Expanded Functional Characterization and Optimization of Protein Expression in <i>Treponema denticola</i> Shuttle Plasmids

Oral spirochetes are among the small group of keystone pathogens contributing to dysregulation of periodontal tissue homeostasis, leading to breakdown of the tissue and bone supporting the teeth in periodontal disease. Of the more than 60 oral Treponema species and phylotypes, Treponema denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is practicable. T. denticola is thus a model organism for studying spirochete behavior, metabolism, and interactions with other microbes and host tissues that are relevant to oral diseases. We recently demonstrated enhanced transformation efficiency using a synthetic shuttle plasmid resistant to T. denticola restriction-modification systems. Here, we report further optimization of the shuttle plasmid system by minimizing its size and by characterizing an array of promoter-gene constructs for plasmid-based genetic complementation, including the first inducible system for controlled expression of potentially toxic plasmid-encoded genes in Treponema. Our results highlight the importance of precise pairing of promoters and genes of interest for obtaining biologically optimal protein expression. This work expands the utility of the T. denticola shuttle plasmid system and will facilitate future studies in the analysis of Treponema physiology and behavior. Rigorous genetic analysis in oral spirochetes has been hampered by the limited utility of available versions of the Escherichia coli-T. denticola shuttle plasmid system. We report expanded characterization, refinement, and minimization of the shuttle plasmid, including relative activity of diverse promoters and the first inducible expression system described for T. denticola. We show that careful customization of the shuttle plasmid for specific applications is crucial for obtaining successful results.