He S Y, Lindeberg M, Chatterjee A K, Collmer A
Department of Plant Pathology, Cornell University, Ithaca, NY 14853.
Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1079-83. doi: 10.1073/pnas.88.3.1079.
The out genes of the enterobacterial plant pathogen Erwinia chrysanthemi are responsible for the efficient extracellular secretion of multiple plant cell wall-degrading enzymes, including four isozymes of pectate lyase, exo-poly-alpha-D-galacturonosidase, pectin methylesterase, and cellulase. Out- mutants of Er. chrysanthemi are unable to export any of these proteins beyond the periplasm and are severely reduced in virulence. We have cloned out genes from Er. chrysanthemi in the stable, low-copy-number cosmid pCPP19 by complementing several transposon-induced mutations. The cloned out genes were clustered in a 12-kilobase chromosomal DNA region, complemented all existing out mutations in Er. chrysanthemi EC16, and enabled Escherichia coli strains to efficiently secrete the extracellular pectic enzymes produced from cloned Er. chrysanthemi genes, while retaining the periplasmic marker protein beta-lactamase. DNA sequencing of a 2.4-kilobase EcoRI fragment within the out cluster revealed four genes arranged colinearly and sharing substantial similarity with the Klebsiella pneumoniae genes pulH, pulI, pulJ, and pulK, which are necessary for pullulanase secretion. However, K. pneumoniae cells harboring the cloned Er. chrysanthemi pelE gene were unable to secrete the Erwinia pectate lyase. Furthermore, the Er. chrysanthemi Out system was unable to secrete an extracellular pectate lyase encoded by a gene from a closely related plant pathogen. Erwinia carotovora ssp. carotovora. The results suggest that these enterobacteria secrete polysaccharidases by a conserved mechanism whose protein-recognition capacities have diverged.
植物病原菌菊欧文氏菌（Erwinia chrysanthemi）的out基因负责多种植物细胞壁降解酶的高效胞外分泌，这些酶包括果胶酸裂解酶的四种同工酶、外切聚-α-D-半乳糖醛酸酶、果胶甲基酯酶和纤维素酶。菊欧文氏菌的Out-突变体无法将这些蛋白质中的任何一种分泌到周质之外，并且毒力严重降低。我们通过互补几个转座子诱导的突变，从菊欧文氏菌中克隆出了out基因，并将其克隆到稳定的低拷贝数黏粒pCPP19中。克隆出的out基因聚集在一个12千碱基的染色体DNA区域，互补了菊欧文氏菌EC16中所有现有的out突变，并使大肠杆菌菌株能够高效分泌由克隆的菊欧文氏菌基因产生的胞外果胶酶，同时保留周质标记蛋白β-内酰胺酶。out基因簇内一个2.4千碱基的EcoRI片段的DNA测序揭示了四个共线排列的基因，它们与肺炎克雷伯菌（Klebsiella pneumoniae）的pulH、pulI、pulJ和pulK基因有很大的相似性，这些基因是支链淀粉酶分泌所必需的。然而，携带克隆的菊欧文氏菌pelE基因的肺炎克雷伯菌细胞无法分泌欧文氏菌果胶酸裂解酶。此外，菊欧文氏菌的Out系统无法分泌由密切相关的植物病原菌胡萝卜软腐欧文氏菌（Erwinia carotovora ssp. carotovora）的一个基因编码的胞外果胶酸裂解酶。结果表明，这些肠杆菌通过一种保守的机制分泌多糖酶，但其蛋白质识别能力已经发生了分化。
