摘要
盐环境中的放线菌不仅能产生多种抗生素,还能产生多种耐盐胞外酶,其中耐盐淀粉酶、耐盐核酸酶、耐盐蛋白酶等有广阔的应用前景。盐环境中的放线菌产生的四氢嘧啶和甜菜碱是高效的保护剂,又是酶类、核酸、细胞膜的稳定剂,在酶技术、医药工业和环境保护领域有重要的应用价值。研究盐环境中的放线菌
多样性,能促进放线菌资源开发,有利于新物种和新产物的发现。位于新疆吐鲁番盆地南部的艾丁湖具有低海拔、高盐度的特点。本研究采集艾丁湖沉积物样品,应用基于16S rDNA基因序列系统发育分析的免培养方法和选择性分离培养方法揭示放线菌多样性,以期获得艾丁湖放线菌物种资源,为开发利用提供技术支撑和菌种支持。
(1)利用放线菌特异性引物扩增16S rDNA基因,构建16S rDNA基因克隆文库,对文库中的克隆片段进行RFLP(Restriction Fragment Length Polymorphism,限制性片段长度多态性)分析。研究分析表明:273个克隆分布于放线菌纲(Actinobacteria)的三个亚纲中,即放线菌亚纲(Actinobacteridae 208个)、酸微菌亚纲(Acidimicrobidae 13个)和红色杆菌亚纲(Rubrobacteridae 52个)。优势放线菌为放线菌亚纲微球菌科的罗斯氏菌属(Rothia),占克隆总数的37%。有45.8%的序列与已有效发表菌株的序列相似性小于97%,部分序列形成了几个独立的进化分支,可能代表着放线菌新的类群。
(2)采用不同盐浓度的9种选择性培养基分离放线菌,共得到55株放线菌,分布在放线菌目的6个亚目中,以链霉菌属(Streptomyces)和拟诺卡氏菌属(Nocardiopsis)为主要类群。还分离到一些放线多孢菌属(Actinopolyspora)、拟无枝酸菌属(Amycolatopsis)、疣孢菌属(Verrucosispora)、糖多孢菌属(Saccharopolyspora)、糖单孢菌属(Saccharomonospora)和微球菌属(Micrococcus)的稀有放线菌。有3株放线菌的16S rDNA基因序列与已有效发表菌株的序列最大相似度小于97%,为潜在的放线菌新分类单元。
(3)对2株为潜在新种的放线菌进行了多相分类,结果表明,菌株TRM46004为Streptoalloteichus属的新种,菌株TRM46012为Streptomyces属的新种。综合分析免培养和分离培养结果,表明艾丁湖放线菌具有较高的多样性,并蕴藏着多种新类型,有进一步研究价值。
Actinomycetes from salt environment is well known for its remarkable antibiotic producing ability and the potential capacity to produce salt tolerant extracellular enzyme such as amylase, nuclease and proteinase. The betaine produced by salt tolerant actinobacteria is efficient preservative and stabilizer, which has potential application in the fields of medical industry, biological chemistry and environmental protection. Studies on the actinobacterial diversity in salt environment would promote the development and utilization of actinomycetes resources. This study aims at investigating the diversity of actinobacteria in Aiding Lake, a hypersaline lake and the lowest land point in China. The diversity of actinobacteria in the sediment from Aiding Lake was investigated by culture-independent method based on phylogenetic analysis of 16S rDNA gene sequences and selective isolation method.
Specific primers were used to amplify the actinobacterial 16S rDNA gene, and corresponding clone libraries were constructed for the sediment samples. Different clones selected on the basis of HaeIII digestion patterns were sequenced. Nine selective media with different salinities were used to isolate actinobacteria from the sediment samples. The analysis of 16S rDNA gene sequences showed that 273 clone sequences belonged to subclasses Actinobacteridae (208), Acidimicrobidae (13) and Rubrobacteridae (52), respectively. The dominant actinobacteria was genus Rothia, which accounted for 37% of total clones. The similarity between 45.8% of 273 detected sequences and published sequences were less than 97%, which might represent novel taxa. Some sequences, which formed several distinct clades in phylogenetic tree may represent novel taxonomical groups of Actinobacteria.
