软腐欧氏杆菌的蛋白酶与致病作用的关系

THE RELATIONSHIP BETWEEN PROTEASE AND PATHOGENICITY OF ERWINIA CAROTOVORA PV. CAROTOVORA

胡罗卜软腐欧氏杆菌(Erwinia carotovora var.carotovora Dyc)在合成培养基MS中产生两种蛋白酶,但在无细胞滤液中测不到果胶裂解酶和果胶水解酶的活性。用等电聚焦电泳测得这两种蛋白酶都是碱性蛋白酶,等电点分别为8.3和8.9。变性温度为49—50℃,对酸或碱具有较高的稳定性。纯化的蛋白酶单独作用马铃薯块、能使其变软,切片观察表明细胞壁被明显降解。通过滤膜结合法,将供体菌1830/pJB4J1::Tn5的转座子转入到野生型StEcc—12中,从2000个接合子中获得了11个缺少产蛋白酶能力的突变体。这些突变体的分泌性蛋白质等电聚焦图谱与野生型菌株明显不同,其中一个变突体蛋白酶带完全缺失,另外一个则出现8条新的蛋白带,其他5个属部分缺失。这些突变体对马铃薯块组织离析、腐烂的能力都有不同程度的减弱。研究结果认为在由胡罗卜软腐欧氏杆菌引起的软腐病程中蛋白酶和果胶酶是协同作用的,果胶酶降解植物细胞间和胞壁中的果胶质,而蛋白酶则降解植物细胞壁和膜中的蛋白质。

The soft rot bacteria (ECC) were grown in the liquid MS medium containing different carbon sources and compared for the cell wall degration enzymes produced in the culture supernatants. The extracellular pectate lyase and polygalacturonase were not detected but extracellular proteolytic enzyme activity was detected in the supernatants of the same medium. Extracellular proteases produced by StEcc-12 were purified by ammonium sulfate fractionation and diethylaminoethyl cellulose column chromatography (DEAE-52). lsoelectric focusing tests showed that the extracellular protease had two kinds of isoenzymes with isoelectric points of 8.3 and 8.9 respectively.The denatured temperature of the protease is about 50℃.The pH changing did not obviously effect on activity of the protease. Soft-rot was evident when potato tubers were treated by purified proteases.The sections from the tissue treated by purified proteases showed that the cell walls obviously degrated.Transposon Tn5 originated from E.coli 1830/pJB4J1::Tn5 was inserted into wild type strain (StEcc-12) by conjugation.11 mutants which protease activity was defective were obtained from 2000 transconjugates. These mutants did not liquefy gelatin or hydrolyte casein. The ability of these mutants to macerate potato tissue was obviously lower than the ability of wild type. Isoelectric focusing maps of mutants was apparent different from the maps of wild type strain. The results suggest that protease of Erwinia carotovora subsp. carotovora should be in cooperation with pectinase in the process of soft-rot of potato tuber tissues.