Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (11): 2232-2239.doi: 10.3864/j.issn.0578-1752.2013.11.006

• PLANT PROTECTION • Previous Articles     Next Articles

Construction of Bacillus subtilis Bs916 Mutant Libraries by Transposon Tagging and Cloning the Genes to the Organism’s Anti-Bacterial Activities

 ZHOU  Hua-Fei, LUO  Chu-Ping, WANG  Xiao-Yu, ZHANG  Rong-Sheng, CHEN  Zhi-Yi   

  1. 1.College of Plant Protection, Nanjing Agricultural University, Nanjing 210095
    2.Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2013-01-08 Online:2013-06-01 Published:2013-03-07

PDF

863

Abstract: 【Objective】The objective of this study is to construct Bacillus subtilis Bs916 mutant library by introducing transposon into Bs916 genomic DNA, screen mutants which to appear enhanced or decreased apparently in the inhibition zone to Xanthomonas oryzae pv. oryzicola (Xooc), and to obtain the genes which may be related to anti-bacterial activities changed by cloning the DNA sequence around the transposon inserted sites. 【Method】The pMarA shuttle vector which contained transposon TnYLB-1 was introduced into B. subtilis Bs916 through chemical transformation method and build the random inserted mutant library. The enhanced or decreased anti-bacterial activity mutants screened from the library by in vitro assay against Xooc and the mutants were verified by the PCR and Southern Blot. The genes around the insertion sites were amplified by the Inverse PCR and sequence analyzed by bioinformatics.【Result】The random inserted mutant library of B.subtilis 916 was constructed successfully by using TnYLB-1 transposon. The PCR and Southern Blot results showed that the transposon was inserted into Bs916 genomic DNA successfully, about 85% mutants were inserted by a single copy. More than 30 mutants whose antibacterial activities changed against Xooc strongly enhanced or sharply decreased were screened. Twenty-one genes around the insertion site were cloned from the mutants above and sequenced. The bioinformatic analysis results showed that these genes were related to competence development, bio-film formation and secondary metabolites synthesis.【Conclusion】A random transposon inserted mutant library of B. subtilis Bs916 was constructed successfully. These cloned genes in mutation points from the library to Xooc with inhibitory effect significantly changed. It was speculated that these genes may be related to synthesis and regulation of the anti-bacterial compound in B. subtilis Bs916.

Key words: Bacillus subtilis , mutant , Xanthomonas oryzae pv. oryzicola , antibacterial gene , clone

[1]黄海婵, 裘娟萍. 枯草芽胞杆菌防治植物病害的研究进展. 浙江农业科学, 2005(3): 213-215.

Huang H C, Qiu J P. The research progress of plant diseases control by Bacillus subtilis. Journal of Zhejiang Agricultural Sciences, 2005(3): 213-215. (in Chinese)

[2]Broadbent P, Baker K F, Franks N. Effect of Bacillus spp. on increased growth of seedings in streamed and nontreated soil. Phytopathology, 1977, 67: 1027-1034.

[3]罗楚平, 刘邮洲, 吴荷芳, 王晓宇, 刘永锋, 聂亚峰, 张荣胜, 陈志谊. 脂肽类化合物 bacillomycin L抗真菌活性及其对水稻病害的防治. 中国生物防治学报, 2011, 27(1): 76-81.

Luo C P, Liu Y Z, Wu H F, Wang X Y, Liu Y F, Nie Y F, Zhang R S, Chen Z Y. Antifungal activity and rice disease biocontrol performance of lipope antibiotic Bacillomycin L. Chinese Journal of Biological Control, 2011, 27(1): 76-81. (in Chinese)

[4]陈志谊, 刘永锋, 陆凡. 水稻纹枯病生防菌Bs-916产业化生产关键技术. 植物保护学报, 2004, 31(3): 230-234.

Chen Z Y, Liu Y F, Lu F. Study on key technology in the industrialized production of Bacillus subtilis Bs-916, the rice sheath blight control agent. Acta Phytophylacica Sinica, 2004, 31(3): 230-234. (in Chinese)

[5]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. 枯草芽孢杆菌 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 Bs916. Scientia Agricultura Sinica, 2010, 43(22): 4624-4634. (in Chinese)

[6]张卉, 王小珂, 马世俊. 转座元件mariner. 遗传, 2004, 26(5): 756-762.

Zhang H, Wang X K, Ma S J. Transposable element mariner. Hereditas, 2004, 26(5): 756-762. (in Chinese)

[7]罗楚平, 陈志谊, 刘永峰, 张杰, 刘邮洲, 王晓宇, 聂亚锋. 转座子诱变生防枯草芽胞杆菌B-916及抗真菌活性相关基因的克隆. 农业生物技术学报, 2009, 17(4): 728-734.

