BACKGROUND Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing. RESULTS In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response. CONCLUSIONS The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.

中文翻译：

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非必需的Sec途径成分和与细胞包膜相关的蛋白酶对枯草芽孢杆菌分泌高水平酶的相对贡献。

背景技术枯草芽孢杆菌（Bacillus subtilis）是一种重要的工业用马匹，用于生产许多不同的商业相关蛋白质，尤其是酶。实际上，所有这些蛋白质都是通过一般分泌（Sec）途径分泌的。来自不同实验室的研究表明，各种Sec机械组件对枯草芽孢杆菌蛋白质分泌具有重要或非必要的贡献。但是，到目前为止，还没有系统地比较高级蛋白质分泌条件下各个Sec机械组件的影响。结果在本研究中，我们比较了非必需的Sec途径成分和细胞包膜相关蛋白酶对三种高表达蛋白分泌效率的贡献。这与来自B的α-淀粉酶AmyE有关。地衣芽孢杆菌的枯草芽孢杆菌和AmyL，以及解淀粉芽孢杆菌的丝氨酸蛋白酶BPN'。我们通过脉冲追逐标记实验比较了突变菌株在摇瓶培养物中的分泌能力以及各自的分泌动力学。结果表明，secDF，secG或rasP突变会严重影响AmyE，AmyL和BPN'的分泌，但实际效应大小取决于所研究的蛋白质。另外，分子伴侣DnaK对于BPN'的分泌很重要，而AmyE或AmyL的分泌不受dnaK缺失的影响。此外，我们通过检查质量控制蛋白酶HtrA和HtrB的AmyE和AmyL依赖性诱导来评估突变菌株中分泌应激反应的诱导。有趣的是 某些Sip基因的缺失揭示了特定信号肽酶对分泌应激反应幅度的强烈差异影响。结论本研究结果突出了SecDF，SecG和RasP对蛋白质分泌的重要性，并揭示了在不同突变菌株中诱导分泌应激反应的意想不到的差异。