Molecular cloning of an Erwinia chrysanthemi oligogalacturonate lyase gene involved in pectin degradation.

Mutants of Erwinia chrysanthemi 3937 deficient in the pectin catabolic enzyme oligogalacturonate lyase were isolated by chemical and phage Mud(Aplac) insertion mutagenesis. The ogl mutation was biochemically characterized and localized near the trp his markers on the E. chrysanthemi chromosomal map. Analysis of Mud(Aplac) insertions, which generate polar mutations, revealed that oligogalacturonate lyase was the only affected enzyme in the pectin catabolic pathway, indicating that the ogl gene probably forms a separate transcriptional unit. Out of the two Mud(Aplac) insertions obtained, neither was an ogl-lac fusion. We cloned the ogl gene by complementing the mutation using the RP4::miniMu plasmid pULB113. pR'ogl plasmids were analyzed for the presence of other unselected genes of strain 3937. One of them, called pROU2, also carried the kduD and kdgR genes encoding 2-keto-3-deoxygluconate oxidoreductase, an enzyme of the pectin catabolic pathway, and the KdgR repressor, governing the expression of several genes of pectin degradation, respectively. The plasmid pROU2 harbored a chromosomal DNA insert of about 35 kb indicating that ogl, kduD and kdgR are very closely linked. Structural analysis of the ogl gene was carried out in subcloning experiments. This gene was localized on a 3.5-kb PstI fragment.