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Molecular Plant Pathology

Cloning of Genes for Erwinia carotovora subsp. carotovora Pectolytic Enzymes and Further Characterization of the Polygalacturonases. J. W. Willis, Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan 66506; J. K. Engwall(2), and A. K. Chatterjee(3). (2)(3)Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan 66506, (2)(3)Present address: Department of Plant Pathology, 108 Waters Hall, University of Missouri, Columbia 65211. Phytopathology 77:1199-1205. Accepted for publication 11 February 1987. Copyright 1987 The American Phytopathological Society. DOI: 10.1094/Phyto-77-1199.

The pectolytic enzymes produced by Erwinia carotovora subsp. carotovora strain Ecc71 were characterized by thin-layer isoelectric focusing (IEF), activity gel overlays, and qualitative enzyme assays. Ecc71 produced five pectate lyases (Pel1, pI ? 10.0; Pel2, pI 9.7; Pel3, pI 9.2; Pel4, pI 8.0; and Pel5, pI 6.6), one endo-polygalacturonase (Peh 1, pI ? 10.0), and at least one exo-polygalacturonase (Peh2) in culture. The levels of pectolytic activity could be increased by induction with citrus pectin, but the enzyme profile remained constant. Four of the pel genes (pel1, pel2, pel3, and pel4) and the peh1 gene were recovered from an Ecc71 gene library constructed in E. coli HB101. The simultaneous recovery of two or more of the pectolytic enzyme genes on single cosmid clones indicated clustering of these genes within the genome. The closely linked genes encoding Peh 1 and Pel3 were resolved by Tn5 mutagenesis. Lack of polar effects in the Tn5 mutants demonstrated that the genes were distinct transcriptional units. Enzyme preparations from HB101(pAKC213::Tn5-2), which had pel3 inactivated by Tn5 insertion, were used to characterize Peh1. The enzyme had a pH optimum of 5.5, an inactivation temperature of 60 C, and was stimulated by Na+, Li+, K+, and NH4+ ions. Peh1 activity resulted in loss of plant cell membrane integrity, as evidenced by release of polyphenol oxidase, and maceration of potato tuber tissue. Tn5 mutagenesis followed by marker exchange was used to construct an Ecc71 peh1::Tn5 mutant. This mutant maintained Peh2 activity and, like the parent Ecc71, was virulent under aerobic and anaerobic conditions.