Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Genetic manipulation of Chlamydia trachomatis is hindered by extremely low transformation frequencies.
• Development of efficient genetic systems is crucial for understanding Chlamydia biology and pathogenesis.
• Self-replicating transposon delivery vectors offer a potential solution for stable genetic modification.

Purpose of the Study:

• To engineer a self-replicating transposon delivery vector for Chlamydia trachomatis.
• To enable expansion of the vector prior to transposase induction for improved transformation efficiency.
• To investigate the role of the Himar1 C9 transposase and its control elements in vector stability.

Main Methods:

• Construction of E. coli/C. trachomatis shuttle vectors containing the Himar1 C9 transposase and transposon.
• Utilized tet promoter control for inducible transposase expression.
• Assessed transposase activity via immunoblot and DNA sequencing; evaluated plasmid recovery in C. trachomatis.

Main Results:

• The designed self-replicating plasmid (pSW2-mCh-C9) could not be recovered in C. trachomatis.
• Deletion constructs (pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase) were functional in E. coli and recoverable in C. trachomatis independently.
• Attempts at lateral gene transfer via transformation and mixed infection were unsuccessful in recovering dual-plasmid progeny.

Conclusions:

• The self-replicating plasmid pSW2-mCh-C9, while functional in E. coli, is unstable in C. trachomatis.
• Low-level expression from the tet-inducible promoter leads to premature transposition and early plasmid loss.
• This premature transposition is identified as the primary bottleneck preventing successful genetic manipulation in C. trachomatis using this vector system.