Wilhelm M, Hollenberg C P
EMBO J. 1984 Nov;3(11):2555-60. doi: 10.1002/j.1460-2075.1984.tb02173.x.
A fragment of Bacillus subtilis DNA coding for xylose isomerase and xylulokinase was isolated from a BamHI restriction pool by complementation of an isomerase-defective Escherichia coli strain. The spontaneous insertion of IS5, which occurred during the very slow growth of the E. coli xyl- cells on xylose, allowed the expression of the cloned Bacillus genes in E. coli. Without IS5 insertion, the xylose genes were inactive in E. coli. Deletion experiments indicated that the control of the expression resides within a 270-bp long region at the right end of IS5. Deletion of this region led to a loss of expression, which could be restored by insertion of the lacUV5 promoter fragment at the deletion site. Sequence analysis showed that the site of IS5 insertion is 195 bp upstream from the putative ATG initiation codon of the xylose isomerase structural gene. This ATG is preceded by a ribosome binding sequence and two hexamers also found in promoter regions of other Bacillus genes. Deletion and mutagenesis analysis led to a preliminary map of the Bacillus xylose operon.
通过对一株异构酶缺陷型大肠杆菌菌株进行互补，从BamHI酶切文库中分离出一段编码木糖异构酶和木酮糖激酶的枯草芽孢杆菌DNA片段。在木糖上生长非常缓慢的大肠杆菌木糖利用缺陷型（xyl-）细胞生长过程中发生的IS5自发插入，使得克隆的芽孢杆菌基因在大肠杆菌中得以表达。没有IS5插入时，木糖基因在大肠杆菌中无活性。缺失实验表明，表达的控制位于IS5右端一个270 bp长的区域内。缺失该区域导致表达丧失，通过在缺失位点插入lacUV5启动子片段可恢复表达。序列分析表明，IS5插入位点在木糖异构酶结构基因推定的ATG起始密码子上游195 bp处。该ATG之前有一个核糖体结合序列以及在其他芽孢杆菌基因启动子区域也发现的两个六聚体。缺失和诱变分析得出了芽孢杆菌木糖操纵子的初步图谱。
