Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli

Abstract

A genomic library of Erwinia chrysanthemi EC16 was constructed in plasmid pHC79, and seven putative pectate lyase (PL) clones in Escherichia coli were selected on pectate agar. Six of the recombinant cosmids contained a common PstI fragment of ca. 8.2 kilobases (kb). Subcloning of this fragment in either orientation into the PstI site of plasmid pBR329 resulted in E. coli transformants that produced a PL of pI 9.8 which was indistinguishable from one of two PLs produced by strain EC16. A 6.6-kilobase PstI fragment from the remaining cosmid clone caused production of an Erwinia PL of pI 8.8 when the fragment was subcloned in either orientation into plasmid pBR329 and transformed into E. coli. Selected pBR329 subclones for the 8.2- and 6.6-kilobase PstI fragments showed no similarity in their restriction maps and did not cross-hybridize. All of the E. coli cosmid clones that produced large amounts of PL also caused soft-rot of potato tubers and tuber slices, thus confirming the role of the enzymes in plant tissue maceration. The E. coli cosmid clones and plasmid pBR329 subclones produced the PLs constitutively, unlike Erwinia chrysanthemi, which made the enzymes inducibly. However, catabolite repression appeared to function in the E. coli clones, and almost all of the PL activity occurred in the periplasm and culture fluids. Thus, the Erwinia PL clones appear to contain signal peptide sequences, transcription and translation signals, and a recognition sequence for the catabolite activator protein, all of which function efficiently in E. coli.