Abstract
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 β-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.
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Acknowledgments
This work was supported by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) to T. Kawata (no. 2150230, 24510307), and a Faculty of Science Special Grant for Promoting Scientific Research at Toho University to T. Kawata (301–12). We thank Dr. Tamao Saito, Sophia University, Japan for her helpful comments on the manuscript. We are grateful to Dr. Margaret K. Nelson, Allegheny College, PA, USA, for proofreading the manuscript.
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Communicated by Sureshkumar Balasubramanian
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Kunii, M., Yasuno, M., Shindo, Y. et al. A Dictyostelium cellobiohydrolase orthologue that affects developmental timing. Dev Genes Evol 224, 25–35 (2014). https://doi.org/10.1007/s00427-013-0460-x
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DOI: https://doi.org/10.1007/s00427-013-0460-x