Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes |
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Authors: | Allaway D Schofield N A Leonard M E Gilardoni L Finan T M Poole P S |
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Institution: | Division of Microbiology, School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK.;Faculty of Veterinary Science, Chorroarin 280 CP 1472, University of Buenos Aires, Argentina.;Life Sciences Rm 539, 1280 Main Street West, McMasters University, Hamilton, Ontario, Canada L8S 4K1. |
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Abstract: | The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp . Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase ( thiE ) and cyclic glucan synthase ( ndvB ) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX , and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression. |
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