218 微生物学通报 2009, Vol.36, No 活性检测菌 : Staphylococcus aureus CMCC Escherichia Coli CMCC Pseudomonas aeruginosa CMCC Bacillus su

Size: px

Start display at page:

Download "218 微生物学通报 2009, Vol.36, No 活性检测菌 : Staphylococcus aureus CMCC Escherichia Coli CMCC Pseudomonas aeruginosa CMCC Bacillus su"

骧耗猛辟
5 years ago
Views:

1 微生物学通报 FEB 20, 2009, 36(2): 217~222 Microbiology 2009 by Institute of Microbiology, CAS 研究报告一株杀黄孢链霉菌 IMB-14 的分离与鉴定 * 邹懿刘莉胡昌华 ( ) 摘要 : 采用薄膜过滤法从武当山采集土壤中分离到一株具有广谱抗菌活性的链霉菌, 命名为 IMB-14 通过对其抗菌活性形态特征培养特征生理生化特性和 16S rdna 序列的多相分类研究, 结合代谢产物的性质鉴定该菌株为杀黄孢链霉菌 (Streptomyces xanthocidicus) 该杀黄孢链霉菌的分离鉴定为新型抗菌及其抗癌活性物质的筛选奠定了基础关键词 : 薄膜过滤法, 多相分类, 杀黄孢链霉菌, 抗生素 FR The Isolation and Identification of Strain Streptomyces xanthocidicus IMB-14 ZOU Yi LIU Li HU Chang-Hua * (Institute of Modern Biopharmaceuticals, School of Pharmaceutical Sciences, Southwest University, Chongqing , China) Abstract: The strain Streptomyces sp., nominated IMB-14, was isolated from the soil sample of WuDang Mountain by the method of cellulose ester membrane filter. The studies on antibiotic activities, morphological characteristics, cultural characteristics, physiological characteristics, 16S rdna sequence analysis and the metabolite of strain IMB-14 showed that the strain IMB-14 was accordance with Streptomyces xanthocidicus. The study on the isolation and identification of strain Streptomyces xanthocidicus establishes a foundation on screening of novel antibacterial and antitumor agents. Keywords: Cellulose ester membrane filter method, Polyphasic taxonomy, Streptomyces xanthocidicus, Antibiotic FR Waksman,, 70%, [1],,,, IMB-14, 16S rdna (Streptomyces xanthocidicus) 1 材料与方法 1.1 材料土壤样品 :, * 通讯作者 :Tel: ; : chhhu@swu.edu.cn 收稿日期 : ; 接受日期 :

218 微生物学通报 2009, Vol.36, No.2 1.1.2 活性检测菌 : Staphylococcus aureus CMCC 26003 Escherichia Coli CMCC 44102 Pseudomonas aeruginosa CMCC 10104 Bacillus subtilis CMCC 63501 Candida albicaus CMCC 98001

2 218 微生物学通报 2009, Vol.36, No 活性检测菌 : Staphylococcus aureus CMCC Escherichia Coli CMCC Pseudomonas aeruginosa CMCC Bacillus subtilis CMCC Candida albicaus CMCC Mycobacterium Smegmatis MC2 Micrococcus luteus CNCC(B) 分离培养基 ISP2,, 50 μg/ml 1.2 土壤样品分离, 110 C 30 min, Hirsch [2], 0.22 μm 1.3 抗菌活性测试 1.4 形态及培养特征观察 ISP2,, [3] 1.5 生理生化特征观察 [4] 1.6 基因组 DNA 的提取和 16S rdna 的 PCR 扩增 Chun [5] DNA PCR 16S rdna, 27f 1495r S rdna 测序及序列分析 GenBank Blast, 1.8 菌株的发酵与发酵产物的 TLC 鉴定 Kuroda [6], 3 L, 6 mol/l HCl 100 C 6 htlc TLCGF 254 (); = 6:4(V/V); 0.1% 2 结果与分析 2.1 菌株的分离及其抗菌活性的测定,, 32,,, C. albicaus CMCC M. smegmatis MC, IMB-14, 1 1 图 1 薄膜法分离放线菌流程 Fig. 1 Membrane filter isolation procedure for actinomycetes A ; B 3 d ; C; D 2 d. Note A After spoting; B Cultured three days; C Unveil the membrane; D Cultured two days after unveiling the membrane. 表 1 IMB-14 的抗菌活性结果 Table 1 The antimicrobial activities of IMB-14 Test strain Frist secreening Second screening S. aureus CMCC E. coli CMCC P. aeruginosa CMCC B. subtilis CMCC C. albicaus CMCC M. smegmatis MC M. luteus CNCC(B) ; +;. Note: ++ Positive; + Weakly positive; Negative. 2.2 形态及培养特征观察 IMB-14 ISP ,,,,,, ( 2),

