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 pl 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 pl 8.8 when the fragment was subcloned in either orientation into plasmid pBR329 and transformed into E. coli. Selected pBR329 subclones for the 8.2and 6.6kilobase 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.

Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomicron

“…The mineral medium of Lennox (1955) containing thiamine hydrochloride (2 p g ml-'), amino acids (leucine plus proline; 20 p g ml-' each) and a carbon source, was also used to test pectinolytic activities of the recombinant E. coli clones. Preliminary detection of the latter was achieved with the complex solid YC medium of Keen et al (1984), which was also adapted for Bacteroides by adding the following compounds: haemin dihydrochloride, 5 mg I-' ; vitamin B,, , 10 pg 1-'; cysteine hydrochloride, 0.5 g 1-' and resazurin, 1 mg I-'. Plates inoculated with Bacteroides were incubated a t 37°C in an anaerobic station (Forma Scientific, Marietta, USA).…”

“…Plates inoculated with Bacteroides were incubated a t 37°C in an anaerobic station (Forma Scientific, Marietta, USA). For all the pectin-or polygalacturonate-containing solid media, pectinolytic activities were revealed around the colonies by flooding with 1 YO N-cetyl-N,N,N-trimethylammonium bromide (CTAB), 10% copper acetate or 0.05% ruthenium red (Keen et al 1984;Collmer et al 1988).…”

“…K-PGA. Under these conditions an increase of 1.73 absorbance unit min-' represented the formation of 1 pmol min-' of unsaturated uronide, and one unit of activity was defined as the release of 1 pmol of product (Keen et al 1984;Kotoujansky et al 1985).…”

“…Several genes encoding depolymerizing pectic enzymes have been cloned in Escherichia coli, mainly from plant-pathogenic species of Erwinia and Pseudomonas. These studies, together with the isolation of mutants Condemine et al 1986), have provided considerable information about their chromosomal organization (Reverchon et al 1986;Plastow 1988;Tamaki et al 1988), structure (Tamaki et al 1988;He and Colmer 1990;Spok et al 1991), regulation (Keen et al 1984;Zink and Chatterjee 1985), role in phytopathogenicity (Collmer et al 1985;Lei et al 1988) and evolutionary relationships by sequence comparisons (Tamaki et al 1988;Liao 1991).…”

Cloning, Expression, Purification and Initial Analysis of a Novel Pectate Lyase Pcpel1 from Phytophthora capsici

“…Application of molecular genetic techniques to bacterial pathogens has permitted direct evaluation of the role of pectolytic enzymes in disease development. In the soft-rotting bacterium Erwinia chrysanthemi, PELs exist in multiple gene family (Pel1-7) and virulence of pathogens has been related to specific PEL isozymes (Keen et al 1984). Then, insertion directed mutagenesis in E. chrysanthemi strain 3937, they obtained five mutant strains for each of the pel genes (pelA, pelb, pelC, pelD and pelE), and the pathogenicity testing indicated that the acidic PLa and the basic PLd and PLe isozymes were the most important for pathogenicity (Boccara et al 1988).…”