Fujita Y, Fujita T
Proc Natl Acad Sci U S A. 1987 Jul;84(13):4524-8. doi: 10.1073/pnas.84.13.4524.
The gluconate (gnt) operon of Bacillus subtilis consists of four gnt genes; the second and third genes code for gluconate kinase (gluconokinase, EC 2.7.1.12) and gluconate permease, respectively. A fragment carrying the promoter of this operon (gnt promoter) and the first gene (gntR) was subcloned into a promoter probe vector (pPL603B). Repression of the expression of cat-86 gene, encoded in the vector portion of a constructed plasmid (pgnt21), that is under the control of the gnt promoter was removed by gluconate. The results of deletion analysis and of insertional inactivation of the gntR gene cloned in pgnt21 suggested that the product of the gntR gene, actually synthesized as a 29-kDa protein in vivo, is involved in repression of the gnt promoter. A 4-base-pair insertional mutation within the gntR gene constructed in vitro was introduced into the B. subtilis chromosomal gnt operon by use of linkage of the 4 base pairs to gntK10 in transformation. The introduced mutation gntR1 caused the constitutive expression of the gluconate kinase and gluconate permease genes. S1 nuclease analysis indicated that the mRNA of this operon is synthesized in the gntR1 strain and amounts of mRNA are not changed very much by gluconate, which acts as an inducer in the wild-type gene. These results strongly indicate that the gntR gene codes for a transcriptional negative regulator for the gnt operon.
枯草芽孢杆菌的葡萄糖酸盐（gnt）操纵子由四个gnt基因组成；第二个和第三个基因分别编码葡萄糖酸盐激酶（葡糖激酶，EC 2.7.1.12）和葡萄糖酸盐通透酶。携带该操纵子启动子（gnt启动子）和第一个基因（gntR）的片段被亚克隆到一个启动子探针载体（pPL603B）中。构建的质粒（pgnt21）载体部分编码的cat - 86基因的表达受gnt启动子控制，其表达的抑制作用被葡萄糖酸盐消除。对克隆在pgnt21中的gntR基因进行缺失分析和插入失活的结果表明，gntR基因的产物在体内实际合成的是一种29 kDa的蛋白质，它参与gnt启动子的抑制作用。通过在转化过程中将4个碱基对与gntK10连接，将体外构建的gntR基因内的一个4碱基对插入突变引入枯草芽孢杆菌染色体gnt操纵子中。引入的突变gntR1导致葡萄糖酸盐激酶和葡萄糖酸盐通透酶基因的组成型表达。S1核酸酶分析表明，该操纵子的mRNA在gntR1菌株中合成，并且mRNA的量不会因葡萄糖酸盐而发生很大变化，而葡萄糖酸盐在野生型基因中起诱导剂的作用。这些结果有力地表明，gntR基因编码gnt操纵子的转录负调控因子。
