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 30kb 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 2.5kb, 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.

Characterization of transposon insertion out- mutants of Erwinia carotovora subsp. carotovora defective in enzyme export and of a DNA segment that complements out mutations in E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi

“…carotovora 71, however, indicate a fundamental difference in the arrangement of these genes in E. carotovora and E. chrysanthemi. Previous work with E. chrysanthemi has demonstrated that at least three diaferent chromosomal loci control the Out phenotype (22,23,43,45). The data presented here strongly suggest a clustering of the out genes …”

Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida

“…This information will be important for us to understand whether Ca2+ is required for translocation of PL from cytosol to periplasm or from periplasm to culture fluids. In Erwinia chrysanthemi (5) and P. viridiflava (24), the genes (out) controlling the export of PL have been identified, and one of them has recently been cloned and characterized (13 support the notion that divalent ions play a role in regulation of extracellular enzyme production, none of the foregoing studies included experiments to determine whether the increase in enzyme production is due to the improvement in enzyme-exporting efficiency.…”

Expanded linkage map of Erwinia chrysanthemi strain 3937

“…The kdgTger\e, coding for the KDG permease is located near ogl and kduD (Allen et al, 1989). The gene outJ, involved in the specific export of pectinases and cellulases toward the external mediunn, is located between ade-377 and the pro locus (Ji et al, 1989). We recently isolated Mu-lac (Km") insertions in the pectinolytic enzyme genes pelD, peIC and pme in strain 3937 (N. Hugouvieux-Cotte-Pattat, submitted for publication).…”