Institut für Industrielle Genetik, Universität Stuttgart, Germany.
Microb Cell Fact. 2011 Oct 20;10:83. doi: 10.1186/1475-2859-10-83.
Several vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis.
Regulation of the promoters of mtlAFD operon (P(mtlA)) and mtlR (P(mtlR)) encoding the activator were investigated by fusion to lacZ. Identification of the P(mtlA) and P(mtlR) transcription start sites revealed the σ(A) like promoter structures. Also, the operator of P(mtlA) was determined by shortening, nucleotide exchange, and alignment of P(mtlA) and P(mtlR) operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in P(mtlA) constitutive expression demonstrating the inhibitory effect of EIICB(Mtl) and EIIA(Mtl) on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both P(mtlA) and P(mtlR) were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in P(mtlR) catabolite repression. Similarly, using P(groE) as a constitutive promoter, putative cre sites of P(mtlA) and P(mtlR) slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of P(mtlA) and P(mtlR) was completely abolished in a ΔptsG mutant and significantly reduced in a MtlR (H342D) mutant.
The mtl operon promoter (P(mtlA)) is a strong promoter that reached a maximum of 13,000 Miller units with lacZ as a reporter on low copy plasmids. It is tightly regulated by just one copy of the mtlR gene on the chromosome and subject to CCR. CCR can be switched off by mutations in MtlR and the glucose transporter. These properties and the low costs of the inducers, i.e. mannitol and glucitol, make the promoter ideal for designing regulated expression systems.
已经开发了几种载体系统来表达想要在枯草芽孢杆菌中研究的任何基因。其中，参与碳水化合物利用的转录调控启动子是研究的重点。已经成功构建了基于枯草芽孢杆菌木糖、麦芽糖和甘露糖利用的启动子以及异源大肠杆菌乳糖启动子的表达系统，以及用于利用甘露醇的 mtlAFD 操纵子的启动子，是在表达载体中使用的另一个有前途的候选者。在这项研究中，我们研究了 mtl 基因的调控，以确定构建枯草芽孢杆菌中强甘露醇诱导表达系统所需的元件。
通过融合到 lacZ 来研究 mtlAFD 操纵子（P(mtlA)）和编码激活物的 mtlR（P(mtlR)）启动子的调控。鉴定 P(mtlA)和 P(mtlR)转录起始位点揭示了 σ(A)样启动子结构。此外，通过缩短、核苷酸交换和 P(mtlA)和 P(mtlR)操纵区的对齐来确定 P(mtlA)的操纵子。甘露醇特异性 PTS 基因（mtlAF）的缺失导致 P(mtlA)组成型表达，表明在没有甘露醇的情况下 EIICB(Mtl)和 EIIA(Mtl)对 MtlR 的抑制作用。mtlD 的破坏使细胞对甘露醇和山梨醇敏感。P(mtlA)和 P(mtlR)均受碳分解代谢物阻遏（CCR）的影响。然而，CcpA 缺陷突变体仅显示 P(mtlR)分解代谢物阻遏略有降低。同样，使用 P(groE)作为组成型启动子，P(mtlA)和 P(mtlR)的推定 cre 位点在存在葡萄糖时略微降低了启动子活性。相比之下，P(mtlA)和 P(mtlR)在 ptsG 突变体中完全消除了葡萄糖抑制，在 MtlR（H342D）突变体中显著降低。
mtl 操纵子启动子（P(mtlA)）是一个强启动子，当以 lacZ 作为报告基因在低拷贝质粒上时，其最大可达 13000 个 Miller 单位。它仅受染色体上一个 mtlR 基因的调控，并且受到 CCR 的调控。通过 MtlR 和葡萄糖转运蛋白的突变可以关闭 CCR。这些特性和诱导物甘露醇和山梨醇的低成本使该启动子成为设计调控表达系统的理想选择。
