PERIPLASMIC DISULFIDE BOND FORMATION IS ESSENTIAL FOR CELLULASE SECRETION BY THE PLANT PATHOGEN ERWINIA-CHRYSANTHEMI

Secretion to the cell exterior of cellulase EGZ and of at least six pe ctinases enables the Gram-negative Erwinia chrysanthemi to cause sever e plant disease. The C-terminal cellulose-binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys-325 and Cys-382. Dithiothreitol (DTT)-treatment o f native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultur es led to immediate arrest of secretion of EGZ which accumulated in th e periplasm where the CBD was eventually proteolysed. Site-directed mu tagenesis that affected Cys residues involved in disulphide bond forma tion resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted. Instead they accumulate d in the periplasm. Interestingly, the region around EGZ Cys-325 is co nserved in two pectinases secreted by the same pathway as EGZ. We conc lude that the conserved Cys, and possibly adjacent residues, bear esse ntial information for EGZ to be secreted and that periplasmic disulphi de bond formation is an obligatory step which provides a pre-folded fu nctional form of EGZ with secretion competence.