Antelmann H, Tjalsma H, Voigt B, Ohlmeier S, Bron S, van Dijl J M, Hecker M
Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universiät Greifswald, D-17487 Greifswald, Germany.
Genome Res. 2001 Sep;11(9):1484-502. doi: 10.1101/gr.182801.
The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.
完整基因组序列的可得性使得利用专门算法预测相应生物体的所有输出蛋白成为可能。尽管众多研究报道了基于基因组对信号肽和细胞保留信号的预测，但它们缺乏蛋白质组学验证。例如，最近预测革兰氏阳性真细菌枯草芽孢杆菌有180个分泌信号肽和114个脂蛋白信号肽。在本研究中，采用蛋白质组学方法来确定枯草芽孢杆菌分泌蛋白组的细胞外成分。利用不同的生长条件和一个超分泌突变体，通过二维（2D）凝胶电泳可视化了大约200种细胞外蛋白，其中82种通过质谱鉴定。这些蛋白包括41种具有I型信号肽酶（SPase）切割位点的潜在信号肽且缺乏保留信号的蛋白。引人注目的是，其余41种蛋白此前因明显缺乏信号肽（22种）或除输出信号外还存在特定细胞保留信号（19种）而被预测为与细胞相关。为了测试枯草芽孢杆菌的五种I型SPase和独特的脂蛋白特异性SPase的重要性，分析了（多个）SPase突变体的细胞外蛋白质组。令人惊讶的是，只有多跨膜蛋白YfnI的加工在Spase I突变体中受到强烈抑制，首次表明一种天然真细菌膜蛋白是真正 的Spase I底物。此外，一个影响脂蛋白修饰和加工的突变导致至少23种（脂）蛋白释放到培养基中。总之，我们的观察结果表明，基于基因组的预测大约反映了细胞外蛋白质组实际组成的50%。目前预测缺乏信号肽的细胞外蛋白（包括细胞质蛋白）和脂蛋白时遇到了主要问题。
