Use of differential fluorescence induction and optical trapping to isolate environmentally induced genes.
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.
Allaway
D
D
Division of Microbiology, School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK.
Schofield
N A
NA
Leonard
M E
ME
Gilardoni
L
L
Finan
T M
TM
Poole
P S
PS
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015342:DNA Probes; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000382:microbiology; D018517:Plant Roots / Q000382:microbiology; D016961:Rhizobium leguminosarum / Q000166:cytology / Q000235:genetics* / Q000502:physiology*; D012988:Soil Microbiology
D001426:Bacterial Proteins; D015342:DNA Probes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins