Functional analysis of chitinases of Aspergillus nidulans

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Chitin is a β-1, 4-linked polymer of N-acetyl-D-glucosamine (GlcNAc) and is one of the major cell wall components of many filamentous fungi. We cloned two chi genes (chiA and chiB) encoding chitinase-like proteins from Aspergillus nidulans. chiA gene encodes a protein of 961 amino acids which is homologous to fungal-type chitinases and has secretary signal-like sequence and long Ser/Thr/Pro rich sequence at its N- and C-termini, respectively. chiB encodes a protein of 416 amino acids homologous to bacterial-type chitinases. Disruptants of chiA could not grow on the medium containing 0.01% of SDS in the pyrGィイD1+ィエD1 background. Moreover, severe growth defects were observed under low osmotic condition in the pyrGィイD1-ィエD1 background even when uridine was added to the medium at the final concentration of 10 mM. Hyphal morphology of the double mutant of chiA and pyrG was aberrant and chitin distribution detected by Calcofluor white staining was abnormal under that condition. Lysis of hyphae at sub apical region was occasionally detected. On the other hand, chiB disruptants exhibited almost the same morphological phenotypes as the wild-type strain under various conditions. In the chiB-null mutant, however, the intracellular and extracellular chitinase activities were considerably reduced. Decrease in hyphal dry weight during autolysis occurred more slowly in the mutant than in the wild-type strain. Western blot analysis using anti-ChiB antiserum indicated that the synthesis of ChiB was largely induced when mycelia were transferred to media lacking carbon sources, a condition which induced hyphal autolysis. From these results, it is suggested that chiB plays a role in the process of autolysis in A. nidulans. Thus, it is suggested that the product of chiA is very important for formation of hyphae under some conditions, while contribution of that of chiB to it is small.