Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes

Abstract

Erwinia chrysanthemi 3937 secretes four major pectate lyase isoenzymes (PL, EC 4.2.2.2) and one endocellulase (Cx, EC 3.2.1.4). A genomic library of this strain was constructed in the Lambda L47‐1 vector, and screened for the presence of PL and Cx on pectate and caboxymethylcellulose agar. Among the seven Cx‐positive phage clones, three were shown to encode an enzyme of the same mol. wt. as the one found in the culture supernatant of strain 3937. The 34 PL‐positive phage clones were analyzed by electrofocusing and could, according to the PL they produced, be arranged in five classes. Phages from three classes produced three different single PL, named PLb, c and d. No common fragment was evidenced between the inserts of the phages of these three classes. This demonstrated that, in strain 3937, PLb, C, and d were encoded by three different genes called pelB, C, and D. Furthermore, our results suggest the existence of two additional genes encoding PLa and e. In addition, a pectin methylesterase gene was found closely linked to pelD.

Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli

“…Our pI 9.8 enzyme also conforms to the same pectate lyase isolated from strain EC16 by Thurn et al (Abstract, VI International Conference on Plant Pathogenic Bacteria, Beltsville, Md., 1985) and reported by them to have an isoelectric point of 9.9. This agrees closely with our value, but neither matches well with the pl values reported for the pelE gene products from certain other strains of E. chrysanthemi (14,28). The basis of these differences has not been established.…”

“…Although sequence data have not been reported for pel genes from other E. chrysanthemi strains, the EC16 gene coding for the pl 9. pelE gene defined in other strains of E. chrysanthemi (14,28,34). Our pI 9.8 enzyme also conforms to the same pectate lyase isolated from strain EC16 by Thurn et al (Abstract, VI International Conference on Plant Pathogenic Bacteria, Beltsville, Md., 1985) and reported by them to have an isoelectric point of 9.9.…”

“…Confirming this, E. coli cells containing the cloned pectate lyase genes macerated plant tissue, albeit less efficiently than E. chrysanthemi (13). Several groups subsequently cloned similar genes from other strains of E. chrysanthemi (5,14,28,34) and the related bacterium Erwinia carotovora (18,29, 40). The genes that we cloned did not cross-hybridize (13), but coded for enzymes with similar physical properties (molecular weights of ca.…”

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane

“…This hypothesis is supported by two observations: (i) when genes encoding E. chrysanthemi pectic enzymes and cellulases are expressed in E. coli, the proteins are found in the periplasm with their signal peptides removed (2,3,7,15,27,31,40), and (ii) mutations in the out genes result in the accumulation of these extracellular proteins in the periplasm of E. chrysanthemi, again with their signal peptides removed (1,26,50). Since (25).…”

Purification of the acidic pectate lyase and nucleotide sequence of the corresponding gene (pelA) of Erwinia chrysanthemi strain 3937

“…PLs cleave internal glycosidic linkages in pectic polymers by /?-elimination. Five pel genes organized in two clusters encode these five PL activities (Kotoujansky et al, 1985 ;Reverchon et al, 1986). The genes pelB and pelC are adjacent and encode the two 'neutral' isoenzymes, PLb and PLc.…”