天目山自然保护区产抗微生物资源及其系统学研究
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摘要
本文从天目山自然保护区分10条路径采集土样,对20份土样进行微生物系统分离和筛选,共获得霉菌145株,细菌65株。以Ⅰ型聚酮合成酶(PKSⅠ)基因和非核糖体肽合成酶(NRPS)基因这两个抗性相关基因为筛选标记进行筛选,并研究抗菌活性,并用16S rDNA和蛋白编码基因(gyrA和gyrB)序列分析相结合,确定它们的系统发育地位。
共得到基因组中含有PKSⅠ基因或NRPS基因的细菌18株,其中8株检测到抗菌活性。筛选到的细菌全部鉴定到属,其中8株鉴定到种,3株(B5、B10、B27)可能是尚未被鉴定的菌株。
选取有抑菌能力且可能为新菌株的B27进行形态观察、培养条件、生理生化分析和16S rDNA系统发育分析,并研究了其NRPS基因序列。从一系列特征的分析得出,B27是一个未被鉴定的类芽孢杆菌属(Paenibaeillus)菌株。由其NRPS基因部分序列预测其产生的抗性物质可能属于环脂肽类,并给出了进一步研究的建议。
结果表明,天目山自然保护区的原始环境中有着丰富的微生物资源。以抗性相关基因PKSⅠ和NRPS基因为筛选标记,并在分子水平上进行菌株快速鉴定,具有与前人报道方法不同的较好的筛选效果。为最终挑选到的几个有理论与应用价值的菌株之深入研究提供了重要基础。
Soil samples were collected from 10 different routes in Tianmu Mountain Natural Reserve, and 20 of them were studied for microbe diversity. 145 mildews and 65 bacteria were isolated. All bacterial stains were directed selected with the standard whether there were polyketide synthases I (PKS I ) or nonribosomal peptide synthetases (NRPS) genes in there genomes. The selected stains were tested for antimicrobial activities. There phylogenetic relationships were obtained with sequences analysis of 16S rDNA, gyrA and gyrB. Eighteen strains with PKS I or NRPS genes in there genomes were selected, and eight of them have inhibitory activities towards test stains. All eighteen strains were identified to genus level, and ten of them were identified to species, another three have some differences with those stains in public database, and they may have not been studied or identified. B27 which have remarkable resistant activities was studied further, and the phenotype, cultivation situation, physiological and biochemical characteristics, and phylogenesis was tested in detail.On the basis of these results, B27 was identified as a member of genus Paenibacillus. However, there are differences between B27 and other Paenibacillus stains in 16S rDNA sequences and physiological and biochemical characteristics, thus B27 was considered as a new strain of genus Paenibacillus. Its partial NRPS gene sequence indicated that its antimicrobial products might be cyclo-lipopeptide. All there results showed that Tianmu Mountain Natural Reserve has a great storage of microbe resources. Directed selection with PKS I and NRPS genes as the marker, which is followed with fast identification on molecular level, is a effective way of isolation. Ultimately, several valuable stains were picked out, the stain B27 is a good example.
