Immuno-capture differential display method (IDDM) for the detection of environmentally induced promoters in rhizobacteria

A rapid immunological method for trapping and selection of functionally reg ulated prokaryotic promoters is described. The method is based on applicati on of a novel mini-Tn5 derived promoter probe (pUTTKZY-promoterless lacZY a s a reporter and kanamycin resistance) to mutagenise a plant growth promoti ng fluorescent pseudomonad, Pseudomonas fluorescens 54/96. The transposon a llows selection of operon fusion mutants (lacZY(+)) directly on media conta ining lactose as a sole carbon source as well as selection for kanamycin an d lacZ (beta-galactosidase) expression on X-gal indicator media. We have ex tended the technique to target the surface expression of the induced lactos e permease gene (lacY) from mutagenised libraries and the immune-capture of bacteria with magnetic beads and anti-LacY monospecifc antisera. The benef its of the lacZY reporter are that a library can be rapidly generated and s creened in vitro to isolate non-expressed mutants for further in situ scree ning. Here we demonstrate the development and utility of the technique and its potential as a differential display method for the isolation of promote rs that direct regulated gene expression in the phytosphere, or under other imposed conditions. (C) 2000 Elsevier Science B.V. All rights reserved.