Maria Fedatto Luciana, Silva-Stenico Maria Estela, Etchegaray Augusto, Pacheco Flávia T H, Rodrigues Jorge L M, Tsai Siu Mui
Laboratório de Biologia Celular e Molecular, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo-CP 96, 13400-970 Piracicaba, SP, Brazil.
Microbiol Res. 2006;161(3):263-72. doi: 10.1016/j.micres.2005.10.001. Epub 2005 Nov 11.
Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively.
木质部难养菌是一种在多种植物中发现的致病细菌。这些细菌会向培养液中分泌胞外蛋白酶，这在含有明胶作为共聚底物的十二烷基硫酸钠聚丙烯酰胺活性凝胶中可以观察到。柑橘菌株产生了三条主要蛋白带，摩尔质量（MM）分别为122、84和65 kDa。葡萄菌株9713产生了两条大约84和64 kDa的条带。这些微生物在用明胶、酪蛋白和血红蛋白改良的琼脂平板上产生了水解区域。明胶是这些蛋白酶的最佳底物。十二烷基硫酸钠-聚丙烯酰胺电泳（SDS-PAGE）活性凝胶表明，来自柑橘和葡萄的木质部难养菌的蛋白酶被苯甲基磺酰氟（PMSF）完全抑制，被乙二胺四乙酸（EDTA）部分抑制。蛋白酶活性的最适温度为30℃，最适pH值为7.0。在这种植物病原体分泌的蛋白水解酶中，在木质部难养菌（柑橘）培养物中也检测到了几丁质酶和β-1,3-葡聚糖酶活性。从这些结果表明，来自柑橘和葡萄的木质部难养菌菌株产生的蛋白酶分别属于丝氨酸蛋白酶和金属蛋白酶组。
