Department of Biology, Laboratory for Microbiology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, Marburg D-35043, Germany.
Interfaculty Institute of Genetics and Functional Genomics, Department Functional Genomics, University Medicine Greifswald, Friedrich-Ludwig-Jahn-Str. 15, Greifswald D-17475, Germany.
Environ Microbiol. 2017 Sep;19(9):3700-3720. doi: 10.1111/1462-2920.13870. Epub 2017 Aug 24.
The ProJ and ProH enzymes of Bacillus subtilis catalyse together with ProA (ProJ-ProA-ProH), osmostress-adaptive synthesis of the compatible solute proline. The proA-encoded gamma-glutamyl phosphate reductase is also used for anabolic proline synthesis (ProB-ProA-ProI). Transcription of the proHJ operon is osmotically inducible whereas that of the proBA operon is not. Targeted and quantitative proteome analysis revealed that the amount of ProA is not limiting for the interconnected anabolic and osmostress-responsive proline production routes. A key player for enhanced osmostress-adaptive proline production is the osmotically regulated proHJ promoter. We used site-directed mutagenesis to study the salient features of this stress-responsive promoter. Two important features were identified: (i) deviations of the proHJ promoter from the consensus sequence of SigA-type promoters serve to keep transcription low under non-inducing growth conditions, while still allowing a finely tuned induction of transcriptional activity when the external osmolarity is increased and (ii) a suboptimal spacer length for SigA-type promoters of either 16-bp (the natural proHJ promoter), or 18-bp (a synthetic promoter variant) is strictly required to allow regulation of promoter activity in proportion to the external salinity. Collectively, our data suggest that changes in the local DNA structure at the proHJ promoter are important determinants for osmostress-inducibility of transcription.
枯草芽孢杆菌的 ProJ 和 ProH 酶与 ProA（ProJ-ProA-ProH）一起催化，适应渗透压应激合成相容溶质脯氨酸。ProA 编码的谷氨酰磷酸还原酶也用于合成代谢脯氨酸（ProB-ProA-ProI）。ProHJ 操纵子的转录受渗透压诱导，而 ProBA 操纵子的转录不受渗透压诱导。靶向和定量蛋白质组分析表明，ProA 的数量对于相互连接的合成代谢和渗透压应激反应脯氨酸生产途径不是有限的。增强渗透压应激适应性脯氨酸生产的关键因素是渗透压调节的 proHJ 启动子。我们使用定点突变来研究这个应激反应启动子的显著特征。确定了两个重要特征：（i）ProHJ 启动子与 SigA 型启动子的保守序列的偏差有助于在非诱导生长条件下保持转录水平低，而当外部渗透压增加时，仍然允许转录活性的精细调节；（ii）SigA 型启动子的亚最优间隔长度为 16 个碱基（天然 proHJ 启动子）或 18 个碱基（合成启动子变体）对于允许启动子活性根据外部盐度进行调节是严格必需的。总之，我们的数据表明，ProHJ 启动子上局部 DNA 结构的变化是转录渗透压诱导性的重要决定因素。
