芽孢杆菌LM基于全基因组的分类鉴定及抑菌原理的研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1303

摘要/Abstract

摘要：

对一株抗黑曲霉活性的芽孢杆菌进行了基因组和超高效液相色谱质谱（UPLC-Q-TOF-MS）分析研究。结合形态特征、16S rRNA序列和gyrA基因聚类分析,初步鉴定菌株为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）;全基因组分析表明菌株基因组大小为 4 000 885 bp,GC含量为46.24%;antiSMASH分析表明基因组中含11个次级代谢产物基因簇,大多编码抗菌物质;UPLC-Q-TOF-MS分析发现10个化合物的分子量与antiSMASH预测的3个基因簇编码的化合物分子量相同,为表面活性素（surfactin）、伊枯草菌素（iturin）和芬荠素（fengycin）等脂肽类抗生素同系物,推测解淀粉芽孢杆菌的抗黑曲霉活性与这3类物质有关。

关键词: 解淀粉芽孢杆菌, 抑菌活性, 基因组, 次级代谢基因簇, 超高效液相色谱质谱

Abstract:

Bacillus LM,which inhibits Aspergillus niger,was analyzed with genomics and UPLC-Q-TOF-MS. Combined with morphological characteristics,16S rRNA sequence and gyrA gene cluster analysis,the strain was primarily identified as Bacillus amyloliquefaciens. The analysis of whole genome showed that the genome size of the strain was 4 000 885 bp and GC content was 46.24%. The analysis of antiSMASH demonstrated that there were 11 secondary metabolite gene clusters and most of them encoded antibacterial substances. UPLC-Q-TOF-MS analysis revealed that the molecular weights of 10 chemical compounds were consistent with those by 3 predicted gene clusters via antiSMASH,which were lipopeptide antibiotics-like surfactin,iturin and fengycin. It could be speculated that the B. amyloliquefaciens activity of suppressing A. niger was associated with those 3 substances.

Key words: Bacillus amyloliquefaciens, antibacterial activity, genome, secondary metabolism gene cluster, UPLC-Q-TOF-MS

蔡国磊, 陆小凯, 娄水珠, 杨海英, 杜刚. 芽孢杆菌LM基于全基因组的分类鉴定及抑菌原理的研究[J]. 生物技术通报, 2021, 37(8): 176-185.

CAI Guo-lei, LU Xiao-kai, LOU Shui-zhu, YANG Hai-ying, DU Gang. Classification and Identification of Bacillus LM Based on Whole Genome and Study on Its Antibacterial Principle[J]. Biotechnology Bulletin, 2021, 37(8): 176-185.

