Kuwana Ritsuko, Kasahara Yasuhiro, Fujibayashi Machiko, Takamatsu Hiromu, Ogasawara Naotake, Watabe Kazuhito
Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan1.
Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan2.
Microbiology (Reading). 2002 Dec;148(Pt 12):3971-3982. doi: 10.1099/00221287-148-12-3971.
The spores of Bacillus subtilis have characteristic properties and consist of complex structures including various types of proteins. To perform comprehensive analysis of the protein composition of the spores, the proteins extracted from the spore were analysed by a combination of one-dimensional PAGE and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using Turboquest SEQUEST software interfaced with the DNA sequence database of B. subtilis. A total of 154 proteins were identified, and 69 of them were novel. The remaining 85 proteins have been previously reported as sporulation-specific proteins or as proteins that are synthesized in vegetative cells. The expression pattern of each gene deduced to encode novel spore proteins was analysed using a series of strains carrying a lacZ reporter gene. The results revealed that the expression of 26 genes was dependent on sporulation-specific sigma factors, namely sigma(F), sigma(E), sigma(G) and sigma(K). In this study, it is demonstrated that the combination of the techniques of SDS-PAGE and LC-MS/MS, with the mutant library of B. subtilis, is an effective tool for the analysis of complicated cellular structures.
枯草芽孢杆菌的孢子具有独特的特性，由包括各种蛋白质类型的复杂结构组成。为了对孢子的蛋白质组成进行全面分析，从孢子中提取的蛋白质通过一维聚丙烯酰胺凝胶电泳（PAGE）和液相色谱-串联质谱联用（LC-MS/MS）相结合的方法进行分析，使用与枯草芽孢杆菌DNA序列数据库接口的Turboquest SEQUEST软件。总共鉴定出154种蛋白质，其中69种是新发现的。其余85种蛋白质先前已被报道为孢子形成特异性蛋白质或在营养细胞中合成的蛋白质。使用一系列携带lacZ报告基因的菌株分析了每个推导编码新孢子蛋白的基因的表达模式。结果表明，26个基因的表达依赖于孢子形成特异性σ因子，即σ（F）、σ（E）、σ（G）和σ（K）。在本研究中，证明了SDS-PAGE和LC-MS/MS技术与枯草芽孢杆菌突变体文库相结合，是分析复杂细胞结构的有效工具。
