Hirose Isao, Sano Kazuyoshi, Shioda Izumi, Kumano Miyuki, Nakamura Kouji, Yamane Kunio
Institute of Biological Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki 305, Japan1.
Microbiology (Reading). 2000 Jan;146 ( Pt 1):65-75. doi: 10.1099/00221287-146-1-65.
To analyse the proteome of Bacillus subtilis extracellular proteins, extracellular protein samples were prepared from culture media (minimal medium containing 0.4% glucose) of parental B. subtilis 168, a secA-temperature sensitive mutant and an ffh conditional mutant, and examined by two-dimensional gel electrophoresis. Approximately 100 to 110 spots were visualized in a gel of B. subtilis 168 extracellular proteins. Over 90% and 80% of these disappeared in the absence of SecA and Ffh, respectively. Thirty-eight obvious spots on the gel of the B. subtilis 168 preparation were selected and compared with spots obtained under SecA- or Ffh-deficient conditions. The appearance of 36 of these 38 spots depended on SecA and Ffh. Nineteen additional extracellular proteins were detected in cultures maintained in cellobiose, maltose and soluble starch. Among 23 proteins of which the N-terminal amino acid sequences were determined, 17 were extracellular proteins having signal peptides in their precursor form. Two membrane proteins, Yfnl and YflE, were cleaved behind 226Ala-Tyr-Ala228 and 213Ala-Leu-Ala215, respectively, and of which products seemed to be liberated into the culture medium. The production of Yfnl and YflE were also dependent on SecA and Ffh. These results indicate that most extracellular proteins target to and translocate across the cytoplasmic membrane by co-operation between the signal-recognition particle and Sec protein-secretion pathways. In contrast, a spot for Hag appeared independent from SecA and Ffh. Intracellular proteins Gap, SodA and KatA were identified in the extracellular protein samples. On the basis of these results and computer searches, it was predicted that B. subtilis produces 150 to 180 proteins extracellularly.
为分析枯草芽孢杆菌胞外蛋白的蛋白质组，从亲本枯草芽孢杆菌168、secA温度敏感突变体和ffh条件突变体的培养基（含0.4%葡萄糖的基本培养基）中制备胞外蛋白样品，并通过二维凝胶电泳进行检测。在枯草芽孢杆菌168胞外蛋白的凝胶中可观察到约100至110个斑点。其中分别超过90%和80%的斑点在缺乏SecA和Ffh时消失。从枯草芽孢杆菌168制备物的凝胶中挑选出38个明显的斑点，并与在SecA或Ffh缺陷条件下获得的斑点进行比较。这38个斑点中的36个斑点的出现依赖于SecA和Ffh。在以纤维二糖、麦芽糖和可溶性淀粉培养的菌株中还检测到另外19种胞外蛋白。在已确定N端氨基酸序列的23种蛋白质中，有17种是在前体形式中具有信号肽的胞外蛋白。两种膜蛋白Yfnl和YflE分别在226Ala-Tyr-Ala228和213Ala-Leu-Ala215之后被切割，其产物似乎被释放到培养基中。Yfnl和YflE的产生也依赖于SecA和Ffh。这些结果表明，大多数胞外蛋白通过信号识别颗粒和Sec蛋白分泌途径之间的协作靶向并转运穿过细胞质膜。相比之下，Hag的一个斑点的出现独立于SecA和Ffh。在胞外蛋白样品中鉴定出了胞内蛋白Gap、SodA和KatA。基于这些结果和计算机搜索，预测枯草芽孢杆菌可胞外产生150至180种蛋白质。