共得到基因组中含有PKSⅠ基因或NRPS基因的细菌18株,其中8株检测到抗菌活性。筛选到的细菌全部鉴定到属,其中8株鉴定到种,3株(B5、B10、B27)可能是尚未被鉴定的菌株。
选取有抑菌能力且可能为新菌株的B27进行形态观察、培养条件、生理生化分析和16S rDNA系统发育分析,并研究了其NRPS基因序列。从一系列特征的分析得出,B27是一个未被鉴定的类芽孢杆菌属(Paenibaeillus)菌株。由其NRPS基因部分序列预测其产生的抗性物质可能属于环脂肽类,并给出了进一步研究的建议。
结果表明,天目山自然保护区的原始环境中有着丰富的微生物资源。以抗性相关基因PKSⅠ和NRPS基因为筛选标记,并在分子水平上进行菌株快速鉴定,具有与前人报道方法不同的较好的筛选效果。为最终挑选到的几个有理论与应用价值的菌株之深入研究提供了重要基础。
Soil samples were collected from 10 different routes in Tianmu Mountain Natural Reserve, and 20 of them were studied for microbe diversity. 145 mildews and 65 bacteria were isolated. All bacterial stains were directed selected with the standard whether there were polyketide synthases I (PKS I ) or nonribosomal peptide synthetases (NRPS) genes in there genomes. The selected stains were tested for antimicrobial activities. There phylogenetic relationships were obtained with sequences analysis of 16S rDNA, gyrA and gyrB. Eighteen strains with PKS I or NRPS genes in there genomes were selected, and eight of them have inhibitory activities towards test stains. All eighteen strains were identified to genus level, and ten of them were identified to species, another three have some differences with those stains in public database, and they may have not been studied or identified. B27 which have remarkable resistant activities was studied further, and the phenotype, cultivation situation, physiological and biochemical characteristics, and phylogenesis was tested in detail.On the basis of these results, B27 was identified as a member of genus Paenibacillus. However, there are differences between B27 and other Paenibacillus stains in 16S rDNA sequences and physiological and biochemical characteristics, thus B27 was considered as a new strain of genus Paenibacillus. Its partial NRPS gene sequence indicated that its antimicrobial products might be cyclo-lipopeptide. All there results showed that Tianmu Mountain Natural Reserve has a great storage of microbe resources. Directed selection with PKS I and NRPS genes as the marker, which is followed with fast identification on molecular level, is a effective way of isolation. Ultimately, several valuable stains were picked out, the stain B27 is a good example.
引文
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[3] Donadio S, Monciardini P and Sosio M. Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics. Nat. Prod. Rep., 2007,24:1073-1109.
[4] Minowa Y, Araki M and Kanehisa M. Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial
[5]徐平，李文均，张永光，唐蜀昆，高惠英，徐丽华，贺秉坤，姜成林．产生大环聚酮类天然产物放线菌的分子筛选研究。中国抗生素杂志，2003，28(6)：321-324．
[6]王世媛．非核糖体肽合成酶(NRPSs)作用机理与应用的研究进展．微生物学报，2007，47(4)：734-737。
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[30]刘伯宁，石磊，蒋沁．环脂肽类抗生素研究进展．中国抗生素杂志．2007， 32(9)：520-524．
[31]杨世忠，牟伯中，吕应年，陈涛．环脂肽氨基酸顺序的质谱测定．化学学报，2004，62(21)：2200-2204。
[32]．史峰．生物化学实验．2002，浙江大学出版社。
[33]．陈涛，杨世忠，牟伯中．微生物发酵液中脂肽类生物表面活性剂的测定。油田化学，2004，21(4)：385．390．
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[2]李粉霞，王幼芳，刘丽，杨淑贞。浙江西天目山苔藓植物物种多样性的研究．应用生态学报，2006，17(2)：192-196．
[3] Donadio S, Monciardini P and Sosio M. Polyketide synthases and nonribosomal peptide synthetases: the emerging view from bacterial genomics. Nat. Prod. Rep., 2007,24:1073-1109.
[4] Minowa Y, Araki M and Kanehisa M. Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial
[5]徐平，李文均，张永光，唐蜀昆，高惠英，徐丽华，贺秉坤，姜成林．产生大环聚酮类天然产物放线菌的分子筛选研究。中国抗生素杂志，2003，28(6)：321-324．
[6]王世媛．非核糖体肽合成酶(NRPSs)作用机理与应用的研究进展．微生物学报，2007，47(4)：734-737。
[7] Dahllof I, Baillie H, and Kjelleberg S. rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl. Environ. Microbiol., 2000,66:3376-3380.
[8]周德庆．微生物学实验手册．上海：科学技术出版社．1983
[9]奥斯伯等主编，马学军等译校．精编分子生物学实验指南．科学出版社．2005。
[10] Izumikawa M, Murata M, Tachibana K, Ebizuka Y, and Fujii. Cloning of modular type I polyketide synthase genes from salinomycin producing strain of Streptomyces albus, I. Bioorg. Med. Chem., 2003, 11: 3401-3405.
[11] Busti E., Monciardini P, Cavaletti L, Bamonte R, Lazzarini A, Sosio M, and Donadio S. Antibiotic producing ability by representatives of a newly discovered
[12] Mignard S and Flandrois J P. 16S rRNA sequencing in routine bacterial identification: A 30-month Experiment. J. Microbiol. Meth., 2006,67 :574—581
[13] Baker GC, Smith JJ and Cowan DA. Review and re-analysis of domain-specific 16S primers. J. Microbiol. Meth., 2003, 55:541- 555.