Luo C P, Chen Z Y, Liu Y F, Zhang J, Liu Y Z, Wang X Y, Nie Y F. Screening mutants of biocontrol Bacillus subtilis B-916 by transposon tagging and cloning of the genes relative to the organism's antifungal activities. Journal of Agricultural Biotechology, 2009, 17(4): 728-734. (in Chinese)

[8]Le Breton Y, Mohapatra N P, Haldenwang W G. In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon. Applied and Environmental Microbiology, 2006, 72(1): 327-333.

[9]Xia Y F, Xie S S, Ma X, Wu H J, Wang X, Gao X W. The purl gene of Bacillus subtilis is associated with nematicidal activity. FEMS Microbiology Letters, 2011, 322(2): 99-107.

[10]Saifuddin N, Wong C W, Nur Yasumira A. Rapid biosynthesis of silver nanoparticles using culture supernatant of bacteria with microwave irradiation. E-Journal of Chemistry, 2009, 6(1): 61-70.

[11]马欣, 刘俊, 乔俊卿, 伍辉军, 高学文. 利用转座子TnYLB-1构建枯草芽胞杆菌的突变株文库. 南京农业大学学报, 2011, 34(6): 77-81.

Ma X, Liu J, Qiao J Q, Wu H J, Gao X W. Construction of transposition insertion library of Bacillus subtilisby using transposon TnYLB-1. Journal of Nanjing Agricultural University, 2011, 34(6): 77-81. (in Chinese)

[12]罗楚平, 陈志谊, 刘永锋, 张杰, 刘邮洲, 王晓宇, 聂亚峰. 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)

[13]Liu J, He D, Li X Z, Gao S F , Wu H J, Liu W Z, Gao X W, Zhou T. γ-polyglutamic acid (γ-PGA) produced by Bacillus amyloliquefaciens C06 promoting its colonization on fruit surface. International Journal of Food Microbiology, 2010, 142(1/2): 190-197. 

[14]Cui L K, Yin W X, Dong S M, Wang Y C. Analysis of polymorphism and transcription of the effector gene Avr1b in Phytophthora sojae isolates from China virulent to Rps1b. Molecular Plant Pathology, 2011, 13(2): 114-122.

[15]WeinrauchY, Msadek T, Kunst F, Dubnau D. Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis. Journal of Bacteriology, 1991, 173(18): 5685-5693.

[16]Piazza F, Tortosa P, DubnauD. Mutational analysis and membrane topology of ComP, a quorum-sensing histidine kinase of Bacillus subtilis controlling competence development. Journal of Bacteriology, 1999, 181(15): 4540-4548.

[17]王晓宇, 罗楚平, 陈志谊, 刘永锋, 刘邮洲, 聂亚峰, 于俊杰, 尹小乐. 枯草芽胞杆菌 Bs-916 的全基因组分析. 中国农业科学, 2011, 44(23): 4807-4814.

Wang X Y, Luo C P, Chen Z Y, Liu Y F, Liu Y Z, Nie Y F, Yu J J, Yin X L. The complete genome sequence of the gram-positive bacterium Bacillus subtils Bs-916. Scientia Agricultura Sinica, 2011, 44(23): 4807-4814. (in Chinese)

[18]Mata M T, Baquero F, Pérez D. A multidrug efflux transporter in Listeria monocytogenes. FEMS Microbiology Letters, 2000, 187(2): 185-188.

[19]Kurihara S,  Suzuki H,  Oshida M,  Benno Y. A novel putrescine importer required for type 1 Pili-driven surface motility induced by extracellular putrescine in Escherichia coli K-12. The Journal of Biological Chemistry, 2011, 286(12): 10185-10192. 

[20]Iyoda S, Kamidoi T, Hirose K, Kutsukake K, Watanabe H. A flagellar gene fliZ regulates the expression of invasion genes and virulence phenotype in Salmonella enterica serovar Typhimurium. Microbial Pathogenesis, 2001, 30(2): 81-90. 

[21]Lampe D J, Akerley B J, Rubin E J, Mekalanos J J, Robertson H M. Hyperactive transsposase mutants of the Himar1 mariner transposition. Proceedings of the Natural Academic of Sciences of the United States of America, 1999, 96(20): 11428-11433.

[22]Picardeau M. Transposition of fly mariner elements into bacteria as a genetic tool for mutagenesis. Genetica, 2010, 138: 551-558. 

