Cloning of Genes forErwinia carotovorasubsp.carotovoraPectolytic Enzymes and Further Characterization of the Polygalacturonases

Publisher Website
Google Scholar
Abstract
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 (Pell, pI.gtoreq. 10.0; Pe12, pI 9.7; Pe13, pI 9.2; Pe14, pI 8.0; and Pe15, pI 6.6), one endo-polygalacturonase (Peh1, pI .gtoreq. 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 pe1 genes (pe11, pe12, pe13, and pe14) 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 Peh1 and Pe13 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 pe13 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 polyphenyl 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.