Wieland K P, Wieland B, Götz F
Universität Tübingen, Germany.
Gene. 1995 May 26;158(1):91-6. doi: 10.1016/0378-1119(95)00137-u.
We describe a series of plasmid vectors for DNA cloning in staphylococci. pPS11 is a promoter probe plasmid containing a promoterless lipase (Lip)-encoding gene (lip). Insertion of a promoter-bearing DNA fragment at the single BamHI site turns on lip expression. Lip activity can be easily determined to estimate the strength of the inserted promoter. pPS11 served also as a basis for the construction of vectors which allow xylose-inducible gene expression in Staphylococcus carnosus (Sc). Using plasmid pCX15, we studied xylose-inducible lip expression in Sc. The lip expression is under transcriptional control of the repressor, XylR. The xylR gene, the XylR target sequence and the xylA promoter/operator sequence with the cis-acting catabolite-responsive element (cre) are derived from the xyl operon of S. xylosus. The single BamHI site in front of the lip ribosome-binding site (RBS) also makes it possible to put other promoterless genes under transcriptional control of XylR. To facilitate the controlled expression of genes which merely start with the start codon and have no RBS, or to insert genes with their own RBS, pCX26 and pCX26 delta lip were constructed. The influence of xylose and glucose on lip expression was studied both in a batch culture and in a fermentor under controlled pH conditions. With pCX15, the presence of xylose led to a 40-fold increase in extracellular Lip activity, while the presence of glucose caused a repression of lip expression. The results suggest that the xylA promoter is subject to two different regulatory mechanisms, one of which involves the repression of the xylA promoter by XylR in the absence of xylose, and the other involves a glucose-mediated catabolite repression which dominates over the xylose induction.
我们描述了一系列用于葡萄球菌DNA克隆的质粒载体。pPS11是一种启动子探针质粒，含有一个无启动子的脂肪酶（Lip）编码基因（lip）。在单个BamHI位点插入一个带有启动子的DNA片段可开启lip的表达。通过测定Lip活性能够轻松评估插入启动子的强度。pPS11也是构建载体的基础，这些载体可使木糖诱导型基因在肉葡萄球菌（Sc）中表达。利用质粒pCX15，我们研究了Sc中木糖诱导型lip的表达。lip的表达受阻遏蛋白XylR的转录控制。xylR基因、XylR靶序列以及带有顺式作用分解代谢物反应元件（cre）的xylA启动子/操纵序列均源自木糖葡萄球菌的xyl操纵子。lip核糖体结合位点（RBS）前的单个BamHI位点也使得将其他无启动子基因置于XylR的转录控制之下成为可能。为便于仅以起始密码子开始且无RBS的基因的可控表达，或者插入具有自身RBS的基因，构建了pCX26和pCX26 delta lip。在分批培养和pH值受控的发酵罐中研究了木糖和葡萄糖对lip表达的影响。使用pCX15时，木糖的存在导致细胞外Lip活性增加40倍，而葡萄糖的存在则抑制lip的表达。结果表明，xylA启动子受两种不同调控机制的影响，其中一种机制涉及在没有木糖的情况下XylR对xylA启动子的抑制，另一种机制涉及葡萄糖介导的分解代谢物阻遏，该阻遏作用强于木糖诱导作用。