55 strains were isolated by different selective media. They were belonged to 6 suborders of the order Actinomycetales, of which Streptomyces and Nocardiopsis were the dominant groups. Some rare actinomycetes were isolated, in genus of Actinopolyspora, Amycolatopsis, Verrucosispora, Saccharopolyspora, Micrococcus and Saccharomonospora. 3 potential novel species were obtained.
Aiding lake harbors abundant actinobacteria, including large number of unknown actinobacterial groups, is worth while for further study.
引文
[1]姜成林,徐丽华.微生物资源学.第一版.北京:科学出版社, 1997.
[2]康丽华.木麻黄苗期接种弗兰克氏菌效应及其与营养元素的关系.林业科学研究, 1994, 7(3): 129-132.
[3] Rothschild LJ, Manicinelli RL. Life in extreme environments. Nature, 2001, 409: 1092-1101.
[4] Oliver N, Nadege B, David M. Diversity of functional genes of methanogens, methanotrophs and sulfate reducers in deep-sea hydrothermal environments. Environmental microbiology, 2005, 7(1): 118-132.
[5] Gerike U, Danson MJ, Russel NJ. Sequencing and express of a cold-active citrate Synthase from an antarctic bacterium. European Journal of Biochemistry, 1997, 248: 49-57.
[6] Hondt S, Rutherford S, Spivack AJ. Metabolic activity of subsurface life in deep-sea sediments. Science, 2002, 295(5562): 2067-2070.
[7] Yayanos AA, Dietz AS. Isolation of a Deep-Sea Barophilic Bacterium and Some of Its Growth Characteristics. Science, 1979, 205(4408): 808-810.
[8] Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Reviews, 1995, 59(1): 143-169.
[9] Head IM, Saunders JR, Pickup RW. Microbial Evolution, Diversity, and Ecology: A Decade of Ribosomal RNA Analysis of Uncultivated Microorganisms. Microbial Ecology, 1998, 35: 1-21.
[10] Woese CR, Kandler O, Wheelis ML. Towards a Natural System of Organisms: Proposal for the Domains Archaea, Bacteria, and Eucaryal. In Proceedings of the National Academy of Sciences, 1990, 87: 4576-4579.
[11] Stackebrandt E, Rainey FA, Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. International Journal of Systematic Bacteriology, 1997, 47:479-491.
[12] Garrity GM, Holt JG. (2001). "Phylum BVII. Thermomicrobia phy. nov.". In Boone DR, Castenholz RW, Garrity GM. Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 1, The Archaea and the Deeply Branching and Phototrophic Bacteria. Springer.
[13]职晓阳,蔡曼,杨玲玲,李文均,徐丽华,刘志恒,姜成林.建立放线菌门的证据.微生物学通报, 2006, 33(3): 181-183.
[14] Yan-Ming Wang, Zhen-Shui Zhang, Xiao-Li Xu, Ji-Sheng Ruan. Actinopolymorpha singaporensis gen. nov., sp. nov., a novel actinomycete from the tropical rainforest of Singapore. International Journal of Systematic and Evolutionary Microbiology, 2001, 51: 467-473.
[15] Wen-Jun Li, Ping Xu, Shu-Kun Tang. Kroppenstedt, Erko Stackebrandt, Cheng-Lin Jiang. Prauserella halophila sp. nov. and Prauserella alba sp. nov., moderately halophilic actinomycetes from saline soil. International Journal of Systematic and Evolutionary Microbiology, 2003, 53: 1545-1549.
[16] Hua-Hong Chen, Wen-Jun Li, Shu-Kun Tang. Corynebacterium halotolerans sp. nov., isolated from saline soil in the west of China. Int J Syst Evol Microbiol, 2004, 54: 779-782.
[17] Ming-Gang Li, Wen-Jun Li, Ping Xu. Nocardiopsis xinjiangensis sp. nov., a halophilic actinomycete isolated from a saline soil sample in China. International Journal of Systematic and Evolutionary Microbiology, 2003, 53: 317-321.
[18]岳秀娟,余利岩,张月琴.自然界中处于VBNC状态微生物的研究进展.微生物学通报,2004, 31(2): 108-111.
