ena-STUDY-UNIVERSITY OF SOUTHAMPTON-07-10-2021-10:59:43:380-24
The development of a transposon mutagenesis system in Chlamydia trachomatis, using a self-replicating transposon-delivery vector, enabling induction of transposase expression at a defined time point.
Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods. The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of an inducible riboswitch). Chlamydia transformed with this vector (pSW2-C9-RiboA-Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly controlled to stabilise the plasmid prior to transposition. Results. Here we present the preliminary sequencing results of transposon mutant pools of the urogenital strain  C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled us to obtain 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusion. This exciting advance provides the ideal method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term this will provide an efficient method of identifying mutable genes and will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.
Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods. The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of an inducible riboswitch). Chlamydia transformed with this vector (pSW2-C9-RiboA-Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly controlled to stabilise the plasmid prior to transposition. Results. Here we present the preliminary sequencing results of transposon mutant pools of the urogenital strain  C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled us to obtain 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusion. This exciting advance provides the ideal method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term this will provide an efficient method of identifying mutable genes and will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.