Shimizu Takeshi, Shima Kensuke, Yoshino Ken-ichi, Yonezawa Kazuyoshi, Shimizu Tohru, Hayashi Hideo
Department of Microbiology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Ten-nohdai, Tsukuba 305-8575, Japan.
J Bacteriol. 2002 May;184(10):2587-94. doi: 10.1128/JB.184.10.2587-2594.2002.
The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.
利用野生型和virR突变体培养上清液的二维凝胶电泳，对产气荚膜梭菌中受双组分系统VirR/VirS调控的蛋白质进行了分析。基于基质辅助激光解吸电离飞行时间质谱，鉴定出7种正调控蛋白和8种负调控蛋白。转录组分析证实，15种蛋白中的7种在转录水平上受VirR/VirS系统调控，但其余蛋白在翻译后和分泌水平上由VirR/VirS导向的蛋白酶进行修饰。我们从培养上清液中纯化并鉴定了VirR/VirS导向的蛋白酶，确定其为一种梭菌蛋白酶。由于这种蛋白水解活性受到亮抑蛋白酶肽和抗蛋白酶的强烈抑制，因此得出结论，该蛋白酶是产气荚膜梭菌半胱氨酸蛋白酶家族的一员。
