro Akeda |
| author_sort | Kimihiro Abe |
| collection | DOAJ |
| description | Porphyromonas gingivalis is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of P. gingivalis; however, these techniques require specialized equipment such as high-voltage electroporators, conjugative plasmids and donor strains. In this study, we present a simple, cost-effective transformation method for P. gingivalis without any special equipment by exploiting its natural DNA competence. P. gingivalis ATCC 33277 was grown to the early-exponential phase and mixed with a donor DNA cassette. This mixture was then spotted onto a BHI-HM blood-agar plate and incubated for one day to promote colony biofilm formation. The resulting colony biofilm was suspended in a liquid medium and spread onto antibiotic-containing agar plates. Transformants appeared within 4 to 5 days, achieving a maximum efficiency of 7.7 × 106 CFU/μg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other P. gingivalis strains, W83 and TDC60. Additionally, we discovered that deletion of PGN_0421 or PGN_0519, encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence. |
| format | Article |
| id | doaj-art-aa90dfaf7ef14e2a8e84d02f4cb37a2e |
| institution | OA Journals |
| issn | 1664-302X |
| language | English |
| publishDate | 2024-10-01 |
| publisher | Frontiers Media S.A. |
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| spelling | doaj-art-aa90dfaf7ef14e2a8e84d02f4cb37a2e2025-08-20T01:50:45ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-10-011510.3389/fmicb.2024.14761711476171A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competenceKimihiro Abe0Kimihiro Abe1Hiroko Yahara2Ryoma Nakao3Takehiro Yamaguchi4Yukihiro Akeda5Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, JapanResearch Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, JapanGenome Medical Science Project, Research Institute, National Center for Global Health and Medicine, Tokyo, JapanDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, JapanDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, JapanDepartment of Bacteriology I, National Institute of Infectious Diseases, Tokyo, JapanPorphyromonas gingivalis is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of P. gingivalis; however, these techniques require specialized equipment such as high-voltage electroporators, conjugative plasmids and donor strains. In this study, we present a simple, cost-effective transformation method for P. gingivalis without any special equipment by exploiting its natural DNA competence. P. gingivalis ATCC 33277 was grown to the early-exponential phase and mixed with a donor DNA cassette. This mixture was then spotted onto a BHI-HM blood-agar plate and incubated for one day to promote colony biofilm formation. The resulting colony biofilm was suspended in a liquid medium and spread onto antibiotic-containing agar plates. Transformants appeared within 4 to 5 days, achieving a maximum efficiency of 7.7 × 106 CFU/μg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other P. gingivalis strains, W83 and TDC60. Additionally, we discovered that deletion of PGN_0421 or PGN_0519, encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1476171/fullPorphyromonas gingivalisnatural competencetransformationhorizontal gene transfergenetic engineering |
| spellingShingle | Kimihiro Abe Kimihiro Abe Hiroko Yahara Ryoma Nakao Takehiro Yamaguchi Yukihiro Akeda A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence Frontiers in Microbiology Porphyromonas gingivalis natural competence transformation horizontal gene transfer genetic engineering |
| title | A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence |
| title_full | A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence |
| title_fullStr | A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence |
| title_full_unstemmed | A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence |
| title_short | A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence |
| title_sort | simple and cost effective transformation system for porphyromonas gingivalis via natural competence |
| topic | Porphyromonas gingivalis natural competence transformation horizontal gene transfer genetic engineering |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1476171/full |
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