Molecular Cloning and Mutagenesis in Escherichia Coli of Pectinase Genes from Erwinia Chrysanthemi

Abstract

Erwinia chrysanthemi strains 3937 and B374 were shown to secrete 5 major pectate lyase (PL) isozymes with isoelectric points ranging from 4.4 to more than 9.5. To determine the number of pel genes encoding the PL and to study them, two gene libraries were constructed in Escherichia coli, using the Lambda vector L47-1 and the broad host range mobilizable cosmid pMMB33. These gene libraries provided several clones producing the PL. The pel genes of the strain B374 were also cloned by an in vivo procedure promoted by the pULB113 plasmid. Subcloning experiments have shown the existence of 5 pel genes, each with its own promoter, clustered in two regions on the chromosome of E. chrysanthemi. The restriction maps of these regions from 3937 and B374 were similar. In addition, a pectin methylesterase gene of 3937 was found closely linked to one pel gene. Different pel genes were mutagenized in E. coli using a mini-Mu phage able to promote translational fusions with lacZ. One fusion in the pelC gene was introduced by marker exchange into the chromosome of E. chrysanthemi 3937, giving rise to a strain lacking the PLc isozyme and allowing study of the regulation of pelC.KeywordsGene LibraryBroad Host RangePectate LyaseTranslational FusionHindIII FragmentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.