The stringent response does not influence ribosome pausing in Bacillus subtilis [Ribo-Seq]

The stringent response represses translation and is activated when amino acid levels drop, for example during the stationary phase. Bacillus subtilis, a well-known industrial enzyme production organism, secretes most enzymes during the stationary phase. We were curious whether the stringent response affects ribosome pausing, and whether its absence improves protein production yields in the stationary phase. To investigate this, genome-wide ribosome profiling was used using a stringent response mutant that overexpressed the α-amylase AmyM. Although, blocking the stringent response did increases overall protein synthesis, the secretion of AmyM was actually reduced. The ribosome profiling data revealed that this was not caused by a reduction in translation. In fact, absence of the stringent response did not seem to markedly influence ribosome pausing. However, late in stationary phase an increased ribosome pausing at tryptophane codons emerges, suggesting a depletion of tryptophane. A strong suppression of tryptophane biosynthesis and acquisition genes, under control of the trp RNA-binding attenuation protein TRAP, likely accounts for this, although TRAP does not belong to the stringent response regulon of B. subtilis. Finally, the ribosome profiles revealed several genes with unusually low translation activities, illustrating the importance of translation initiation as a regulatory element of expression. A high-copy expression plasmid was used to overexpress the heterologous α-amylase AmyM from G. stearothermophilus. Throughout this study this strain is referred to as the wild type (WT) strain. To eliminate the stringent response, we took this strain and deleted rel, sasA and sasB. This mutant is referred to as the ppGpp0 strain. Both strains were grown in rich medium for 16 h at 30 °C with continuous shaking.

严谨反应（stringent response）可抑制翻译过程，其在氨基酸水平下降时被激活，例如在细菌稳定生长期。枯草芽孢杆菌（Bacillus subtilis）是一类广为人知的工业酶生产宿主，其绝大多数酶类均在稳定期分泌。本研究旨在探究严谨反应是否会影响核糖体暂停，以及严谨反应的缺失是否能够提升稳定期的蛋白质生产产量。为开展相关研究，本研究采用过表达α-淀粉酶AmyM的严谨反应突变株，进行全基因组范围的核糖体谱分析（ribosome profiling）。尽管阻断严谨反应确实能够提升整体蛋白质合成水平，但AmyM的分泌量反而出现下降。核糖体谱分析数据表明，该现象并非由翻译水平降低所致；事实上，严谨反应的缺失似乎并未显著影响核糖体暂停。然而在稳定期后期，色氨酸密码子处的核糖体暂停现象有所增加，提示色氨酸发生了耗竭。尽管色氨酸RNA结合衰减蛋白（TRAP）并不属于枯草芽孢杆菌的严谨反应调控子（regulon），但受TRAP调控的色氨酸生物合成与摄取基因的强烈抑制，或是该现象的成因。最后，核糖体谱分析还发现了若干翻译活性异常低下的基因，阐明了翻译起始作为基因表达调控元件的重要性。本研究使用高拷贝表达质粒，过表达来自嗜热脂肪地芽孢杆菌（G. stearothermophilus）的异源α-淀粉酶AmyM；本研究中，该菌株被定义为野生型（WT）菌株。为消除严谨反应，本研究对该野生型菌株的rel、sasA及sasB基因进行敲除，所得突变株被命名为ppGpp0菌株。两种菌株均于丰富培养基中，在30℃、持续振荡条件下培养16小时。