Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
J Bacteriol. 2010 Apr;192(8):2128-39. doi: 10.1128/JB.01673-09. Epub 2010 Feb 5.
The mannose operon of Bacillus subtilis consists of three genes, manP, manA, and yjdF, which are responsible for the transport and utilization of mannose. Upstream and in the same orientation as the mannose operon a regulatory gene, manR, codes for a transcription activator of the mannose operon, as shown in this study. Both mannose operon transcription and manR transcription are inducible by mannose. The presence of mannose resulted in a 4- to 7-fold increase in expression of lacZ from the manP promoter (P(manP)) and in a 3-fold increase in expression of lacZ from the manR promoter (P(manR)). The transcription start sites of manPA-yjdF and manR were determined to be a single A residue and a single G residue, respectively, preceded by -10 and -35 boxes resembling a vegetative sigma(A) promoter structure. Through deletion analysis the target sequences of ManR upstream of P(manP) and P(manR) were identified between bp -80 and -35 with respect to the transcriptional start site of both promoters. Deletion of manP (mannose transporter) resulted in constitutive expression from both the P(manP) and P(manR) promoters, indicating that the phosphotransferase system (PTS) component EII(Man) has a negative effect on regulation of the mannose operon and manR. Moreover, both P(manP) and P(manR) are subject to carbon catabolite repression (CCR). By constructing protein sequence alignments a DNA binding motif at the N-terminal end, two PTS regulation domains (PRDs), and an EIIA- and EIIB-like domain were identified in the ManR sequence, indicating that ManR is a PRD-containing transcription activator. Like findings for other PRD regulators, the phosphoenolpyruvate (PEP)-dependent phosphorylation by the histidine protein HPr via His15 plays an essential role in transcriptional activation of P(manP) and P(manR). Phosphorylation of Ser46 of HPr or of the homologous Crh protein by HPr kinase and formation of a repressor complex with CcpA are parts of the B. subtilis CCR system. Only in the double mutant with an HPr Ser46Ala mutation and a crh knockout mutation was CCR strongly reduced. In contrast, P(manR) and P(manP) were not inducible in a ccpA deletion mutant.
枯草芽孢杆菌的甘露糖操纵子由三个基因组成，manP、manA 和 yjdF，它们负责甘露糖的运输和利用。在甘露糖操纵子的上游和相同方向上，有一个调节基因 manR，它编码甘露糖操纵子的转录激活因子，如本研究所示。甘露糖操纵子转录和 manR 转录均可被甘露糖诱导。甘露糖的存在导致来自 manP 启动子（P(manP)）的 lacZ 表达增加 4-7 倍，来自 manR 启动子（P(manR)）的 lacZ 表达增加 3 倍。manPA-yjdF 和 manR 的转录起始位点分别为单个 A 残基和单个 G 残基，分别由类似于营养型 sigma(A)启动子结构的-10 和-35 框所前导。通过缺失分析，确定了 P(manP)和 P(manR)上游 ManR 的靶序列位于两个启动子的转录起始位点上游约-80 至-35bp 处。缺失 manP（甘露糖转运蛋白）导致两个启动子的 P(manP)和 P(manR)均组成型表达，表明磷酸转移酶系统（PTS）组件 EII(Man)对甘露糖操纵子和 manR 的调节具有负效应。此外，P(manP)和 P(manR)均受碳分解代谢阻遏（CCR）的影响。通过构建蛋白序列比对，在 ManR 序列的 N 端末端鉴定出一个 DNA 结合基序、两个 PTS 调节结构域（PRD）和一个 EIIA-和 EIIB 样结构域，表明 ManR 是一个包含 PRD 的转录激活因子。与其他 PRD 调节剂的发现一样，磷酸烯醇丙酮酸（PEP）依赖性磷酸化通过组氨酸蛋白 HPr 通过 His15 对 P(manP)和 P(manR)的转录激活起着至关重要的作用。HPr 激酶对 HPr 的 Ser46 或同源 Crh 蛋白的磷酸化以及与 CcpA 形成阻遏复合物是枯草芽孢杆菌 CCR 系统的一部分。只有在 HPr Ser46Ala 突变和 crh 敲除突变的双重突变体中，CCR 才会受到强烈抑制。相反，在 ccpA 缺失突变体中，P(manR)和 P(manP)均不能诱导。
