THE CLONING AND CHARACTERIZATION OF PHAGE PROMOTERS, DIRECTING HIGH EXPRESSION OF LUCIFERASE IN PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA, ALLOWING SINGLE-CELL AND MICROCOLONY DETECTION

Regions of DNA containing promoter sequences from a Pseudomonas syring ae pv. phaseolicola-specific phage (phi11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When u sed in conjunction with the luciferase reporter genes, one of these DN A fragments, 19H, directed gene expression at a level which enabled th e subsequent light output (bioluminescence) of single cells of P. syri ngae pv. phaseolicola to be detected and visualized using a charge-cou pled device (CCD). The P. syringae pv. phaseolicola phi11P, 19H and P. aeruginosa phiPLS27, HcM promoters gave a 50-fold increase in biolumi nescence (maximum relative light output) compared to similar construct s containing other well-characterized promoters, for example, tetracyc line. Similar bioluminescent characteristics of the transformed bacter ium, 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 biolumi nescent and during phylloplane colonization by the lux+ bacterium, bio luminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increas ed 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.