Taxonomic Identification Of A Biocontrol Agent Streptomyces Sp. Men-myco-93-63 Against Verticillium Dahliae And Characterization Of Its Chitinase Genes

Streptomyces sp. Men-myco-93-63, with screwy aerial hyphae and oval spore, grows well in nine different media and produces ivory, yellow, or bean yellow soluble pigments in three of the nine media, respectively. Its survival temperature and pH range from 15℃ to 37℃ and from pH5 to pH12. Streptomyces sp. Men-myco-93-63 can not grow under the environmental temperature of 45 . Streptomyces sp. Men-myco-93-63 in specific media can fluidify the glutin, curdle the milk, hydrolyze the amylum and cellulose, but do not deoxidize nitrate, do not produce H2S and melanin. It can utilize included glucose, maltose, glycerol, xylose, fructose, sucrose, inositol, arabinose and rhamnose as carbon sources, but not raffmose, mannitol and sodium acetate.It is found that most of its morphological, cultural, physiological and biochemical characteristics are the same or very similar to those of Streptomyces roseoflavus. In addition, BLAST analysis of its 16S rDNA sequence suggests the identity of 99.93% with Streptomyces roseoflavus, and of 100% between the 120bp-y regions of 16S rDNA of those two strains. In light of the polyphasic taxonomical principle, Streptomyces sp. Men-myco-93-63 is accordingly named as Streptomyces roseoflavus Men-myco-93-63.To align the sequences of chitinase genes published in GeneBank using Clustal V progeamme, 39 Streptomyces chitinase genes are mainly clustered into 3 groups, i.e. Group A, B, and C. Based on the conservative sequence, two sets of PCR primers have been designed from alignments of the nucleotide sequences of the group B and group C chitinase genes and used to amplify the genomic DNA from the 5 strains of Streptomyces spp., in vitro antagonistic to Verticillium dahliae of cotton. The results indicate that all of the 5 strains of Streptomyces contain not only the Streptomyces griseus c///C-like gene (family 19) but also the S. lividans chiC -like gene (family 18). The PCR products of the genomic DNA from Streptomyces roseoflavus Men-myco-93-63 using those two pairs of primers are designated as chi 19-1 and chiC. Sequencing has shown these genes to have high sequence identity within the region amplified to the catalytic domain of Streptomyces griseus chiC and S. lividans chiC genes, with 85.42% and 92.02%, respectively.The genomic library of Streptomyces roseoflavus Men-myco-93-63 is constructedin E. coli LE392 using the E. coli-Streptomyces cosmid shuttle vector pKC505. The size of the DNA fractions inserted in the 3168 transformants ranges from 20kb to 30 kb, and covered 99.93% of the total genomic DNA of Streptomyces roseoflavus Men-myco-93-63. A 445bp DNA fragment is amplified from the genomic DNA with the c/zz'C-specific PCR primers. Using this 441bp chiC partial gene as probe, 5 positive cosmids are fished out by in situ hybridization of the genomic library. It suggests that the genomic library is completed and qualified enough for further study on antibiotic biosynthesis metabolism of Streptomyces roseoflavus Men-myco-93-63.Sequencing of the 1179bp fragment of Streptomyces roseoflavus chiC by chromosome-walking shows it contains a Pfam match to entry PF00704, Glycosyl hydrolases family 18 and a Prosite hit to PS01095 Chitinases family 18 active site, which encodes 354 amino acid residues and has identity of 87.2% to the catalytic domain of S. coelicolor chiD genes in nucleic acid overlap and of 86.2% in amino acid overlap.