Oral 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 that Treponema 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 oral Treponema 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 an Escherichia coli–T. denticola shuttle plasmid and its utility for expression in T. denticola of an exogenous fluorescent protein that is active under anaerobic conditions. Here, we expand on this work by characterizing T. denticola Type I and Type II restriction–modification (R–M) systems and designing a high-efficiency R–M-silent “SyngenicDNA” shuttle plasmid resistant to all T. denticola ATCC 35405 R–M systems. Resequencing of the ATCC 33520 genome revealed an additional Type I R–M system consistent with the relatively low transformation efficiency of the shuttle plasmid in this strain. Using SyngenicDNA approaches, we optimized shuttle plasmid transformation efficiency in T. denticola and used it to complement a defined T. denticola ΔfhbB mutant strain. We further report the first high-efficiency transposon mutagenesis of T. denticola using an R–M-silent, codon-optimized, himarC9 transposase-based plasmid. Thus, use of SyngenicDNA-based strategies and tools can enable further mechanistic examinations of T. denticola physiology and behavior.

中文翻译：

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缺乏限制性修饰目标基序的基于 SyngenicDNA 的质粒提高了齿垢密螺旋体的转化效率

口腔螺旋体是一小群关键病原体之一，它会导致组织稳态过程失调，导致牙周病中支撑牙齿的组织和骨骼崩溃。此外，我们的小组最近证明，密螺旋体是在口腔鳞状细胞癌患者的肿瘤标本中细胞内检测到的优势微生物属之一。虽然已检测到超过 60 种口腔密螺旋体的物种和系统型，但齿垢密螺旋体是少数可在培养物中生长的密螺旋体之一，也是唯一一种定期进行基因操作的密螺旋体。因此，齿垢螺旋体是研究螺旋体代谢过程、与其他微生物的相互作用、与口腔疾病以及性病和非性病密螺旋体病相关的宿主细胞和组织反应的关键模型生物，其病原体缺乏可行的遗传系统。我们之前证明了使用大肠杆菌- T. denticola穿梭质粒可以提高转化效率，及其在T. denticola中表达在厌氧条件下有活性的外源荧光蛋白的实用性。在这里，我们通过表征T. denticola I 型和 Type II 限制性修饰 (R–M) 系统并设计对所有T. denticola ATCC 35405具有抗性的高效 R–M 沉默“SyngenicDNA”穿梭质粒来扩展这项工作R-M 系统。ATCC 33520 基因组的重测序揭示了一个额外的 IR-M 型系统，与该菌株中穿梭质粒相对较低的转化效率相一致。使用 SyngenicDNA 方法，我们优化了T. denticola中的穿梭质粒转化效率，并将其用于补充定义的T. denticola Δ fhbB突变株。我们进一步报告了使用 R-M 沉默、密码子优化、基于himarC9转座酶的质粒对T. denticola进行的第一个高效转座子诱变。因此，使用基于 SyngenicDNA 的策略和工具可以对T. denticola生理学和行为进行进一步的机械检查。