Studies on plasmid-based genetic tools for C. muridarum: progress towards a replicating vector for transposon mutagenesis

Abstract

Plasmid-based genetic tools for transposon (Tn)-mutagenesis using a ‘suicide’ vector approach to achieve genome saturation in Chlamydia are hindered by low transformation efficiencies. Progress toward achieving saturation was recently made with a self-replicating vector carrying an inducible Tn-system that facilitates Tn-transposition at the end of the developmental cycle, but this approach remains undeveloped in C. muridarum, and would require a stable replication-proficient vector to deliver the transposon-transposase cassette. No such vector has been identified. Identifying such a stably replicating vector in C. muridarum faces the additional challenge of a lower plasmid copy number (≤1) and a tendency for in-vitro plasmid loss even with positive selection using the available selectable markers.
The extent to which in vitro plasmid loss would inhibit the development of plasmid-based genetic tools in C. muridarum remained unknown due to the lack of a standardised, accurate method for measuring plasmid stability in Chlamydia. Development of a robust means for measuring plasmid stability was required to investigate the stability of available vectors and identify an appropriate vector backbone that is stable in C. muridarum. This was achieved by generating standard inocula for transformants, where every cell contained at least one plasmid, using bactericidal concentrations of chloramphenicol to kill plasmid-free cells. Both direct and indirect measures of plasmid loss were then applied in three generations of chlamydial elementary bodies passaged with and without selection.
The E. coli-C. muridarum shuttle vector pGFP::Nigg remained stable under bactericidal concentrations of chloramphenicol, which caused destabilisation of plasmid copy number when removed from the growth media. These results identified a potential mechanism for plasmid elimination, which is required in Tn-systems to reduce the likelihood of secondary transposition events. A minimal-gene vector derived from pGFP::Nigg with coding sequences CDS5 and -6 deleted was stably maintained under penicillin selection at a higher plasmid copy number (~4), comparable to plasmid copy numbers reported for C. trachomatis.
The replication-proficient Tn-delivery vector in C. muridarum, pNiggHimar, was developed using pGFP::Nigg and a Tet-inducible Himar1 Tn-system containing an erythromycin-resistance marker, and the wild type Himar1 transposase. Inducible expression of transposase from pNiggHimar was demonstrated in E. coli. pNiggHimar could not be recovered in C. muridarum indicating weak repression of transposase expression by the Tet promoter in the “off”-state, also demonstrated with the Tet-inducible Tn-system in C. trachomatis. Transposition-negative constructs derived from pNiggHimar also failed to be recovered in C. muridarum. Together these findings suggest that a replicating Tn-system for C. muridarum will require a stable vector backbone with a higher plasmid copy number than pGFP::Nigg and tighter control over transposase expression.

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