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.

Extracellular proteolytic enzyme activity has been detected in cultures of Erwinia chrysanthemi. This activity, which appears when the cells are grown in the presence of peptides, is rather unstable. A hyperproteolytic mutant was isolated which produces two proteases of apparent polypeptide molecular mass of 50 and 55 kDa, respectively. The 50 kDa protease, which is produced in the largest amounts, has been purified to near homogeneity. It has the properties of a neutral serine protease. The 50 and 55 kDa proteases are unrelated antigenically. Preliminary evidence suggests that both proteases are also produced by the wild-type strain, but that they are either produced in much smaller quantities or are much less stable.

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.

Erwinia chrysanthemi produced several pectate lyases (EC 4.2.2.2) and endocellulases (EC 3.2.1.4) which were largely secreted into the culture medium. Mutants deficient in the secretion mechanism for these enzymes were obtained by chemical and insertion mutagenesis. Further study of one such mutant revealed that both enzyme activities were retained simultaneously within the periplasmic space.

The cup-plate technique makes it possible to detect enzyme activities after diffusion into buffered, substrate-containing agar gels. This technique has been used after nondenaturing blotting transfer in order to detect depolymerizing enzyme activities once analytical protein separation (e.g., by electrophoresis, electrofocusing, or titration curves) has been completed. This rapid and very sensitive method was successfully applied to the enzymes polygalacturonate lyase, polygalacturonate hydrolase, endoglucanase, and xylan hydrolase. Other possible applications are presented.