A Dictyostelium cellobiohydrolase orthologue that affects developmental timing

Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound - tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug - late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2-4 h. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-myc (oe) strain displayed 4-methylumbelliferyl beta-d-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native-polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development.