: IMB-14 219 图 2 IMB-14 的气生菌丝 (A) 及其基内菌丝 (B) 的形态特征 ( 1000 倍 ) Fig. 2 The aerial hypha (A) and basal filament (B) morphological characteristics of strain IMB-14 A: ; B.

3 : IMB 图 2 IMB-14 的气生菌丝 (A) 及其基内菌丝 (B) 的形态特征 ( 1000 倍 ) Fig. 2 The aerial hypha (A) and basal filament (B) morphological characteristics of strain IMB-14 A: ; B. Note A: Aerial hypha; B: Basal filament., 2.3 生理生化特性及其碳源利用情况 IMB-14 3,,,,,, H 2 S ; ; IMB-14 Medium 表 2 IMB-14 菌株在不同培养基上的培养特征 Table 2 Cultural characteristics of the strain IMB-14 in different medium Aerial hypha Basal filament Soluble pigment 7 d 14 d 7 d 14 d 7 d 14 d,,,,,,,,,,,,,,,, Table 3 表 3 IMB-14 的生理生化特征及其碳源利用情况 The partial physiological and biochemical characteristics and the carbon utilization of strain IMB-14 Physiological characteristics IMB-14 IMB-14 strain Carbon utilization IMB-14 IMB-14 strain 7 d 14 d (Gelatin liquefaction) + (xylose) + + (Milk solidation) + (fructose) + + (Milk peptonization) (arabinose) + + (Melanin generation) + (maltose (Amylohydrolysis) + (sucrose) H 2 S(H 2 S generation) (mannose) + + (Optimum growth temperature) 28 C~30 C (glucose) ph(optimum growth ph value) 7.2~7.4 (inose) (sorbitol) (lactose) ± ± ++ ; +; ; ±. Note ++ Positive; + Weakly positive; Negative; ± Doubtful.

4 220 微生物学通报 2009, Vol.36, No.2 28 C ~30 C, ph 7.2~ S rdna 的序列分析及其系统进化树的建立, IMB-14 16S rdna 1398 bp GenBank BLAST, 16S rdna, IMB-14 97%, ClustalX MEGA neighbor-joining 16S rdna, Bootstrap analysis, , IMB-14 16S rdna (Streptomyces kitasatoensis) (Streptomyces xanthocidicus) (Streptomyces tsukiyonensis) 99%, 2.5 IMB-14 菌株代谢产物的 TLC 分析 IMB-14 TLC, R f 0.69,,, 4 FR [6] 3 讨论,, [7], 0.22 μm,,,, Fig. 3 图 3 菌株 IMB-14 及其从 GenBank 数据库中调集的相关属种构建的以 16S rdna 序列为基础的系统发育树状关系图 Neighbor-Joining tree constructed showing the phylogenetic relaionships among the strain IMB-14 and otherrelated strains downloaded from GenBank

: IMB-14 221,, C-Cl,,, IMB-14, 5 Yasuda [11] FR-900148,, 6, Respinomycins [12 14],, 图 4 IMB-14 菌株发酵液 TLC 检测结果 Fig. 4 The TLC detect result of the IMB-14 strain 1; 2; 3 6 mol/l.

