Gering M, Brückner R
Mikrobielle Genetik, Universität Tübingen, Germany.
J Bacteriol. 1996 Jan;178(2):462-9. doi: 10.1128/jb.178.2.462-469.1996.
In Staphylococcus xylosus, scrB is one of two genes necessary for sucrose utilization. It encodes a sucrase that hydrolyzes intracellular sucrose-6-phosphate generated by the uptake of sucrose via the sucrose-specific enzyme II of the phosphotransferase system, the gene product of scrA. ScrB sucrase activity is inducible by the presence of sucrose in the culture medium. Primer extension experiments demonstrated that the observed regulation is achieved at the level of scrB transcription initiation. The protein mediating sucrose-specific regulation of scrB was found to be encoded immediately upstream of the sucrase gene. The nucleotide sequence of the regulatory gene scrR comprises an open reading frame that specifies a protein of 35.8 kDa. This protein exhibits similarity to transcriptional regulators of the GalR-LacI family. Inactivation of the scrR reading frame in the genome of S. xylosus led to the constitutive expression of scrB at a high level, identifying ScrR as a repressor of transcription. Sucrose-specific regulation of scrB was also lost upon deletion of 4 bp of a palindromic sequence (OB) covering positions +6 to +21 downstream of the scrB transcriptional start site. These results suggested a direct interaction of the ScrR repressor and the operator OB. Accordingly, a fusion protein consisting of the maltose-binding protein of Escherichia coli and the ScrR protein was able to interact with an scrB promoter fragment in gel mobility shift experiments but failed to bind an scrB fragment carrying the 4-bp deletion derivative of OB. An scrR promoter fragment, which dose not contain a sequence resembling OB, was not shifted by the fusion protein. This result corroborates scrR primer extension analyses showing that transcription of the repressor gene itself is not regulated. Therefore, the sucrase gene operator OB is the target sequence through which the ScrR protein exerts its negative effect on transcription initiation. In the promoter region of scrA, the gene essential for sucrose transport, two palindromic sequences that are similar to the scrB operator are found. Their presence in scrA suggests that ScrR controls a sucrose-specific regulon in S. xylosus.
在木糖葡萄球菌中，scrB是蔗糖利用所需的两个基因之一。它编码一种蔗糖酶，该酶可水解通过磷酸转移酶系统的蔗糖特异性酶II（scrA的基因产物）摄取蔗糖所产生的细胞内蔗糖-6-磷酸。ScrB蔗糖酶活性可被培养基中蔗糖的存在诱导。引物延伸实验表明，观察到的调控是在scrB转录起始水平实现的。发现介导scrB蔗糖特异性调控的蛋白质编码在蔗糖酶基因的紧邻上游。调控基因scrR的核苷酸序列包含一个开放阅读框，该阅读框指定一个35.8 kDa的蛋白质。该蛋白质与GalR-LacI家族的转录调节因子具有相似性。木糖葡萄球菌基因组中scrR阅读框的失活导致scrB的高水平组成型表达，确定ScrR为转录阻遏物。在scrB转录起始位点下游+6至+21位的回文序列（OB）缺失4 bp后，scrB的蔗糖特异性调控也丧失了。这些结果表明ScrR阻遏物与操纵子OB直接相互作用。因此，在凝胶迁移率变动实验中，由大肠杆菌麦芽糖结合蛋白和ScrR蛋白组成的融合蛋白能够与scrB启动子片段相互作用，但不能结合携带OB的4 bp缺失衍生物的scrB片段。不包含类似OB序列的scrR启动子片段不会被融合蛋白迁移。这一结果证实了scrR引物延伸分析，表明阻遏物基因本身的转录不受调控。因此，蔗糖酶基因操纵子OB是ScrR蛋白对转录起始发挥负向作用的靶序列。在蔗糖转运必需基因scrA的启动子区域，发现了两个与scrB操纵子相似的回文序列。它们在scrA中的存在表明ScrR控制木糖葡萄球菌中的蔗糖特异性调控子。
