Identification, Characterization, and Regulation of a Novel Antifungal Chitosanase Gene ( cho ) in Anabaena spp

Two contrasting cyanobacterial species (Anabaena fertilissima andAnabaena sphaerica ) were selected based on differences in antifungal behavior in order to study the mechanism for production of an antifungal enzyme and the genes responsible for this production. InA. fertilissima , chitosanase and antifungal activities were increased significantly under of growth-limiting conditions (8 of light and 16 h of darkness). The lack of such activities inA. sphaerica was associated with high levels of protein that accumulated during the stationary phase (at 28 days) under the same light conditions. The gene putatively responsible for chitosanase and antifungal activities was amplified using specific primers, and sequence analysis of the amplified products (1.086 and 1.101 kb inA. sphaerica andA. fertilissima , respectively) showed that they belong to the glycoside hydrolase 3 (GH3)-like family ofAnabaena variabilis ATCC 29413. Pairwise alignment of the corresponding protein sequences identified a putative signal peptide (amino acids 1 to 23) and some amino acid changes in the sequence ofA. fertilissima which may be responsible for functioning of the chitosanase and the observed antifungal activity. Hydrolysis of the chitosan oligosaccharide (GlcN)5 to (GlcN)2 and (GlcN)3 confirmed the presence of chitosanase activity inA. fertilissima . Site-directed mutagenesis of theA. fertilissima chitosanase-encoding gene (cho ) led to identification of catalytic residues (Glu-121 and Glu-141) important for the antifungal effect of thecho product. The level of expression ofcho was monitored by quantitative real-time reverse transcription-PCR, which indicated that transcription of this gene is significantly enhanced under conditions that retard growth, such as a long dark period.