Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus

Abstract

A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid. The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance. A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose. This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10. Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments. Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation. NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence. Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance. Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences.