Title

An inducible transposon mutagenesis approach for the intracellular human pathogen

DOI

10.12688/wellcomeopenres.16068.1

Document Type

Journal Article

Publication Date

1-1-2021

Publication Title

Wellcome open research

Keywords

Chlamydia trachomatis, MinION, TraDIS, anhydrotetracycline, dual-control regulon, genetics, inducible promoter, riboswitch, theophylline, transposon mutagenesis

Abstract

is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in 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 using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the genome. The low transformation efficiency of 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 promoter system with the addition of a riboswitch). transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Here we present the preliminary sequencing results of transposon mutant pools of both biovars, using two plasmid-free representatives: urogenital strain SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled '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. This significant advance provides an efficient method for assaying gene function and will enable the identification of the essential gene set of . In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

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