Phytopathology. 2004 Nov;94(11):1228-34. doi: 10.1094/PHYTO.2004.94.11.1228.
ABSTRACT Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens strain A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas Pantoea agglomerans strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pantoea agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zinc- or calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, partially purified extracellular protease inactivated the antibiotics mccEh252 and herbicolin O, which are produced by Pantoea agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. Strain A506 inactivated mccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously unknown role of the extracellular protease produced by Pseudomonas fluorescens A506 in interactions among plant-associated microbes.
摘要 荧光假单胞菌 A506 和成团泛菌 Eh252 及 C9-1 菌株是抑制火疫病的生物防治因子，火疫病是一种由韧皮部杆菌引起的重要梨树和苹果树病害。荧光假单胞菌 A506 主要通过在花表面与韧皮部杆菌竞争排斥来抑制疾病，花表面是主要的感染部位，而成团泛菌 Eh252 和 C9-1 菌株则产生对韧皮部杆菌有毒的抗生素。在本研究中，对 A506 产生的一种细胞外蛋白酶进行了特征描述和评价，以评估其使成团泛菌菌株产生的抗生素失活的能力。细胞外蛋白酶的活性在 pH9 时最佳，并被锌或钙螯合剂抑制，表明该蛋白酶是一种碱性金属蛋白酶。在琼脂平板生物测定中，部分纯化的细胞外蛋白酶使成团泛菌菌株 Eh252 产生的抗生素 mccEh252 和 C9-1 产生的抗生素 herbicolin O 失活。通过转座子诱变获得了缺乏细胞外蛋白酶产生能力的 A506 衍生物，并克隆和测序了编码该蛋白酶的 aprX 基因。在琼脂平板生物测定中，A506 菌株可使 mccEh252 和 herbicolin O 失活，而 aprX 突变株则不能使抗生素失活。A506 和 aprX 突变株均对 C9-1 和 Eh252 的抑菌作用不敏感；因此，蛋白酶对于保护 A506 免受抑菌作用并非必需。这些数据突出了荧光假单胞菌 A506 产生的细胞外蛋白酶在植物相关微生物相互作用中的一个先前未知的作用。