5 : IMB ,, C-Cl,,, IMB-14, 5 Yasuda [11] FR ,, 6, Respinomycins [12 14],, 图 4 IMB-14 菌株发酵液 TLC 检测结果 Fig. 4 The TLC detect result of the IMB-14 strain 1; 2; 3 6 mol/l. 1 Fermentation broth; 2 Valine solution; 3 Hydrolytic broth by 6 mol/l HCl., [2] IMB-14,, IMB-14 [6] IMB-14,,, [8] FR , Yoshio Kuroda,,,, [6], IMB-14,, IMB-14, C. albicaus, [9] Kuroda [10] FR 图 5 先前报道的 FR 结构 Fig. 5 Previous structure of FR 图 6 真正的 FR 结构 Fig. 6 Accurate Structure of FR 致谢 : 感谢柳云帆同学在土壤采集中提供的帮助 ; 感谢冉茜同学在菌种分离过程中提供的帮助参考文献 [1] Berdy J. Bioactive microbial metabolites. J Antibiot (Tokyo), 2005, 58(1): [2] Hirsch CF, Christensen DL. Novel method for selective isolation of actinomycetes. Appl Environ Microbiol, 1983, 46(4): [3].. :, 2006, pp [4],,,.. :, 2007, pp

6 222 微生物学通报 2009, Vol.36, No.2 [5] Chun J, Goodfellow M. A phylogenetic analysis of the genus Nocardia with 16S rrna gene sequences. Int J Syst Bacteriol, 1995, 45(2): [6] Kuroda Y, Okuhara M, Goto T, et al. FR , a new antibiotic. I. Taxonomy, fermentation, isolation and characterization. J Antibiot (Tokyo), 1980, 33(3): [7] von Bubnoff A. Seeking new antibiotics in nature's backyard. Cell, 2006, 127(5): [8] Neumann CS, Fujimori DG, Walsh CT. Halogenation strategies in natural product biosynthesis. Chem Biol, 2008, 15(2): [9] Ferron MAV, Lopez JLC, Perez JAS, et al. Rapid screening of Aspergillus terreus mutants for overproduction of lovastatin. World Journal of Microbiology & Biotechnology, 2005, 21(2): [10] Kuroda Y, Okuhara M, Goto T, et al. FR , a new antibiotic.. Structure determination of FR J Antibiot (Tokyo), 1980, 33(3): [11] Yasuda N, Sakane K. Revised structure and the chemical transformations of FR J Antibiot (Tokyo), 1991, 44(7): [12] Ubukata M, Tanaka C, Osada H, et al. Respinomycin A1, a new anthracycline antibiotic. J Antibiot (Tokyo), 1991, 44(11): [13] Ubukata M, Osada H, Kudo T, et al. Respinomycins A1, A2 B, C and D, a novel group of anthracycline antibiotics. I. Taxonomy, fermentation, isolation and biological activities. J Antibiot (Tokyo), 1993, 46(6): [14] Ubukata M, Uzawa J, Osada H, et al. Respinomycins A1, A2, B, C and D, a novel group of anthracycline antibiotics.. Physico-chemical properties and structure elucidation. J Antibiot (Tokyo), 1993, 46(6): 编辑部公告关于微生物学通报专题刊申请的通知,,,,,,,,, 2008,,,,,,,, 1., ; 2. () ;, tongbao@im.ac.cn, ; 3.,, tongbao@im.ac.cn

: 379 (Bacillus thuringiensis) (Bacillus subtilis) (Salmonella typhi) (Shigella flexneri) (Proteus vulgaris) (Pseudomonas aeruginosa) (Enterobacter ae

: 379 (Bacillus thuringiensis) (Bacillus subtilis) (Salmonella typhi) (Shigella flexneri) (Proteus vulgaris) (Pseudomonas aeruginosa) (Enterobacter ae 微生物学通报 MAR 20, 2008, 35(3): 378~383 Microbiology tongbao@im.ac.cn 研究报告 2008 by Institute of Microbiology, CAS 一株具有广谱抗菌活性小单孢菌的分离和鉴定 * 龙中儿朱跃进黄运红付学琴 ( 330022) 摘要 : 从南昌瑶湖的农田土壤样品中分离到一株具有广谱抗菌活性的稀有放线菌通过形态特征