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The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv. phaseolicola , allowing single cell and microcolony detection
Author(s) -
WATERHOUSE R. N.,
SILCOCK D. J.,
WHITE H. L.,
BUHARIWALLA H. K.,
GLOVER L. A.
Publication year - 1993
Publication title -
molecular ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.619
H-Index - 225
eISSN - 1365-294X
pISSN - 0962-1083
DOI - 10.1111/j.1365-294x.1993.tb00021.x
Subject(s) - pseudomonas syringae , biology , luciferase , cloning (programming) , promoter , genetics , microbiology and biotechnology , computational biology , bacteria , gene , gene expression , transfection , programming language , computer science
Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola ‐specific phage (φ11P) were identified by shotgun cloning into a broad‐host‐range promoter‐probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge‐coupled device (CCD). The P. syringae pv. phaseolicola φ11P, 19H and P. aeruginosa φPLS27, HcM promoters gave a 50‐fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well‐characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic‐selection. When lux + bacteria were inoculated onto French bean leaf ( Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux + bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.