Isolation and sequencing of gene fusions carried by lambda placMu specialized transducing phage.

Abstract

Using Mudl and XplacMu mutator phage to fuse a transposonborne lacZ reporter gene to the promoter of a target gene has proven to be invaluable for the study of gene regulation in Escherichia coli. Mudl derivatives have also been used to delineate gene regulons that respond to a specific condition such as carbon starvation stress (1) or phosphate deprivation (2). These phage integrate into the bacterial chromosome via Mu transposition functions which are encoded by the mutator phage itself or, in the case of XplacMu, by a helper phage. Upon integration into a gene of interest, a gene fusion is produced in which the flanking ends of the prophage are the Mu S and Mu c integration sequences. The use ofXplacMu derivatives provide the additional capability of in vivo cloning ofadjacent regulatory sequences that drive lacZ expression in the resident prophage since UV irradiation ofXplacMu lysogens produces transducing phage that carry varying amounts of adjacent host DNA. This DNA, located between the Mu S andMu c attachment sites, can be subsequently cloned and sequenced (Fig. 1). While this latter property should, in theory, allow the sequence of the 5' regulatory region to be easily determined, sequencing of the promoter-proximal region has proven difficult. The structure of the Mu S end ofMud phage