Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product
1990
Brooks, A.D. | He, S.Y. | Gold, S. | Keen, N.T. | Collmer, A. | Hutcheson, S.W.
The ability of Erwinia chrysanthemi to cause soft-rotdiseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase. E.chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectatelyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a genelibrary of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone(pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase(ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame fora 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5alpha(pPPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6.Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL-ExoPL- mutant of EC16 was constructed that exhibited reducedgrowth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicatethat ExoPL does not contribute to the residual macerating activity of UM1005.
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