No abstract

Bioinformatic tools guided PCR amplification assays were employed for analyzing two Anabaena strains A. laxa and A. iyengarii which exhibited chitosanase activity, allelopathic and fungicidal activity. Sequencing of a 297 bp fragment obtained by amplification with primers directed towards mcy A gene (involved in the production of microcystins), revealed significant similarity with the condensation domain, while amplification with specific primers towards N-methyltransferase (NMT) domain showed 59% similarity with a homologous domain in a toxic strain of Microcystis aeruginosa. An amplified product of 172 bp obtained using specific primers derived from the coding region of chitinase (chi IS) gene in Streptomyces sp., showed 100% similarity with hydrogenbyrinic acid a, cdiamide cobaltochelatase gene in Anabaena, and significant similarity with chi IS gene of Streptomyces sp. under less stringent conditions. The 663 bp sequence obtained by employing specific primers for chitosanase (choA) derived from Mitsuaria chitosanitabida 3001 strain, showed 100% similarity with glycoside hydrolase family three domain like protein(s). This study is a first time report on the presence of homologues of chitosanase in cyanobacteria which can play a role in allelopathic activity exhibited by these oxygenic photosynthetic prokaryotes.

Two contrasting cyanobacterial species (Anabaena fertilissima and Anabaena 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. In A. 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 in A. 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 in A. sphaerica and A. fertilissima, respectively) showed that they belong to the glycoside hydrolase 3 ( Cyanobacteria comprise a heterogeneous assemblage of photosynthetic prokaryotes having extraordinary biosynthetic potential and a repertoire of diverse metabolic activities. They are an important source of novel antifungal, antibacterial, and herbicidal or weedicidal compounds, which have been implicated in allelopathic interactions in water and soil (29). A majority of these metabolites are biologically active and are products of either nonribosomal polypeptide (NRP) or mixed polyketide-NRP biosynthetic pathways. The toxins produced by cyanobacteria are greatly influenced by various physiological and environmental factors, including light, temperature, nutrients, and pH (5).The antifungal properties of cyanobacterial metabolites, most of which have not been exploited, have immense potential in agriculture for use against fungal plant pathogens. In bacteria, lytic enzymes, such as chitinases, chitosanases, proteases, and ␤-1,3-glucanases, are known to have key roles in biocontrol of various soilborne fungal pathogens. Chitin is a linear polymer of 1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues, and the deacetylated derivative of chitin is chitosan. In contrast to chitin, chitosan has been found in very few organisms, but it has been found in members of the Zygomycota, such as Mucor rouxii (42), Absidia coerulea (22), and Rhizopus oryzae (8). The filamentous fungi Lentinus edodes andPleurotus sajo-caju were investigated to determine their abilities to produce chitosan (30). Chitosan and chitosan-glucan complexes have been found in the mycelia of Aspergillus niger, Humicola lutea, and Fusarium moniliforme (39). Chitosanases (EC 3.2.1.132) produced by bacteria are classified into five glycoside hydrolase (GH) families (families 5, 8, 46, 75, and 80) (6, 10, 11, 12, 13). Families 5 and 8 are composed of enzymes that are hydrolytic with glycosides, and the family 46, 75, and 80 enzymes studied so far are chitosanases. This classification of chitosanases is based on amino acid sequence similarities of the catalytic domains. Recently, the family 46 chitosanase of Amycolatopsis sp. CsO-2 responsible for antifungal activity against Rhizopus oryzae ...

No abstract

A cyanobacterial strain (Anabaena laxa RPAN8) exhibiting fungicidal activity and β-1,3 and 1,4 endoglucanase activities was selected for identifying the gene(s) involved. Functional analyses of the genomic library revealed that four clones (8, 64, 116, and 248) of RPAN8 exhibited fungicidal activity and induced structural deformities in the cell wall of the growing mycelia of Pythium aphanidermatum. Higher expression of fungicidal and β-1,4 endoglucanase activities, along with low expression of β-1,3 endoglucanase activity, was recorded in two E. coli clones (8 and 64). Clones 8 and 64 exhibited identical sequences while clones 116 and 248 were also similar. Bioinformatic analyses were undertaken only for the two nonidentical clones 8 and 116 which showed open reading frames (ORFs) of 348 (end 1) and 656 amino acid residues (end 2), respectively. The amino acid sequence analyses revealed that the end 1 encoding endoglucanases belonged to peptidase M20 family while end 2 showed significant similarities with several known genes. The putative promoters and ribosomal binding sites were identified and amino acid exchanges were observed in both end 1 and 2. The presence of signal peptides of 24 and 20 amino acid residues respectively revealed the secretory nature of these proteins.