Abstract
A rapid immunological method for trapping and selection of functionally regulated prokaryotic promoters is described. The method is based on application of a novel mini-Tn 5 derived promoter probe (pUTTKZY-promoterless lacZY as a reporter and kanamycin resistance) to mutagenise a plant growth promoting fluorescent pseudomonad, Pseudomonas fluorescens 54/96. The transposon allows selection of operon fusion mutants ( lacZY +) directly on media containing lactose as a sole carbon source as well as selection for kanamycin and lacZ (β-galactosidase) expression on X-gal indicator media. We have extended the technique to target the surface expression of the induced lactose permease gene ( lacY) from mutagenised libraries and the immuno-capture of bacteria with magnetic beads and anti-LacY monospecifc antisera. The benefits of the lacZY reporter are that a library can be rapidly generated and screened in vitro to isolate non-expressed mutants for further in situ screening. Here we demonstrate the development and utility of the technique and its potential as a differential display method for the isolation of promoters that direct regulated gene expression in the phytosphere, or under other imposed conditions.
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