A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

Oral pathogens, includingTreponema denticola , initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles ofT. 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 studiedT. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems inT. denticola contributed to these problems. To facilitate further molecular genetic analysis ofT. 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 ofT. 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 inT. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to theTreponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism.