Characterization of Tn 10d-Cam: A transposition-defective Tn 10 specifying chloramphenicol resistance

We have constructed a small, transposition-defective derivative of the transposon Tn 10 that carries the chloramphenicol acetyltransferase gene of pACYC184. This new genetic element, Tn 10d-Cam, transposes when Tn 10 transposase is provided from a multi-copy plasmid. Transposon insertion mutagenesis of Salmonella typhimurium was performed by using a strain carrying a Tn 10d-Cam insertion in an Escherichia coli F' episome as the donor in transductional crosses into recipients that carried a plasmid expressing Tn 10 transposase. Tn 10d-Cam insertion mutations were also generated by complementation in cis of Tn 10d-Cam by a cotransducible Tn 10 element that overproduces transposase. Here, transposase was provided only transiently, and the Tn 10d-Cam insertion mutations were recovered in a transposase-free strain. Cis complementation was used for mutagenesis of a plasmid target. The site specificity of insertion and the effect of insertions on expression of a downstream gene were investigated, using Tn 10d-Cam insertions in a plasmid carrying a segment of the histidine operon.