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 regulated prokaryotic promoters is described. The method is based on application of a novel mini-Tn5 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 (beta-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.
Timms-Wilson
T M
TM
Molecular Microbial Ecology Laboratory, Natural Environment Research Council, Institute of Virology and Environmental Microbiology, Oxford, UK.
Ellis
R J
RJ
Bailey
M J
MJ
D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005455:Fluorescent Antibody Technique; D005798:Genes, Bacterial; D007106:Immune Sera; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology; D008858:Microscopy, Phase-Contrast; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D011551:Pseudomonas fluorescens / Q000235:genetics*; D011775:Pythium / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D027981:Symporters; D001616:beta-Galactosidase / Q000235:genetics
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D007106:Immune Sera; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C021499:lactose permease; D001616:beta-Galactosidase