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

Abstract

Background
Genetic systems have been developed for Chlamydia but the extremely low transformationfrequency remains a significant bottleneck.  Our goal is to develop aself-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposaseinduction.

Methods
We made E. coli/ C. trachomatis shuttle vectors bearing the Himar1 C9  transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the β-lactamase gene. Activity of the transposase was monitored by immunoblot and by DNA sequencing.

Results
We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as aself-replicating vector carrying both the Himar1 C9  transposase under tet promoter control and its transposon. However, we were unable to recover this plasmid in C. trachomatis following multiple attempts attransformation.
Therefore, we assembled two new deletion plasmidspSW2-mCh-C9-ΔTpon carrying only the Himar1 C9  transposase (under tet promoter control) and a sister vector (samesequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  Wedemonstrated that the biological components that make up both pSW2-mCh-C9-ΔTponand pSW2-mCh-C9-ΔTpase are active in E. coli.  Both these plasmids could be independentlyrecovered in C. trachomatis.
We attempted to perform lateral gene transfer bytransformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success inachieving mixed infections, it was not possible to recover progeny bearing bothversions of these plasmids.

Conclusions
We have designed a self-replicating plasmid vectorpSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9  transposase under tet promoter control.  Whilst this can betransformed into E. coli it cannot be recovered in C. trachomatis.  Based on selected deletions and phenotypicanalyses we conclude that low level expression from the tet inducible promoter is responsible forpremature transposition and hence plasmid loss early on in the transformationprocess.

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