[14] Yamada S, Ohashi E, Agata N, and Venkateswaran K. Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B.mycoides, and B. anthracis and their application to the detection of B. cereus in rice. Appl. Environ. Microbiol., 1999,65:1483-1490.
[15] Chun J, and Bae K S. Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Antonie Leeuwenhoek, 2000,78:123-127.
[16] Saitou N and Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 1987,4:406-425.
[17] Kaneko T, Nozaki R, and Aizawa K. Deoxyribonucleic-acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol. Immunol., 1978,22:639-641.
[18] Lee J S, Pyun Y R and Bae K S. Transfer of Bacillus ehimensis and Bacillus chitinolyticus to the genus Paenibacillus with emended descriptions of Paenibacillus ehimensis comb. nov. and Paenibacillus chitinolyticus comb, nov., Int J Syst. Bacteriol., 2004,54:929-933.
[19] Kuroshima K I, Sakane T, Takata R and Yokota A. Bacillus ehimensis sp. nov. and Bacillus chitinolyticus sp. nov., new chitinolytic members of the genus Bacillus. Int J Syst. Bacteriol., 1996,46:76-80.
[20] Kim D S, Bae C Y, Jeon J J, Chun S J, Oh H W, Hong S G, Baek K S, Moon E Y and Bae K S. Paenibacillus elgii sp. nov., with broad antimicrobial activity. Int. J. Syst. Evol. Micr., 2004, 54:2031-2035.
[21] Park M J, Kim H B, An D S, Yang H C, Oh S T, Chung H J and Yang D C. Paenibacillus soli sp. nov., a xylanolytic bacterium isolated from soil. Int. J. Syst. Evol. Micr., 2007, 57:146-150.
[22] Daane L L, Harjono I, Barns S M, Launen L A, Palleroni N J and Haggblom M M. PAH-degradation by Paenibacillus spp. And description of Paenibacillus
naphthalenovorans sp. nov., a naphthalene-degrading bacterium from the rhizosphere of salt marsh plants. Int. J. Syst. Evol. Micr., 2002, 52,131-139.
[23] William R S. The role of natural products in a modern drug discovery program. Drug. Discov. Today 2000,5(2):39-41.
[24] Crameri R and Davies J E. Increased production of amino glycosides associated with amplified antibiotic resistance genes. J. Antibiot., 1986,39:128-135.
[25]刘晶晶，陈全震，曾江宁，高爱根，廖一波．海洋微生物活性物质的研究进展．海洋学研究，2007，25(1)：56-65．
[26] Case R J, Boucher Y, Dahllof I, Holmstrom C, Doolittle W F and Kjelleberg S. Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies. Appl. Environ Microb., 2007,73, (1): 278-288
[27]. Walsh D A, Bapteste E, Kamekura M and Doolittle W F. Evolution of the RNA polymerase B subunit gene (rpoB) in Halobacteriales: a complementary molecular marker to the SSU rRNA gene. Mol. Biol. Evol., 2004,21:2340-2351.
[28] Mota F F, Gomes E A, Paiva E, Seldin L. Assessment of the diversity of Paenibacillus species in environmental samples by a novel rpoB-based PCR-DGGE method. FEMS Microbiol. Ecol., 2005, 53:317-328.
[29] An S Y, Asahara M, Goto K, Kasaiand H and Yokota A. Terribacillus saccharophilus gen. nov., sp. nov. and Terribacillus halophilus sp. nov., spore-forming bacteria isolated from field soil in Japan. Int. J. Syst. Evol. Micr., 2007,57:51-55
[30]刘伯宁，石磊，蒋沁．环脂肽类抗生素研究进展．中国抗生素杂志．2007， 32(9)：520-524．
[31]杨世忠，牟伯中，吕应年，陈涛．环脂肽氨基酸顺序的质谱测定．化学学报，2004，62(21)：2200-2204。
[32]．史峰．生物化学实验．2002，浙江大学出版社。
[33]．陈涛，杨世忠，牟伯中．微生物发酵液中脂肽类生物表面活性剂的测定。油田化学，2004，21(4)：385．390．
[1]Zuckerkandl E,and Pauling L.Molecules as documents of evolutionary history.J.Theor.Biol.,1965,8:357-366.
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