图/表 11

参考文献 23

[1]	梁晓琳, 孙莉, 张娟, 等. 利用 Bacillus amyloliquefaciensSQR9 研制复合微生物肥料[J]. 土壤, 2015, 47(3):558-563.
	Liang XL, Sun L, Zhang J, et al. Utilization of Bacillus amyloliquefaciensSQR9 for developing compound microbial fertilizer[J]. Soils, 2015, 47(3):558-563.
[2]	张楠, 吴凯, 沈怡斐, 等. 根际益生菌解淀粉芽孢杆菌SQR9在香蕉根表的定殖行为研究[J]. 南京农业大学学报, 2014, 37(6):59-65.
	Zhang N, WU K, Shen YF, et al. Investigation of the colonization patterns of plant growth-promoting rhizobacteria Bacillus amyloliquefaciensSQR9 on banana roots[J]. Journal of Nanjing Agricultural University, 2014, 37(6):59-65.
[3]	杜丽华, 张维瑞, 袁王俊. 解淀粉芽孢杆菌次生代谢产物的研究进展[J]. 科技资讯, 2018, 16(6):128-130.
	Du LH, Zhang WR, Yuan WJ. Research progress of secondary metabolites of Bacillus amyloliquefaciens[J]. Science and Technology Information, 2018, 16(6):128-130.
[4]	高学文, 姚仕义, Huong P, 等. 枯草芽孢杆菌B2菌株产生的抑菌活性物质分析[J]. 中国生物防治, 2003, 19(4):175-179.
	Gao XW, Yao SY, Huong P, et al. Analysis of antibacterial substances produced by Bacillus subtilis B2 strain[J]. Chinese Journal of Biological Control, 2003, 19(4):175-179.
[5]	Priest FG, Goodfellow M, Shute LA, et al. Bacillus amyloliquefaciens. sp. nov. nom. rev[J]. International Journal of Systematic and Evolutionary Microbiology, 1987, 37(1):69-71.
[6]	夏京津, 陈建武, 宋怿, 等. 解淀粉芽孢杆菌HE活性成分鉴定及抗菌特性分析[J]. 南方水产科学, 2019, 15(3):41-49.
	Xia JJ, Chen JW, Song Y, et al. Identification of antibacterial substances from Bacillus amyloliquefaciens HE and analysis of antibacterial characteristics[J]. Southern Fisheries Science, 2019, 15(3):41-49.
[7]	戚家明, 孙杉杉, 张东旭等. 芽孢杆菌BS-6基于全基因组数据的分类鉴定及拮抗能力分析[J]. 生物技术通报, 2019, 35(10):111-118.
	Qi JM, Sun SS, Zhang DG, et al. Identification and biocontrol activity analysis of Bacillus sp. BS-6 based on genome-wide data[J]. Biotechnology Bulletin, 2019, 35(10):111-118.
[8]	Arima K, Kakinuma A, Tamura G. Surfactin a crystalline peptidelipid surfactant produced by Bacillus subtilis:isolation characterization and its inhibition of fibrin clot formation[J]. Biochem Biophys Res Commun, 1968, 31(3):488-494. doi: 10.1016/0006-291X(68)90503-2 URL
[9]	Kakinuma A, Sugino H, Isono M, et al. Determination of fatty acid in surfactin and elucidation of the total structure of surfactin[J]. Agr Biol Chem, 1969, 33(6):973-976. doi: 10.1080/00021369.1969.10859409 URL
[10]	王志远, 吴兴兴, 吴毅歆, 等. 解淀粉芽孢杆菌B9601-Y2抗性基因标记及其在作物根部的定殖能力[J]. 华中农业大学学报, 2012, 31(3):313-319.
	Wang ZY, Wu XX, Wu YX, et al. Resistance genes labeling and colonization ability of a biocontrol agent B9601-Y2 of Bacillus amyloliquefaciens in crop rhizospheres[J]. Journal of Huazhong Agricultural University, 2012, 31(3):313-319.
[11]	Nguyen ATV, Nguyen DV, Tran MT, et al. Isolation and characterization of Bacillus subtilis CH16 strain from chicken gastrointestinal tracts for use as a feed supplement to promote weight gain in broilers[J]. Letters in Applied Microbiology, 2015, 60(6):580-588. doi: 10.1111/lam.12411 pmid: 25754534
[12]	Kim BJ, Lee SH, Lyu MA, et al. Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene(rpoB)[J]. Journal of Clinical Microbiology, 1999, 37(6):1714. pmid: 10325313
[13]	Wang LT, Lee FL, Tai CJ, et al. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group[J]. International Journal of Systematic and Volutionary Microbiology, 2007, 57(8):1846-1850.
[14]	陈成, 崔堂兵, 于平儒. 一株抗真菌的解淀粉芽孢杆菌的鉴定及其抗菌性研究[J]. 现代食品科技, 2011, 27(1):36-39.
	Chen C, Cui TB, Yu PR. Identification of an anti-fungal strain of Amyloliquefaciens Bacillus and the properties of the antifungal substance[J]. Modern Food Science and Technology, 2011, 27(1):36-39.
[15]	王继华, 徐世强, 张木清. 解淀粉芽孢杆菌的研究进展[J]. 亚热带农业研究, 2017, 13(3):191-195.
	Wang JH, Xu SG, Zhang MQ. Research progress on Bacillus amyloliquefaciens[J]. Subtropical Agricultural Research, 2017, 13(3):191-195.
[16]	李雪飞, 郭丽琼, 叶志伟, 等. 芒果源植物乳杆菌FMNP01的全基因组测序及序列分析[J]. 中国食品学报, 2018, 18(5):232-238.
	Li XF, Guo LQ, Ye ZW, et al. Whole-genome sequencing and analysis of Lactobacillus plantarum FMNP01 from fresh mangifera indica L[J]. Journal of Chinese Institute of Food Science and Technology, 2018, 18(5):232-238.
[17]	贾慧慧, 王超男, 魏艳敏, 等. 解淀粉芽胞杆菌BJ-6基因组测序与抗菌代谢产物合成基因分析[J]. 北京农学院学报, 2020, 35(3):64-68.
	Jia HH, Wang CN, Wei YM, et al. Whole genome sequences of Bacillus amyloliquefaciens BJ-6 and analysis of biosynjournal genes of antibiotic metabolite[J]. Journal of Beijing University of Agriculture, 2020, 35(3):64-68.
[18]	Koumoutsi A, Chen XH, Henne A, et al. Structural and functional characterization of gene clusters directing nonribosomal synjournal of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens Strain FZB42[J]. Journal of Bacteriology, 2004, 186(4):1084-1096. doi: 10.1128/JB.186.4.1084-1096.2004 URL
[19]	陈华, 袁成凌, 蔡克周, 等. 枯草芽孢杆菌JA产生的脂肽类抗生素-iturin A的纯化及电喷雾质谱鉴定[J]. 微生物学报, 2008(1):116-120.
	Chen H, Yuan CL, Cai KZ, et al. Purification and identification of iturin A from Bacillus subtilis JA by electrospray ionization mass spectrometry[J]. Acta Microbiologica Sinica, 2008(1):116-120.
[20]	董伟欣, 李宝庆, 李社增, 等. 脂肽类抗生素 fengycin 在枯草芽胞杆菌 NCD-2 菌株抑制番茄灰霉病菌中的功能分析[J]. 植物病理学报, 2013, 43(4):401-410.
	Dong WX, Li BQ, Li SZ, et al. The fengycin lipopeptides are major components in the Bacillus subtilis strain NCD-2 against the growth of Botrytis cinereal[J]. Acta Phytopathologica Sinica, 2013, 43(4):401-410.
[21]	张荣胜, 王晓宇, 罗楚平, 等. 解淀粉芽孢杆菌Lx-11产脂肽类物质鉴定及表面活性素对水稻细菌性条斑病的防治作用[J]. 中国农业科学, 2013, 46(10):2014-2021.
	Zhang RS, Wang XY, Luo CP, et al. Identification of the lipopeptides from Bacillus amyloliquefaciensLx-11 and biocontrol efficacy of surfactin against bacterial leaf streak[J]. Scientia Agricultura Sinica, 2013, 46(10):2014-2021.
[22]	胡陈云, 李勇, 刘敏, 等. 枯草芽孢杆菌ge25对两种人参病原菌的抑制作用及脂肽类抑菌代谢产物的鉴定[J]. 中国生物防治学报, 2015, 31(3):386-393.
	Hu CY, Li Y, Liu M, et al. Antagonism of Bacillus subtilis ge25 against two kinds of ginseng pathogens and identification of antifungal lipopeptide metabolites[J]. Chinese Journal of Biological Control, 2015, 31(3):386-393.
[23]	胡陈云, 李勇, 刘敏, 等. 枯草芽孢杆菌ge25菌株发酵及其抑菌活性成分的初步研究[J]. 中国中药志, 2014, 39(14):2624-2628.
	Hu CY, Li Y, Liu M, et al. Fermentation of Bacillus subtilis ge25 strain and its antibacterial active components[J]. China Journal of Chinese Materia Medica, 2014, 39(14):2624-2628.

