Molecular cloning of the outJ gene involved in pectate lyase secretion by Erwinia chrysanthemi

Abstract

The phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant-tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non-secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade-22 marker on the E. chrysanthemi 3937 chromosome. An R-prime containing a chromosomal DNA insert of about 30 kb was first obtained; subcloning into pBR325 permitted the isolation of a 4kb ClaI/SspI fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5 kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the ClaI/SspI fragment demonstrated that outJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.