Weickert M J, Chambliss G H
Laboratory of Genetics, University of Wisconsin, Madison 53706.
J Bacteriol. 1989 Jul;171(7):3656-66. doi: 10.1128/jb.171.7.3656-3666.1989.
The amyR2 allele of the Bacillus subtilis alpha-amylase cis-regulatory region enhances production of amylase and transcription of amyE, the structural gene, by two- to threefold over amyR1. The amylase gene bearing each of these alleles was cloned on plasmids of about 10 to 15 copies per chromosome. Transcription of the cloned amylase gene by each amyR allele was activated at the end of exponential growth and was subject to catabolite repression by glucose. The amount of amylase produced was roughly proportional to the copy number of the plasmid, and cells containing the amyR2-bearing plasmid, pAR2, produced two- to threefold more amylase than cells with the amyR1 plasmid, pAMY10. Deletion of DNA 5' to the alpha-amylase promoter, including deletion of the A + T-rich inverted repeat found in amyR1 and amyR2, had no effect on expression or transcription of alpha-amylase. Deletion of DNA 3' to the amyR1 promoter did not impair temporal activation of chloramphenicol acetyltransferase in amyR1-cat-86 transcriptional fusions, but catabolite repression was abolished. When an 8-base-pair linker was inserted in pAMY10 at the same site from which the 3' deletion was made, amylase expression doubled and was repressed less by glucose. Both the deletion and the insertion disrupted four bases at the 3' end of the putative amylase operator region. Site-directed mutagenesis was used to change bases in the promoter-operator region of amyR1 to their amyR2 counterparts. Either change alone increased amylase production twofold, but only the change at +7, next to the linker insertion of 3' deletion site, yielded the increased amylase activity in the presence of glucose that is characteristic of the amyR2 strain. The double mutant behaved most like strains carrying the amyR2 allele.
枯草芽孢杆菌α-淀粉酶顺式调控区的amyR2等位基因可使淀粉酶产量以及结构基因amyE的转录水平比amyR1提高两到三倍。携带这些等位基因的淀粉酶基因被克隆到每个染色体约有10到15个拷贝的质粒上。每个amyR等位基因对克隆的淀粉酶基因的转录在指数生长末期被激活，并受到葡萄糖的分解代谢物阻遏。产生的淀粉酶量大致与质粒的拷贝数成正比，含有携带amyR2质粒pAR2的细胞比含有amyR1质粒pAMY10的细胞产生的淀粉酶多两到三倍。删除α-淀粉酶启动子5'端的DNA，包括删除amyR1和amyR2中发现的富含A+T的反向重复序列，对α-淀粉酶的表达或转录没有影响。删除amyR1启动子3'端的DNA不会损害amyR1-cat-86转录融合体中氯霉素乙酰转移酶的时间激活，但分解代谢物阻遏被消除。当在pAMY10中从进行3'端缺失的相同位点插入一个8碱基对的接头时，淀粉酶表达增加了一倍，并且受葡萄糖的抑制作用减弱。缺失和插入都破坏了推定的淀粉酶操纵区3'端的四个碱基。使用定点诱变将amyR1启动子-操纵区的碱基改变为它们的amyR2对应碱基。单独的任何一种改变都会使淀粉酶产量增加两倍，但只有在+7处（紧邻3'端缺失位点的接头插入处）的改变在葡萄糖存在下产生了amyR2菌株特有的淀粉酶活性增加。双突变体的行为最类似于携带amyR2等位基因的菌株。
