Chryseobacterium Flavum Sp.Nov. A Novel Strain Isolated From The Skin Of Odorrana Grahami With High Extracellular Proteolytic Activity

A bacterial isolate CW-E 2 exhibiting remarkable extracellular proteolytic activity was obtained from the skin of R.Grahami. The CW-E 2 strain was subsequently identified as belonging to family Flavobacteriaceae, genus Chryseobacterium, and the name Chryseobacterium flavum is proposed for the new species on the basis of its physiological and biochemical properties.Isolate CW-E 2 was a kind of gram-negative rod bacterium lacking flagella but has fimbriae on the whole cell. Observed under the transmission electron microscope,the size of the rod ranges from 0.5 to 0.7μm in width and 1.1 to 1.3um in length.The colony was circular with smooth edges around and a diameter of 1-1.5mm.It was ropy,convex, shiny,bright-yellow coloured and smooth on LB agar plates.Cells are non-motile,non-spore-forming,aerobic and Chemo-organotrophic.Physiological and biochemical tests indicated that CW-E 2 is able to decompose D-fructose, D-glucose and D-galactose.Acid is produced from D-fructose,D-maltose, D-trechalose.It shows catalase,β-galactosidase, phenylalanine decarboxylase activity,but not ornithine decarboxylase, lysine decarboxylase urease and DNase activity. Nitrate or nitrite can not be reduced.It hydrolyzes casein, gelatin, Tween 20 but dose not hydrolyze cellulose. The predominant menaquinone is MK-6. Growth occurs at 0-6% NaCl and with 1% NaCl as optimal growth condition. A non-diffusible yellow pigment flexirubin is produced on LB agar. Growth occurs at 5-42℃, pH 5-9, optimal temperature and pH are 30-35℃and 6.0-7.0. Growth is observed on MacConkey agar. It is susceptible to carbenicillin, tetracycline, doxycycline, gentamicin, penicillin G, kanamycin, Vanoomycin, erythromycin, rifampin, neomycin, streptomycin, chloramphenicol and resistant to ampicillin, lincomycin and polymixin B. The DNA G+C content is 37.2 mol%.The fatty acid profiles mainly contain i-C_15:0(35.1%), i-C_17:1ω9c(16.8%) and i-C_17:0 3-OH(18.5%). Phylogenetic analyses displayed that the strain CW-E 2 belongs to the genus Chryseobacterium and showed only 94.9%, 93.9% and 93.9% 16S rRNA gene sequence similarity values to its closest type strains of C. joostei LMG 18212^T, C. gleum ATCC 35910^T and C. indologenes ATCC 29897^T, respectively.Phenotypic,physiological and biochemical characteristics,fatty acid composition, together with the 16S rRNA phylogenetic analysis revealed that isolate CW-E 2 belongs to Phylum Cytoghaga-Flavobacterium-Bacteroides,Class Flavobacteria,Order Flavobacteria, Family Flavobacteriaceae,Genus Chryseobacterium, and the name Chryseobacterium flavum is proposed for it.Extracellular proteolytic activity was preliminarily assayed subsequently after the identification of the taxonomic position of CW-E 2.The casein agar plates on which CW-E 2 has been inoculated showed evident clearance halos indicative of protein degradation.The changes of the level of proteolytic activity in response to the growth of CW-E 2 was clarified also. Proteolytic activity reached maximum at the optimum temperature of 50℃.Some ions at a proximate concentration of 1mM are of great significance for the effectiveness and stability of the protease and prevent it from autolysis as well.EDTA at 2.5mM inhibited the activity up by 60%.The protease adapts to a wide range of pH and the optimum is 4.0.