Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product.
The ability of Erwinia chrysanthemi to cause soft-rot diseases 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 endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library 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 for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) 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 reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005.
Brooks
A D
AD
Department of Botany, University of Maryland, College Park 20742.
He
S Y
SY
Gold
S
S
Keen
N T
NT
Collmer
A
A
Hutcheson
S W
SW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000235:genetics; D004885:Erwinia / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D005798:Genes, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology
D004269:DNA, Bacterial; D010368:Pectins; D012964:Sodium; D011133:Polysaccharide-Lyases; C037051:pectate disaccharide-lyase; D002118:Calcium; C003181:polygalacturonic acid