Novel Twin-Arginine Translocation Pathway-Dependent Phenotypes of <i>Bacillus subtilis</i> Unveiled by Quantitative Proteomics

The twin-arginine translocation (Tat) pathway is known to translocate fully folded proteins across bacterial, archaeal, and organellar membranes. To date, the mechanisms involved in processing, proofreading, and quality control of Tat substrates have remained largely elusive. Bacillus subtilis is an industrially relevant Gram-positive model bacterium. The Tat pathway in B. subtilis differs from that of other well-studied organisms in that it is composed of two complexes operating in parallel. To obtain a better understanding of this pathway in B. subtilis and to identify Tat-associated proteins, the B. subtilis 'Tat proteome' was investigated by quantitative proteomics. Metabolically labeled proteins from cytoplasmic, membrane, and extracellular fractions were analyzed by LC–MS/MS. Changes in the amounts of identified peptides allowed for quantitative comparisons of their abundance in tat mutant strains. The observed differences were suggestive of indirect or direct protein–protein relationships. The rich data set generated was then approached in hypothesis-driving and hypothesis-driven manners. The hypothesis-driving approach led to the identification of a novel delayed biofilm phenotype of certain tat mutant strains, whereas the hypothesis-driven approach identified the membrane protein QcrA as a new Tat substrate of B. subtilis. Thus, our quantitative proteomics analyses have unveiled novel Tat pathway-dependent phenotypes in Bacillus.双精氨酸转运（Twin-arginine translocation, Tat）通路可介导完全折叠的蛋白质穿过细菌、古菌及细胞器膜。截至目前，Tat底物的加工、校对与质量控制相关机制仍不甚明晰。枯草芽孢杆菌（Bacillus subtilis）是一种兼具工业应用价值的革兰氏阳性模式细菌，其Tat通路与其他已被广泛研究的物种存在差异：该通路由两套并行运作的复合物构成。为深入解析枯草芽孢杆菌的Tat通路并鉴定Tat相关蛋白，研究人员采用定量蛋白质组学技术对枯草芽孢杆菌的“Tat蛋白质组”展开了研究。研究人员通过液相色谱-串联质谱（LC-MS/MS）分析了细胞质、膜及细胞外组分中经代谢标记的蛋白质。通过比对鉴定肽段的丰度变化，可实现Tat突变菌株中蛋白质丰度的定量比较。观察到的丰度差异可提示蛋白质之间存在直接或间接的相互作用。研究人员随后分别以假说驱动与预设假说的两种分析方式，对所获得的丰富数据集进行处理。其中假说驱动的分析策略鉴定出特定Tat突变菌株存在全新的生物膜形成延迟表型；而预设假说的分析策略则将膜蛋白QcrA鉴定为枯草芽孢杆菌的新型Tat底物。综上，本研究的定量蛋白质组学分析揭示了枯草芽孢杆菌中全新的Tat通路依赖型表型。