Data_Sheet_2_Thuricin17 Production and Proteome Differences in Bacillus thuringiensis NEB17 Cell-Free Supernatant Under NaCl Stress.xlsx

Bacillus thuringiensis strain NEB17, produces a bacteriocin, thuricin17 (Th17) and is known to promote the growth more effectively under salt stress conditions. In this study, bacterial salt stress tolerance screening and the possible changes in its secretome under two levels of NaCl stress was evaluated. The salt tolerance screening suggested that the bacterium is able to grow and survive in up to 900 mM NaCl. Thuricin17 production at salt levels from 100 to 500 mM NaCl was quantified using High Performance Liquid Chromatography (HPLC). Salt stress adversely affected the production of Th17 at levels as low as 100 mM NaCl; and the production stopped at 500 mM NaCl, despite the bacterium thriving at these salt levels. Hence, a comparative proteomic study was conducted on the supernatant of the bacterium after 42 h of growth, when Th17 production peaked in the control culture, as determined by Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS). Optimal (salt free) bacterial culture served as a control and 200 and 500 mM NaCl as stress conditions. As salt levels increased, the major enzyme classes, transferases, hydrolases, lyases, and ligases showed increased abundance as compared to the control, mostly related to molecular function mechanisms. Some of the notable up-regulated proteins in 500 mM NaCl stress conditions included an S-layer protein, chitin binding domain 3 protein, enterotoxins, phosphopentomutase, glucose 6-phosphate isomerase and bacterial translation initiation factor; while notable down-regulated proteins included hemolytic enterotoxin, phospholipase, sphingomyelinase C, cold shock DNA-binding protein family and alcohol dehydrogenase. These results indicate that, as the salt stress levels increase, the bacterium probably shuts down the production of Th17 and regulates its molecular functional mechanisms to overcome stress. This study indicates that end users have the option of using Th17 as a biostimulant or the live bacterial inoculum depending on the soil salt characteristics, for crop production. The mass spectrometry proteomics data have been deposited to Mass Spectrometry Interactive Virtual Environment (MassIVE) with the dataset identifier PXD024069, and doi: 10.25345/C5RB8T.

苏云金芽孢杆菌（Bacillus thuringiensis）菌株NEB17可产生细菌素thuricin17（Th17），且已知其在盐胁迫条件下能更有效地促进生长。本研究对该细菌的盐胁迫耐受性进行了筛选，并评估了其在两种浓度NaCl胁迫下分泌组的潜在变化。盐胁迫筛选结果显示，该细菌可在最高900 mM的NaCl环境中生长存活。采用高效液相色谱（High Performance Liquid Chromatography, HPLC）对100~500 mM NaCl浓度下的Th17产量进行了定量分析。结果发现，即便在低至100 mM的NaCl浓度下，盐胁迫就已对Th17的产生产生不利影响；且当NaCl浓度达到500 mM时，尽管该细菌仍可在该盐浓度下良好生长，Th17的合成就已完全停止。因此，本研究针对该细菌生长42小时后的上清液开展了比较蛋白质组学研究——此时对照培养物中的Th17产量达到峰值，该分析通过液相色谱-串联质谱（Liquid Chromatography - Tandem Mass Spectrometry, LC-MS/MS）完成。以无盐的最优细菌培养条件作为对照，以200 mM和500 mM NaCl作为胁迫条件。随着盐浓度升高，与分子功能机制密切相关的主要酶类（转移酶类、水解酶类、裂合酶类及连接酶类）的丰度相较于对照组均有所上升。在500 mM NaCl胁迫条件下，显著上调的蛋白包括S层蛋白、几丁质结合域3蛋白、肠毒素、磷酸戊糖变位酶、葡萄糖-6-磷酸异构酶以及细菌翻译起始因子；而显著下调的蛋白则包括溶血性肠毒素、磷脂酶、鞘磷脂酶C、冷休克DNA结合蛋白家族成员以及乙醇脱氢酶。上述结果表明，随着盐胁迫水平升高，该细菌可能会关闭Th17的产生，并调控其分子功能机制以应对胁迫。本研究提示，终端用户可根据土壤盐分特征，选择将Th17作为生物刺激素或活性细菌接种剂用于作物生产。本研究的质谱蛋白质组学数据已上传至质谱交互式虚拟环境（Mass Spectrometry Interactive Virtual Environment, MassIVE），数据集标识符为PXD024069，DOI为10.25345/C5RB8T。