Isolation of chitosomes from taxonomically diverse fungi and synthesis of chitin microfibrils in Vitro

Abstract

Small spheroidal structures (mostly 40–70 nm in diameter) similar in shape, size, and chitin-synthesizing ability to the chitosomes described earlier for yeast cells of Mucor rouxii were isolated from four other fungi: Allomyces macrogynus (Emerson) Emerson and Wilson, Saccharomyces cerevisiae Meyen ex Hansen, Neurospora crassa Shear and Dodge, and Agaricus bisporus (Lange) Imbach. Chitosomes were also found in the mycelial form of M. rouxii. Evidence of zymogenicity of chitin synthetase was found in crude preparations from all five fungal genera in that protease treatment caused an increase in chitin synthetase activity. There were, however, specific differences among the fungi in the response of the zymogen to acid or neutral proteases and in the retention of zymogenicity during chitosome isolation. Upon addition of substrate (uridine diphosphate N-acetyl-D-glucosamine) and activating protease (if needed), the isolated chitosome samples produced chitin microfibrils. Both “fibroid” coils and extended microfibrils were seen. The isolation of functional chitosomes from five widely diverse genera, representing five major fungal groups (Zygomycetes, Chytridiomycetes, Hemiascomycetes, Ascomycetes, and Basidiomycetes) and three distinct developmental forms (hyphase, yeast cells, and basidiocarp), suggests that the chitosomes are a ubiquitous component of the cytoplasmic mechanism that conveys chitin synthetase to the sites of microfibril assembly at the surface of fungal cells.