Enhanced transformation efficiency in Treponema denticola enabled by SyngenicDNA ‐based plasmids lacking restriction–modification target motifs

AbstractOral spirochetes are among a small group of keystone pathogens contributing to dysregulation of tissue homeostatic processes that leads to breakdown of the tissue and bone supporting the teeth in periodontal disease. Additionally, our group has recently demonstrated thatTreponema are among the dominant microbial genera detected intracellularly in tumor specimens from patients with oral squamous cell carcinoma. While over 60 species and phylotypes of oralTreponema have been detected,T. denticola is one of the few that can be grown in culture and the only one in which genetic manipulation is regularly performed. Thus,T. denticola is a key 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 whose agents lack workable genetic systems. We previously demonstrated improved transformation efficiency using anEscherichia coli–T. denticola shuttle plasmid and its utility for expression inT. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizingT. denticola Type I and Type II restriction –modification (R–M) systems and designing a high-efficiency R–M-silent “SyngenicDNA” shuttle plasmid resistant to allT. denticola ATCC 35405 R –M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R–M system consistent with...
Source: Molecular Oral Microbiology - Category: Microbiology Authors: Tags: ORIGINAL ARTICLE Source Type: research