Abstract
Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck. Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. 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 a self-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 at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9 transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon. We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli. Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation 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 in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9 transposase under tet promoter control. Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis. Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.
Highlights
Chlamydia trachomatis is a major human pathogen, causing trachoma, the most prevalent infectious blinding disease[1], and is the main bacterial agent of sexually transmitted infections worldwide[2]
We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar[1] C9 transposase under tet promoter control and its transposon
We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar[1] C9 transposase under tet promoter control. Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis
Summary
Chlamydia trachomatis is a major human pathogen, causing trachoma, the most prevalent infectious blinding disease[1], and is the main bacterial agent of sexually transmitted infections worldwide[2]. Once an EB has been taken up within a nascent inclusion it will, over several hours (the process takes 8–12 hours depending on strain and host cell and culture conditions) differentiate into a reticulate body (RB). During the exponential growth phase of C. trachomatis L2 the RBs multiply at a rate of one bacterial division/chromosomal replication every 4 to 6 hours and during the process most asynchronously differentiate back to become EBs. Once the inclusion reaches a critical late phase of development, the host cell lyses. Once the inclusion reaches a critical late phase of development, the host cell lyses At this point of maturation the infected cell contains around 500 – 1000 EBs which are released to infect further cells and commence a new developmental cycle[5]. Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction
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