[19]郭斌,吴晓磊,钱易.提高微生物可培养性的方法和措施.微生物学报, 2006, 46(3): 504-507.
[20]安德荣,慕小倩,赵文军,刘翠娟,史遂校.土壤放线菌分离中抑制剂的应用研究.西北农业学报, 2002, 11(1): 106-108.
[21]李文均,张忠泽,姜成林.几种主要稀有放线菌的选择性分离.国外医药抗生素分册, 2002, 23(1): 18-22.
[22] Pace NR, Stahl DA, Lane DJ. The analysis of natural microbial populations by ribosomal RNA sequences. Advances in Microbial Ecology, l986, 9: 1-55.
[23] Rondon MR, August PR, Betterman AD. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl Environ Microbiol, 2000, 66(6): 2541-2547.
[24] Claudia SG, Saskia M, Stephan K, Sabine R, Talin BA. Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel is and 16S rDNA sequence analysis. Diagnostic Microbiology and Infections Disease, 2002, 43: 193-199.
[25] Luca C, Giuseppe C. Use of a culture-independent molecular method to study the ecology of Yersinia spp. in food. International Journal of Food Microbiology, 2005, 105:71-82.
[26] Wobus A, Bleul C, Maassen S. Microbial diversity and functional characterization of sediments from reservoirs of different trophic state. Federation of European Microbiological societies, 2003, 46: 331-347.
[27] Mazza P, Monciardini P, Cavaletti L, Sosio M, Donadio S. Diversity of actionplanes and related genera isolated from an Italian soil. Microbial Ecology, 2003, 45(4): 362-372.
[28]张洪勋,王晓谊,齐鸿雁.微生态学研究方法进展.生态学报, 2003, 23(5): 988-995.
[29] Sabine P, Stefanie K, Franks. Succession of microbial communities during hot composting as detected by PCR-Single-Strand-Conformation Polymorphism-Based genetic profiles of mall-subunit rRNA genes. Applied and Environmental Microbiology, 2000, 66(3): 930-936.
[30] Takeshi W, Susumu A, Asumi N, Kazunari N, Makoto K. DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil. Federation of European Microbiology Letters, 2004, 232: 153-163.
[31] Danilo Ercolini. PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. Journal of Microbiological Methods, 2004, 56: 297-314.
[32] Anderson IC, Cairney JW. Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environmental Microbiology, 2004, 6(8): 769-779.
[33]叶姜瑜,罗固源.遗传指纹图谱技术对微生物群落的分析.重庆大学学报, 2003, 26(3): 147-153.
[34] Gochnauer MB, Leppard G, Komaratat P. Isolation and characterisation of Actinopolyspora halophila. Canadian Journal of Microbiology, 1975, 21: 1500-1511.
[35]蔡蔓.嗜盐放线菌链单胞菌属的快速分离筛选和多相分类研究.云南大学微生物研究所硕士学位论文, 2008.
[36]曹兰兰,王芸,唐蜀昆,等.新疆哈密地区盐湖放线菌的多样性及其功能酶的筛选.微生物学报, 2009, 49(3): 287-293.
[37]唐蜀昆,姜怡,职晓阳,等.嗜盐放线菌分离方法.微生物学通报, 2007, 34(2): 390-392.
[38]王栋,唐蜀昆,李文均,等.中度嗜盐或耐盐放线菌生长对阴离子选择性研究.微生物学通报, 2005, 32(1): 1-4.
[39] Colwell RR. Polyphasic Taxonomy of the Genus Vibrio: Numerical Taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and Related Vibrio Species. Journal of Bacteriology, 1970, 104(1): 410-433.
[40] Collins MD, Jones D. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication. Microbiology, 1981, 45: 316-354.
[41] Minnikin DE, Patel PV, Alshamaony L, Goodfellow M. Polar Lipid Composition in the Classification of Nocardia and Related Bacteria. International Journal of Systematic Bacteriology, 1977, 27(2): 104-117.
[42] Stackebrandt E, Liesack W. Nucleic acids and Classification. In: Goodfellow M, O’Donell A G ed. Handbook of New Bactrial Systematics. London: Academic Press Ltd, 1993.
