Functional characterization and optimization of protein expression in Treponema denticola shuttle plasmids

Oral spirochetes are among a 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 greater than sixty oral Treponema species and phylotypes, T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation has been shown to be practicable. T. denticola is thus a model organism for studying spirochete metabolic processes, interactions with other microbes and host cell and tissue responses relevant to oral diseases as well as venereal and nonvenereal treponematoses. We recently demonstrated enhanced transformation efficiency using a SyngenicDNA-based shuttle plasmid resistant to T. denticola restriction-modification systems. Here we expand on this work by further characterizing the shuttle plasmid and optimizing expression of cloned genes using several promoter-gene constructs for genetic complementation and exogenous gene expression, 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 to obtaining biologically optimal protein expression. This work expands the utility of the shuttle plasmid and will facilitate future studies employing shuttle plasmids in analysis of Treponema physiology and behavior.

Rigorous genetic analysis in oral spirochetes has been hampered by the limited utility of available versions of the E. coli-T. denticola shuttle plasmid system. We report expanded characterization 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.