{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T21:47:05Z","timestamp":1782856025306,"version":"3.54.5"},"reference-count":40,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,10,21]],"date-time":"2024-10-21T00:00:00Z","timestamp":1729468800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p><jats:italic>Porphyromonas gingivalis<\/jats:italic> 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 <jats:italic>P. gingivalis<\/jats:italic>; 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 <jats:italic>P. gingivalis<\/jats:italic> without any special equipment by exploiting its natural DNA competence. <jats:italic>P. gingivalis<\/jats:italic> 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\u2009days, achieving a maximum efficiency of 7.7\u2009\u00d7\u200910<jats:sup>6<\/jats:sup>\u2009CFU\/\u03bcg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other <jats:italic>P. gingivalis<\/jats:italic> strains, W83 and TDC60. Additionally, we discovered that deletion of <jats:italic>PGN_0421<\/jats:italic> or <jats:italic>PGN_0519<\/jats:italic>, encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence.<\/jats:p>","DOI":"10.3389\/fmicb.2024.1476171","type":"journal-article","created":{"date-parts":[[2024,10,21]],"date-time":"2024-10-21T04:41:01Z","timestamp":1729485661000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence"],"prefix":"10.3389","volume":"15","author":[{"given":"Kimihiro","family":"Abe","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroko","family":"Yahara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryoma","family":"Nakao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takehiro","family":"Yamaguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukihiro","family":"Akeda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,10,21]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"fiaa031","DOI":"10.1093\/femsec\/fiaa031","article-title":"Biofilms: hot spots of horizontal gene transfer (HGT) in aquatic environments, with a focus on a new HGT mechanism","volume":"96","author":"Abe","year":"2020","journal-title":"FEMS Microbiol. Ecol."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"2312953","DOI":"10.1080\/20008686.2024.2312953","article-title":"Review of antibiotic-resistant bacteria and antibiotic resistance genes within the one health framework","volume":"14","author":"Ajayi","year":"2024","journal-title":"Infect. Ecol. Epidemiol."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"12","DOI":"10.1002\/9780471729259.mc13c02s05","article-title":"Genetic manipulation of Porphyromonas gingivalis","volume":"5","author":"B\u00e9langer","year":"2007","journal-title":"Curr. Protoc. Microbiol. Chapter"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"1126","DOI":"10.1016\/j.cub.2016.08.058","article-title":"Natural competence for transformation","volume":"26","author":"Blokesch","year":"2016","journal-title":"Curr. Biol."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"1622","DOI":"10.1002\/iub.2343","article-title":"Half a century of bacteriophage lambda recombinases: in vitro studies of lambda exonuclease and red-beta annealase","volume":"72","author":"Brewster","year":"2020","journal-title":"IUBMB Life"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1111\/omi.12375","article-title":"Prevalence of antibiotic resistance genes in the oral cavity and mobile genetic elements that disseminate antimicrobial resistance: a systematic review","volume":"37","author":"Brooks","year":"2022","journal-title":"Mol Oral Microbiol"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"241","DOI":"10.1038\/nrmicro844","article-title":"DNA uptake during bacterial transformation","volume":"2","author":"Chen","year":"2004","journal-title":"Nat. Rev. Microbiol."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"1012","DOI":"10.1016\/0006-291X(92)90847-E","article-title":"Conjugal transfer of plasmid and transposon DNA from Escherichia coli into Porphyromonas gingivalis","volume":"186","author":"Dyer","year":"1992","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1128\/aem.62.1.196-202.1996","article-title":"Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed \u03b2-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B14","volume":"62","author":"Gardner","year":"1996","journal-title":"Appl. Environ. Microbiol."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"1969","DOI":"10.1038\/nprot.2011.417","article-title":"Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries","volume":"6","author":"Goodman","year":"2011","journal-title":"Nat. Protoc."},{"key":"ref11","doi-asserted-by":"crossref","first-page":"1176","DOI":"10.1128\/JB.182.4.1176-1180.2000","article-title":"Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems","volume":"182","author":"Haake","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"305","DOI":"10.1021\/acssynbio.2c00565","article-title":"Simple and rapid non-ribosomal peptide synthetase gene assembly using the SEAM-OGAB method","volume":"12","author":"Jagadeesh","year":"2023","journal-title":"ACS Synth. Biol."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"767","DOI":"10.1016\/j.resmic.2007.09.004","article-title":"Natural genetic transformation: prevalence, mechanisms and function","volume":"158","author":"Johnsborg","year":"2007","journal-title":"Res. Microbiol."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"59","DOI":"10.1111\/prd.12429","article-title":"Periodontal microorganisms and Alzheimer disease - a causative relationship?","volume":"89","author":"Jungbauer","year":"2022","journal-title":"Periodontol."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"D587","DOI":"10.1093\/nar\/gkac963","article-title":"KEGG for taxonomy-based analysis of pathways and genomes","volume":"51","author":"Kanehisa","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"154","DOI":"10.1111\/prd.12425","article-title":"Role of Porphyromonas gingivalis in oral and orodigestive squamous cell carcinoma","volume":"89","author":"Lamont","year":"2022","journal-title":"Periodontol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"945523","DOI":"10.3389\/fneur.2022.945523","article-title":"Porphyromonas gingivalis: a key role in Parkinson's disease with cognitive impairment?","volume":"13","author":"Li","year":"2022","journal-title":"Front. 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Biol."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1016\/j.tim.2015.10.008","article-title":"The DNA-uptake process of naturally competent Vibrio cholerae","volume":"24","author":"Matthey","year":"2016","journal-title":"Trends Microbiol."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"318","DOI":"10.3390\/microorganisms11020318","article-title":"The contribution of the human oral microbiome to oral disease: a review","volume":"11","author":"Morrison","year":"2023","journal-title":"Microorganisms"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1093\/dnares\/dsn013","article-title":"Determination of the genome sequence of Porphyromonas gingivalis strain ATCC 33277 and genomic comparison with strain W83 revealed extensive genome rearrangements in P. gingivalis","volume":"15","author":"Naito","year":"2008","journal-title":"DNA Res."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"6145","DOI":"10.1128\/IAI.00261-06","article-title":"Porphyromonas gingivalis galE is involved in lipopolysaccharide O-antigen synthesis and biofilm formation","volume":"74","author":"Nakao","year":"2006","journal-title":"Infect. Immun."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"5591","DOI":"10.1128\/JB.185.18.5591-5601.2003","article-title":"Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis strain W83","volume":"185","author":"Nelson","year":"2003","journal-title":"J. Bacteriol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"2980","DOI":"10.3389\/fmicb.2018.02980","article-title":"Evolution of the natural transformation protein, ComEC, in Bacteria","volume":"9","author":"Pimentel","year":"2018","journal-title":"Front. Microbiol."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"94","DOI":"10.3389\/fcimb.2019.00094","article-title":"Competence in Streptococcus pneumoniae and close commensal relatives: mechanisms and implications","volume":"9","author":"Salvadori","year":"2019","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1111\/mmi.14083","article-title":"Immunoglobulin-like domains of the cargo proteins are essential for protein stability during secretion by the type IX secretion system","volume":"110","author":"Sato","year":"2018","journal-title":"Mol. Microbiol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"336","DOI":"10.1111\/j.1574-6976.2012.00353.x","article-title":"Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental gram-negative bacteria","volume":"37","author":"Seitz","year":"2013","journal-title":"FEMS Microbiol. Rev."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"15","DOI":"10.3390\/microorganisms11010015","article-title":"Modulatory mechanisms of pathogenicity in Porphyromonas gingivalis and other periodontal pathobionts","volume":"11","author":"Sharaf","year":"2022","journal-title":"Microoranisms"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"784","DOI":"10.1038\/nbt1183-784","article-title":"A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria","volume":"1","author":"Simon","year":"1983","journal-title":"Nat. Biotechnol."},{"key":"ref31","first-page":"161","article-title":"Genetic transformation of Bacteroides spp. using electroporation","volume":"47","author":"Smith","year":"1995","journal-title":"Methods Mol. Biol."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"1072","DOI":"10.1073\/pnas.44.10.1072","article-title":"Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate","volume":"44","author":"Spizizen","year":"1958","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref33","doi-asserted-by":"publisher","first-page":"130","DOI":"10.1016\/j.mib.2017.04.006","article-title":"Making serine integrases work for us","volume":"38","author":"Stark","year":"2017","journal-title":"Curr. Opin. Microbiol."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"6382","DOI":"10.1128\/JB.00460-07","article-title":"Conjugal transfer of chromosomal DNA contributes to genetic variation in the oral pathogen Porphyromonas gingivalis","volume":"189","author":"Tribble","year":"2007","journal-title":"J. Bacteriol."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"e00231","DOI":"10.1128\/mBio.00231-11","article-title":"Natural competence is a major mechanism for horizontal DNA transfer in the oral pathogen Porphyromonas gingivalis","volume":"3","author":"Tribble","year":"2012","journal-title":"MBio"},{"key":"ref36","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1007\/BF00264941","article-title":"Restriction and modification in Bacillus species","volume":"152","author":"Uozumi","year":"1977","journal-title":"Mol. Gen. Genet."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"767","DOI":"10.1038\/nmeth.1377","article-title":"Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms","volume":"6","author":"van Opijnen","year":"2009","journal-title":"Nat. Methods"},{"key":"ref38","doi-asserted-by":"publisher","first-page":"822190","DOI":"10.3389\/fmed.2022.822190","article-title":"Oral and gut microbial dysbiosis and non-alcoholic fatty liver disease: the central role of Porphyromonas gingivalis","volume":"9","author":"Wang","year":"2022","journal-title":"Front. Med."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"4259","DOI":"10.1128\/JB.05269-11","article-title":"Complete genome sequence of the bacterium Porphyromonas gingivalis TDC60, which causes periodontal disease","volume":"193","author":"Watanabe","year":"2011","journal-title":"J. Bacteriol."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"208","DOI":"10.1111\/j.1399-302X.1993.tb00561.x","article-title":"Genetic transformation of Porphyromonas gingivalis by electroporation","volume":"8","author":"Yoshimoto","year":"1993","journal-title":"Oral Microbiol. Immunol."}],"container-title":["Frontiers in Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2024.1476171\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,10,21]],"date-time":"2024-10-21T04:41:03Z","timestamp":1729485663000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmicb.2024.1476171\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,21]]},"references-count":40,"alternative-id":["10.3389\/fmicb.2024.1476171"],"URL":"https:\/\/doi.org\/10.3389\/fmicb.2024.1476171","relation":{},"ISSN":["1664-302X"],"issn-type":[{"value":"1664-302X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,21]]},"article-number":"1476171"}}