Periplasmic disulphide 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 pectinases enables the Gram-negative Erwinia chrysanthemi to cause severe 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 of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site-directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted. Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys-325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre-folded functional form of EGZ with secretion competence.
Bortoli-German
I
I
LCB-CNRS, Marseille, France.
Brun
E
E
Py
B
B
Chippaux
M
M
Barras
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides
D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D002482:Cellulose; D002480:Cellulase; D004229:Dithiothreitol