Waterhouse R N, Silcock D J, White H L, Buhariwalla H K, Glover L A
Department of Molecular and Cell Biology, University of Aberdeen, Scotland, UK.
Mol Ecol. 1993 Oct;2(5):285-93. doi: 10.1111/j.1365-294x.1993.tb00021.x.
Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola-specific phage (phi 11P) 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 phi 11P, 19H and P. aeruginosa phi PLS27, 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.
通过鸟枪法克隆到一个广宿主范围的启动子探针载体（pQF70）中，鉴定出了含有丁香假单胞菌菜豆致病变种特异性噬菌体（phi 11P）启动子序列的DNA区域。当与荧光素酶报告基因结合使用时，其中一个DNA片段19H指导基因表达，其水平能够使丁香假单胞菌菜豆致病变种的单细胞随后的光输出（生物发光）通过电荷耦合器件（CCD）进行检测和可视化。与含有其他特征明确的启动子（例如四环素）的类似构建体相比，丁香假单胞菌菜豆致病变种phi 11P、19H和铜绿假单胞菌phi PLS27、HcM启动子使生物发光（最大相对光输出）增加了50倍。在有和没有抗生素选择的生长过程中，均观察到了转化细菌的类似生物发光特征。当将lux +细菌接种到菜豆叶（菜豆）上时，产生的继发性晕疫病斑会发出生物光，并且在lux +细菌在叶表面定殖期间，通过CCD检测并成像叶表面的生物发光。这些新鉴定的启动子的使用，结合CCD对生物发光检测的大大提高的灵敏度，从而为研究植物细菌定殖过程中的自然生态种群提供了一个新的维度。
