Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, PA 19038, USA.
N Biotechnol. 2010 Feb 28;27(1):1-9. doi: 10.1016/j.nbt.2009.12.003. Epub 2009 Dec 23.
A library of genomic DNA fragments of Pseudomonas syringae pv. tomato DC3000 was constructed in a lacZalpha-containing plasmid, pBS29. The library was used in a preliminary alpha-complementation-based screen to identify clones with promoter activity in Escherichia coli. Ten positive clones were sequenced and their locations in the chromosomal DNA of DC3000 strain were mapped. Five positive clones (P2, P3, P4, P6 and P8) were further assayed for promoter activity in three polyhydroxyalkanoate-producing pseudomonads: Pseudomonas resinovorans, P. corrugata and P. chlororaphis. To this end, a green-fluorescent-protein gene (gfp) was cloned downstream from the putative (DC3000) promoter in a shuttle plasmid. We found that only Pseudomonas transformants harboring the gfp-containing plasmid driven by putative promoter P2 showed fluorescence, indicating that this promoter is functioning in the three tested pseudomonads. Results of an in silico analysis of the P2 sequence further support the assignment of P2 as a bona fide promoter by the localization of putative -10 and -35 promoter regions and a transcription-factor-binding site, rpoD17, in this sequence. We successfully applied promoter P2 to drive the expression in P. chlororaphis of a recombinant alpha-galactosidase gene of Streptomyces coelicolor, which should be useful for the utilization of oligosaccharides of soy molasses for the production of polyhydroxyalkanoate biopolymer or rhamnolipid biosurfactant.
构建了一个含有 lacZalpha 的质粒 pBS29 中的番茄丁香假单胞菌 pv. 基因组 DNA 片段文库。该文库用于初步的基于 α 互补的筛选，以鉴定在大肠杆菌中具有启动子活性的克隆。对 10 个阳性克隆进行测序，并将其在 DC3000 菌株的染色体 DNA 中的位置进行定位。对 5 个阳性克隆（P2、P3、P4、P6 和 P8）在 3 种聚羟基烷酸产生假单胞菌（假单胞菌树脂、P. corrugata 和 P. chlororaphis）中进一步检测启动子活性。为此，将绿色荧光蛋白基因（gfp）克隆在穿梭质粒中推定（DC3000）启动子的下游。我们发现，只有携带由推定启动子 P2 驱动的 GFP 基因的假单胞菌转化体显示荧光，表明该启动子在三种测试假单胞菌中均具有功能。P2 序列的计算机分析结果进一步支持了 P2 作为真正启动子的分配，因为在该序列中存在推定的 -10 和 -35 启动子区域以及转录因子结合位点 rpoD17。我们成功地将启动子 P2 应用于驱动重组α-半乳糖苷酶基因在 P. chlororaphis 中的表达，这对于利用大豆糖蜜中的寡糖生产聚羟基烷酸生物聚合物或鼠李糖脂生物表面活性剂应该是有用的。