[43] Stackebrandt E, Goebel BM. Taxonomic note: a place for DNA-DNA reassociation and 16S rDNA sequences analysis in the present species defination in bacteriology. International Journal of Systematic Bacteriology, 1994, 44: 846-849.
[44] Yoon JH, Park YH. Comparative sequence analyses of the ribonuclease P(RNase P) RNA genes from LL-2,6-diaminopimelic acid-containing actinomycetes. International Journal of Systematic and Evolutionary Microbiology, 2000, 50: 2021-2029.
[45] Woese CR. Bacterial evolution. Microbiol Review, 1987, 51: 221-271.
[46] Fox GE, Wisotzkey JD, Jurtshuk P. How Close Is Close: 16S rRNA Sequence Identity May Not Be Sufficient To Guarantee Species Identity. International Journal of Systematic Bacteriology, 1992, 42: 166-170.
[47]李炜,刘志恒.链霉菌分类研究进展.微生物学报, 2001, 41(1): 121-126.
[48]郑喜玉,张明刚,徐旭,李秉孝.中国盐湖志.第一版.北京:科学出版社, 2002.
[49] Newman DK, Banfield JF. How molecular-scale interactions underpin biogeochemical systems. Science, 2002, 296(5570): 1071-1077.
[50]邹静,米琴,马永贵,陈志. Na+, k+, Mg2+, Ca2+浓度对嗜盐放线菌生长的影响.青海农林科技, 2005, 3: 1-3.
[51]鲍士旦.土壤农化分析.第三版.北京:中国农业出版社, 2000.
[52] Zhou JZ, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology, 1996, 62(2): 316-322.
[53] Stach JE, Maldonado LA, Ward AC, Goodfellow M, Bull AT. New primers for the class Actinobacteria: application to marine and terrestrial environments. Environmental Microbiology, 2003, 5(10): 828-841.
[54]图雅,朱伟云,陆承平.东北虎粪细菌区系的16S rRNA基因序列分析.微生物学报, 2005, 45(5): 671-674.
[55] Stach JE, Maldonado LA, Masson DG, Ward AC, Goodfellow M, Bull AT. Statistical approaches for estimating actinobacterial diversity in marine sediments. Applied and Environmental Microbiology, 2003, 69(10): 6189-6200.
[56] Yakimov MM, Lunsdorf H, Golyshin PN. Thermoleophilum album and Thermoleophilum minutum are culturable representatives of group 2 of the Rubrobacteridae. International Journal of Systematic and Evolutionary Microbiology, 2003, 53(2): 377-380.
[57]吴晋元,职晓阳,李岩,关统伟,唐蜀昆,徐丽华,李文均.云南江城和黑井盐矿沉积物未培养放线菌多样性比较.微生物学通报, 2008, 35(10): 1550-1555.
[58]关统伟,吴晋元,职晓阳,唐蜀昆,徐丽华,李文均,张利莉.硝尔库勒湖沉积物中非培养放线菌多样性.微生物学报, 2008, 48(7): 851-856.
[59]关统伟,赵珂,夏占峰,孙红专,张利莉.新疆于田盐池放线菌群落结构.微生物学通报, 2009, 36(4): 515-521.
[60] Takahashi Y, Omura S. Isolation of new actinomycete strains for the screening of new bioactive compounds. The Journal of General and Applied Microbiology, 2003, 49(3): 141-154.
[61]唐蜀昆,姜怡,职晓阳,娄凯,李文均,徐丽华.嗜盐放线菌分离方法.微生物学通报, 2007, 34(2): 390-392.
[62] Shirling EB, Gottlieb D. Methods for characterization of streptomyces species. International Journal of Systematic Bacteriology, 1966, 16(3): 313-340.
[63]徐丽华,李文均,刘志恒,姜成林.放线菌系统学-原理、方法及实践.第一版.北京:科学出版社, 2007.
[64]阮继生.磷酸类脂快速测定方法.微生物学报, 2006, 33(4): 190-192.
[65]曲直,阮继生,洪葵.高效液相色谱和气相色谱在放线菌分类鉴定中的应用.生物技术通报, 2009, (z1): 79-82.
