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

BackgroundGenetic systems have been developed forChlamydiabut the extremely low transformation frequency remains a significant bottleneck. Our goal is to develop a self-replicating transposon delivery vector forC. trachomatiswhich can be expanded prior to transposase induction.MethodsWe madeE. coli/C. trachomatisshuttle vectors bearing theHimar1C9 transposase under control of thetetpromoter and a novel rearrangement of theHimar1transposon with the β-lactamase gene. Activity of the transposase was monitored by immunoblot and by DNA sequencing.ResultsWe constructed pSW2-mCh-C9, aC. trachomatisplasmid designed to act as a self-replicating vector carrying both theHimar1C9 transposase undertetpromoter control and its transposon. However, we were unable to recover this plasmid inC. trachomatisfollowing multiple attempts at transformation.Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only theHimar1C9 transposase (undertetpromoter 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 inE. coli. Both these plasmids could be independently recovered inC. trachomatis.We attempted to perform lateral gene transfer by transformation and mixed infection withC. trachomatisstrains bearingpSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon(a green fluorescent version ofpSW2-mCh-C9-ΔTpase). Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids.ConclusionsWe have designed a self-replicating plasmid vector pSW2-mCh-C9 forC. trachomatiscarrying theHimar1C9 transposase undertetpromoter control. Whilst this can be transformed intoE. coliit cannot be recovered inC. trachomatis. Based on selected deletions and phenotypic analyses we conclude that low level expression from thetetinducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.