生防菌Bs916合成脂肽抗生素泛革素的 操纵子结构功能及生物活性

doi:10.3864/j.issn.0578-1752.2013.24.008

摘要/Abstract

摘要： 【目的】明确枯草芽胞杆菌Bs916（Bacillus subtilis 916）分泌的脂肽化合物Fengycin操纵子的结构、功能和生物活性，阐明枯草芽孢杆菌Bs916防治真菌病害的分子作用机制。【方法】在Bs916全基因组测序基础上，采用LA-PCR方法克隆Fengycin的操纵子Fen；通过生物信息学方法分析Fen操纵子的遗传特征；运用基质辅助解离质谱法测定Fen操纵子合成的脂肽类化合物中同系物的分子量；采用同源重组方法构建Fengycin突变株BSFG；通过抑菌活性和溶血活性试验测定突变株BSFG的生物活性。【结果】克隆到Bs916基因组DNA中全长约37.67 kb的Fen操纵子，含有5个开放阅读框架分别编码FenA、FenB、FenC、FenD和FenE多功能复合酶；与Bs168对应Fen操纵子的同源性为65%。根据该操纵子特征推测其编码的脂肽类化合物为Fengycin；Fen操纵子负责合成的Fengycin包含5个变异体，分属于两类不同同系物。同系物1的质子化峰分子量分别为1 449.8、1 463.9、1 477.9；属于相差1个（-CH2）亚甲基的同系物，一级结构为[cyclo-（[cyclo-（Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln- Tyr-β-amino fatty acid）]；同系物2的质子化峰分子量分别为1 491.9和1 505.9，也属于相差1个（-CH2）亚甲基的同系物，相对于同系物1其第6位的Ala氨酸残基变为Val氨酸残基。生物活性测定结果表明突变株BSFG抗菌活性相对于野生型菌株Bs916得到显著下降，但溶血活性的变化不明显。【结论】本研究克隆了生防菌Bs916中合成Fengycin的完整操纵子，通过对操纵子生物信息学分析和生化试验鉴定了Fengycin的5种变异体的化学结构，并阐明了脂肽Fengycin是Bs916抗真菌活性的重要因子之一。

关键词: 枯草芽孢杆菌 , 泛革素 , 操纵子 , 结构 , 生物活性

Abstract: 【Objective】The objective of this study is to clone and sequence the Fen operon responsible for synthesis of the fengycin from Bacillus subtilis 916 and determine the structure and biological activities of the Fen operon. 【Method】Genome sequence and PCR were performed to clone the Fen operon. Analysis of the Fen operon genetic structure was made by using bioinformatics. The Fen mutant BSFG was constructed by the homologous recombination. Fengycin produced by the Bs916 and mutant was analyzed by the reversed-phase high-performance liquid chromatography (HPLC). The molecular weight of the fengycin was determined by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS). The antifungal activities and hemolytic activities were evaluated in the form of a flat plate. 【Result】The 37.67 kb Fen operon responsible for synthesis of fengycin was identified and cloned from B. subtilis 916. The operon contained five ORFs designated FenA, FenB, FenC, FenD andFenE, respectively. The amino acid sequences encoded by the Bs916 Fen operon share 65% similarity with the counterpart amino acid sequences from the Bs168 Fen operon. The results of HPLC analysis of lipopeptides produced by wild-type Bs916 and mutant BSFG showed the Fen operon responsible for fengycin synthesis. The molecular weights determined by MS were 1 449.8, 1 463.9, 1 477.9 and 1 491.9, 1 505.9 Da showed fengycin produced by Bs916 contained five homologues. The first three homologues were differed by a structure of –CH2 and their peptide moiety primary structure were [cyclo-([cyclo-(Glu-Orn-Tyr-Thr-Glu-Ala-Pro- Gln-Tyr-β-amino fatty acid)]. The last two homologues were also differed by a structure of -CH2 and their peptide moiety primary structure were Val (6) instead Ala (6) compared to homologues above. Although the mutant BSFG decreased clearly in antifungal activities, its hemolytic activities showed no obvious difference compared to wild type Bs916. 【Conclusion】This paper reported the cloning, sequencing and characterization of a whole operon Fen which is responsible for synthesis of fengycin. Through bio-information and chemical analysis, the authors confirmed that the fengycin produced by the Bs916 contains five homologues. The fengycin synthesized by Fen plays a crucial part in Bs916 bio-control activities.

Key words: Bacillus subtilis , fengycin , operon , structure , biological activity

罗楚平1, 2, 王晓宇1, 周华飞1, 2, 刘邮洲1, 陈志谊1, 2. 生防菌Bs916合成脂肽抗生素泛革素的操纵子结构功能及生物活性[J]. 中国农业科学, 2013, 46(24): 5142-5149.

LUO Chu-Ping-1, 2 , WANG Xiao-Yu-1, ZHOU Hua-Fei-1, 2 , LIU You-Zhou-1, CHEN Zhi-Yi-1, 2 . The Operon, Structure and Biological Activities of the Lipopeptide Fengycin Produced by Bacillus subtilis 916[J]. Scientia Agricultura Sinica, 2013, 46(24): 5142-5149.

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参考文献

[1]Stein T. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular Microbiology, 2005, 56(4): 845-857.

[2]Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends in Microbiology, 2007, 16(3): 115-125.

[3]陈中义, 张杰, 黄大昉. 植物病害生防芽孢杆菌抗菌机制与遗传改良研究. 植物病理学报, 2003, 33(2): 97-103.