[23]Tong S, Porco A, Isturiz T, Conway T. Cloning and molecular genetic characterization of the Escherichia coli gntR, gntK, and gntU genes of GntI, the main system for gluconate metabolism. Journal of Bacteriology, 1996, 178(11): 3260-3269.
[1] ZHUANG LiHua, LUO Lei, ZHAO ChunFang, WANG JiZhong, ZHANG YaDong, HE Lei. Identification and Gene Mapping of Rice Grain Shape Mutant sgd13 [J]. Scientia Agricultura Sinica, 2025, 58(24): 5097-5109.
[2] LI YaYu, WANG XinLiang, ZHOU JinHuan, LI ChuXin, LI JiaXin, TIAN XuBin, SONG Zhen. Construction and Infectivity Identification of Genome-Length cDNA of Citrus Psorosis Virus [J]. Scientia Agricultura Sinica, 2025, 58(17): 3451-3460.
[3] LIU ChenXi, ZHAO BingBing, SHI ZhiBin, WANG ShiDa, WANG JingFei. Construction of Infectious Clones for Canine Parvovirus CPV-2c SX-LC Strain and Virus Rescue [J]. Scientia Agricultura Sinica, 2025, 58(17): 3561-3570.
[4] LEI JianFeng, YOU YangZi, ZHANG JinEn, DAI PeiHong, YU Li, DU ZhengYang, LI Yue, LIU XiaoDong. Screening of High-Efficient sgRNA for Targeted Knockout of GhAGL16 Gene in Cotton [J]. Scientia Agricultura Sinica, 2024, 57(6): 1023-1033.
[5] WANG Yuan, DU MengDan, LI ZhengGang, SHE XiaoMan, YU Lin, LAN GuoBing, DING ShanWen, HE ZiFu, TANG YaFei. Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province [J]. Scientia Agricultura Sinica, 2024, 57(12): 2350-2363.
[6] ZHANG Jian, ZHAO BinSen, FENG Hao, HUANG LiLi. Function and Mechanism Analysis of Vm-milRN7 Regulating the Pathogenicity of Valsa mali [J]. Scientia Agricultura Sinica, 2024, 57(10): 1930-1942.
[7] GAO XiaoXiao, TU LiQin, YANG Liu, LIU YaNan, GAO DanNa, SUN Feng, LI Shuo, ZHANG SongBai, JI YingHua. Construction of an Infectious Clone of Tobacco Mild Green Mosaic Virus Isolate Infecting Pepper from Jiangsu Based on Genomic Clone [J]. Scientia Agricultura Sinica, 2023, 56(8): 1494-1502.
[8] WANG ZhuangZhuang, DONG ShaoYun, ZHOU Qi, MIAO Han, LIU XiaoPing, XU KuiPeng, GU XingFang, ZHANG ShengPing. Cloning and Analysis of Key Genes for Vitamin C Synthesis in Cucumber Fruit [J]. Scientia Agricultura Sinica, 2023, 56(3): 508-518.
[9] WANG XiaoYang, PENG Zhen, XING AiShuang, ZHAO YingRui, MA XinLi, LIU Fang, DU XiongMing, HE ShouPu. Identification and Expression Analysis of Fuzz Fiber Development Related Long Noncoding RNAs in Gossypium arboreum [J]. Scientia Agricultura Sinica, 2023, 56(23): 4565-4584.
[10] DU BingChen, WANG Ming, LIU ChunGuo, WANG ShiDa, WEI XinYu, LU YaMan, SUN ZhenZhao, LIU ZaiSi, WEI LiLi, WANG JingFei, YANG DeCheng. Construction of Infectious cDNA Clone of GETV SC483 Strain [J]. Scientia Agricultura Sinica, 2023, 56(17): 3479-3486.
[11] JIANG QiQi,XU JianJian,SU Yue,ZHANG Qi,CAO Peng,SONG ChenHu,LI ZhongAn,SONG Zhen. Construction and Application of Infectious Clone of Citrus Yellow Mosaic Virus [J]. Scientia Agricultura Sinica, 2022, 55(24): 4840-4850.
[12] LI ZhengGang,TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,HE ZiFu. Molecular Characteristics and Pathogenicity Analysis of Youcai Mosaic Virus Guangdong Isolate Infecting Radish [J]. Scientia Agricultura Sinica, 2022, 55(14): 2752-2761.
[13] GUO FengHui,DING Yong,JI Lei,LI XianSong,LI XiLiang,HOU XiangYang. The Response of Leymus chinensis Cloned Offspring to Mowing [J]. Scientia Agricultura Sinica, 2022, 55(11): 2257-2264.
[14] YUAN JingLi,ZHENG HongLi,LIANG XianLi,MEI Jun,YU DongLiang,SUN YuQiang,KE LiPing. Influence of Anthocyanin Biosynthesis on Leaf and Fiber Color of Gossypium hirsutum L. [J]. Scientia Agricultura Sinica, 2021, 54(9): 1846-1855.
[15] JiaQi WANG,YuHong DONG,JuLing JIANG,JianNing QIAN,WenTao WEI,GuoLiang SONG,JinBo JIAO,XinXin GUAN,GuoBiao JI,YeXin ZHANG. Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process [J]. Scientia Agricultura Sinica, 2021, 54(6): 1280-1287.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd