title: A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405.
abstract: Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism.
authors.affiliation: Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
authors.forename: Valentina
authors.initials: V
authors.lastname: Godovikova
authors.affiliation: Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
authors.forename: M Paula
authors.initials: MP
authors.lastname: Goetting-Minesky
authors.affiliation: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
authors.forename: Jae M
authors.initials: JM
authors.lastname: Shin
authors.affiliation: Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA.
authors.forename: Yvonne L
authors.initials: YL
authors.lastname: Kapila
authors.affiliation: Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, Michigan, USA.
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Rickard
authors.affiliation: Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan, USA fenno@umich.edu.
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Fenno
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002478:Cells, Cultured; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D005347:Fibroblasts / Q000382:microbiology; D005486:Flavin Mononucleotide / Q000235:genetics*; D005453:Fluorescence; D005822:Genetic Vectors; D005881:Gingiva / Q000166:cytology / Q000382:microbiology; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D010957:Plasmids; D014169:Transformation, Bacterial; D048373:Treponema denticola / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008164:Luminescent Proteins; D005486:Flavin Mononucleotide; D015252:Deoxyribonucleases, Type II Site-Specific