Chen C Y, Winans S C
Section of Microbiology, Cornell University, Ithaca, New York 14853.
J Bacteriol. 1991 Feb;173(3):1139-44. doi: 10.1128/jb.173.3.1139-1144.1991.
The Agrobacterium VirG protein is normally expressed from two promoters in response to multiple stimuli, including plant-released phenolics (at promoter P1) and acidic growth media (at promoter P2). To simplify the analysis of vir gene induction, we sought to create Agrobacterium strains in which virG could be expressed in a controllable fashion. To study the possibility of using the lac promoter and repressor, we constructed a plasmid containing the lac promoter fused to the lacZ structural gene. A derivative of this plasmid containing the lacIq gene was also constructed. The plasmid not containing lacIq expressed high levels of beta-galactosidase. The plasmid containing lacIq expressed beta-galactosidase at very low levels in the absence of o-nitrophenyl-beta-D-galactoside (IPTG) and at moderate levels in the presence of IPTG. We also fused the lac promoter to a virG::lacZ translational fusion and found that IPTG elevated expression of this translational fusion to moderate levels, though not to levels as high as from the stronger of the two native virG promoters. Finally, the lac promoter was used to express the native virG gene in strains containing a virB::lacZ translational fusion. virB expression in this strain depended on addition of IPTG as well as the vir gene inducer acetosyringone. In a similar strain lacking lacIq, virB expression was greater than in a strain in which virG was expressed from its native promoters. Expression of virG from the lac promoter did not alter the acidic pH optimum for vir gene induction, indicating that the previously observed requirement for acidic media was not due solely to the need to induce P2.
根癌农杆菌VirG蛋白通常由两个启动子响应多种刺激而表达，这些刺激包括植物释放的酚类物质（在启动子P1处）和酸性生长培养基（在启动子P2处）。为了简化对vir基因诱导的分析，我们试图构建能够以可控方式表达virG的根癌农杆菌菌株。为了研究使用lac启动子和阻遏物的可能性，我们构建了一个含有与lacZ结构基因融合的lac启动子的质粒。还构建了该质粒的一个含有lacIq基因的衍生物。不含lacIq的质粒表达高水平的β-半乳糖苷酶。含有lacIq的质粒在没有邻硝基苯基-β-D-半乳糖苷（IPTG）时以非常低的水平表达β-半乳糖苷酶，在有IPTG时以中等水平表达。我们还将lac启动子与virG::lacZ翻译融合体融合，发现IPTG将这种翻译融合体的表达提高到中等水平，尽管不如两个天然virG启动子中较强的那个启动子所表达的水平高。最后，lac启动子用于在含有virB::lacZ翻译融合体的菌株中表达天然virG基因。该菌株中virB的表达取决于IPTG以及vir基因诱导剂乙酰丁香酮的添加。在一个缺乏lacIq的类似菌株中，virB的表达高于virG从其天然启动子表达的菌株。从lac启动子表达virG并没有改变vir基因诱导的最适酸性pH，这表明先前观察到的对酸性培养基的需求并非仅仅是由于诱导P2的需要。