基因组组装 Genome assembly	大小Size
总支架数 Total number of scaffold /个	97
总重叠群数 Total number of contig /个	97
最大长度 The maximum length /bp	365 023
最小长度 The Minimum length /bp	300
支架大小 The scaffold size /bp	4 000 885
重叠群大小The contig size /bp	4 000 885
N50支架大小 Scaffold N50 size /bp	130 035
N50重叠群大小 Contig N50 size /bp	130 035
N90 支架大小Scaffold N90 size /bp	41 219
N90 重叠群大小Contig N90 size /bp	41 219
GC含量 GC content /%	46.24

基因组组装 Genome assembly	大小Size
总支架数 Total number of scaffold /个	97
总重叠群数 Total number of contig /个	97
最大长度 The maximum length /bp	365 023
最小长度 The Minimum length /bp	300
支架大小 The scaffold size /bp	4 000 885
重叠群大小The contig size /bp	4 000 885
N50支架大小 Scaffold N50 size /bp	130 035
N50重叠群大小 Contig N50 size /bp	130 035
N90 支架大小Scaffold N90 size /bp	41 219
N90 重叠群大小Contig N90 size /bp	41 219
GC含量 GC content /%	46.24

菌株 Strain	基因组大小 Genome size /bp	GC含量 GC content /%
Bacillus amyloliquefaciens WF02	4.03	46.50
Bacillus amyloliquefaciens ZJU1	4.06	46.40
Bacillus amyloliquefaciens YP6	4.01	45.90
Bacillus amyloliquefaciens ALB79	3.98	46.40
Bacillus amyloliquefaciens Y14	3.96	46.40
Bacillus amyloliquefaciens WS-8	3.93	46.50
Bacillus amyloliquefaciens MT45	3.90	46.10
Bacillus amyloliquefaciens KC41	4.12	46.00
Bacillus amyloliquefaciens T-5	4.04	46.50
Bacillus amyloliquefaciens LM	4.00	46.24

菌株 Strain	基因组大小 Genome size /bp	GC含量 GC content /%
Bacillus amyloliquefaciens WF02	4.03	46.50
Bacillus amyloliquefaciens ZJU1	4.06	46.40
Bacillus amyloliquefaciens YP6	4.01	45.90
Bacillus amyloliquefaciens ALB79	3.98	46.40
Bacillus amyloliquefaciens Y14	3.96	46.40
Bacillus amyloliquefaciens WS-8	3.93	46.50
Bacillus amyloliquefaciens MT45	3.90	46.10
Bacillus amyloliquefaciens KC41	4.12	46.00
Bacillus amyloliquefaciens T-5	4.04	46.50
Bacillus amyloliquefaciens LM	4.00	46.24

Cluster	Function category
3	Bacilysin biosynthetic（细菌溶素生物合成）
4	Bacillibactin biosynthetic（杆菌素生物合成）
20	Macrolactin H biosynthetic（大环内酰亚胺H生物合成）
9/23/37	Difficidin biosynthetic（大环内酯类抗生素生物合成）
1	Bacillaene biosynthetic（杆菌烯生物合成）
1	Bacillomycin D biosynthetic（杆菌霉素D生物合成）
2	Plantazolicin biosynthetic（车前唑啉生物合成）
28	Plipastatin biosynthetic（巴斯他汀生物合成）
10/33/41	Surfactin biosynthetic（表面活性素生物合成）
30/38/42	Fengycin biosynthetic（芬荠素生物合成）
16	Butirosin A biosynthetic（丁酸普菌素A生物合成）