A Tn10 derivative (T-POP) for isolation of insertions with conditional (tetracycline-dependent) phenotypes

A new Tn10-based transposon has been constructed and used to isolate insertion mutations with tetracycline-conditional phenotypes. Classes of mutants include conditional lethal mutations, conditional auxotrophs, and conditional mutants of the eut (ethanolamine utilization) operon. The described mutations were made with a new derivative of Tn10dTet that we have called Tn10d(T-POP). Others have noted that transposon Tn10dTet directs weak tetracycline-inducible transcripts out of both ends of the element into adjacent sequences. We have increased this level of outward transcription from Tn10dTet by selecting deletion mutations within the element that presumably remove transcription-termination signals. Insertion of the Tn10d(T-POP) element within an operon disrupts the target gene and makes expression of distal genes dependent on induction of outward transcription by tetracycline. Insertion mutations made with Tn10d(T-POP) can cause tetracycline-correctable conditional phenotypes based on expression of distal genes.