Chen Z Y, Zhang J, Huang D F. Reasearch progress on antimicrobial mechanism and genetic engineering of Bacillus subtilis for plant diseases biocontrol. Acta Phytopathologica Sinca, 2003, 33(2): 97-103. (in Chinese)

[4]Lee H, Churey J J, Worobo R W. Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25. Journal of Applied Microbiology, 2008, 105(3): 663-673.

[5]Tsuge K, Akiyama T, Shoda M. Cloning, sequencing and characterization of the iturin A operon. Journal of Bacteriology, 2001, 183(21): 6265-6273.

[6]Moyne A L, Cleveland T E, Tuzun S. Molecular characterization and analysis of the operon encoding the antifugal lipopeptide bacillomycin D. FEMS Microbiology Letters, 2004, 234(1): 43-49.

[7]Koumoutsi A, Chen X H, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. Journal of Bacteriology, 2004, 186(4): 1084-1096.

[8]Eshita S M, Roberto N H, Beale J M, Mamiya B M, Workman R F. Bacillomycin Lc, a new antibiotic of the iturin group: isolations, structures, and antifungal activities of the congeners. The Journal of Antibiotics, 1995, 48(11): 1240-1247.

[9]高学文, 姚仕义, Pham H, Vater J, 王金生. 枯草芽孢杆菌B2菌株产生的表面活性素变异体的纯化和鉴定. 微生物学报, 2003, 43(5): 647-652.

Gao X W, Yao S Y, Pham H, Vater J, Wang J S. Purification and identification of surfactin isoforms produced by Bacillus subtilis B2 strain. Acta Microbiologica Sinica, 2003, 43(5): 647-652. (in Chinese)

[10]Wang X Y, Luo C P, Chen Z Y. Genome sequence of the plant growth promoting growth-promoting rhizobacterium Bacillus sp. strain 916. Journal of Bacteriology, 2012, 194(19): 5467-5468.

[11]罗楚平, 陈志谊, 刘永锋, 张杰, 刘邮洲, 王晓宇, 聂亚锋. 生防菌Bs-916合成脂肽类化合物Bac操纵子突变株构建及功能. 微生物学报, 2009, 49(4): 445-452.

Luo C P, Chen Z Y, Liu Y F, Zhang J, Liu Y Z, Wang X Y, Nie Y F. Construction and function analysis of Bac operon mutants of bio-control strain Bacillus subtilis Bs-916. Acta Microbiologica Sinica, 2009, 49(4): 445-452. (in Chinese)

[12]罗楚平, 王晓宇, 陈志谊, 刘永锋, 张杰, 刘邮洲, 聂亚锋, 于俊杰, 尹小乐. 枯草芽胞杆菌Bs916中脂肽抗生素Bacillomycin L的操纵子结构及生物活性. 中国农业科学, 2010, 43(22): 4624-4634.

Luo C P, Wang X Y, Chen Z Y, Liu Y F, Zhang J, Liu Y Z, Nie Y F, Yu J J, Yin X L. The operon, structure and biological activities of the lipopeptide bacillomycin L produced by Bacillus subtilis 916. Scientia Agricultura Sinica, 2010, 43(22): 4624-4634. (in Chinese)

[13]Wang X Y, Luo C P, Liu Y Z, Nie Y F, Liu Y F, Zhang R S, Chen Z Y. Three non-aspartate amino acid mutations in the ComA response regulator receiver motif severely decrease surfactin production, competence development and spore formation in Bacillus subtilis. Journal of Microbiology and Biotechnology, 2010, 20(2): 301-310.

[14]Leclère V, Béchet M, Adam A, Glue J-S, Wathelet B, Ongena M, Thonart P, Gancel F, Chollet-Imbert M, Jacques P. Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism’s antagonistic and biocontrol activities. Applied and Environmental Microbiology, 2005, 71(8): 4577-4584.

[15]Koumoutsi A, Chen X H, Vater J, Borriss R. DegU and YczE positively regulate the synthesis of bacillomycin D by Bacillus amyloloquefaciens strain FZB42. Applied and Environmental Microbiology, 2007, 73(21): 6953-6964.

[16]Tsuge K, Matsui K, Itaya M. Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment. Journal of Biotechnology, 2007, 129(4): 592-603.

[17]Stachelhaus T, Mootz H D, Marahiel M A. The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chemistry & Biology, 1999, 6(8): 493-505.

[18]Challis G L, Ravel J, Townsend C A. Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chemistry & Biology, 2000, 7(3): 211-224.

[19]Moota H D, Schwarzer D, Marahiel M A. Construction of hybrid peptide synthetases by module and domain fusions. Proceeding of the National Academy of Sciences of the United States of America, 2000, 97(11): 5848-5853.

[20]周华飞, 罗楚平, 王晓宇, 张荣胜, 陈志谊. 枯草芽胞杆菌Bs916突变体库构建和抑制水稻细菌性条斑病菌相关基因克隆. 中国农业科学, 2013, 46(11): 2232-2239.

Zhou H F, Luo C P, Wang X Y, Zhang R S, Chen Z Y. Construction of Bacillus subtilis Bs916 mutant libraries by transposon tagging and cloning the genes to the organism’s anti-bacterial activities. Scientia Agricultura Sinica, 2013, 46(11): 2232-2239. (in Chinese)

[21]Henry G, Deleu M, Jourdan E, Thonart P, Ongena1 M. The bacterial lipopeptide surfactin targets the lipid fraction of the plant plasma membrane to trigger immune-related defence responses. Cellular Microbiology, 2011, 13(11): 1824-1837.