• ISSN 1000-0615
  • CN 31-1283/S
Volume 46 Issue 2
Feb.  2022
Article Contents
Turn off MathJax

Citation:

Genomic analysis of Bacillus velezensis LF01 strain and the biocontrol effect of its secondary metabolites

  • Bacillus velezensis is a novel species in the Bacillus genus which exhibits a broad spectrum of antimicrobial activities because it produces various secondary metabolites. The current research on B. velezensis mainly focuses on its use for the promotion of the growth of animals and plants, for antagonizing pathogens, and on investigating its gene cluster which plays significant roles in biological control, drug research and development, and food fermentation. The aims of this study are to explore the gene clusters related to antagonistic substances of B. velezensis LF01 strain based on the whole genome sequencing, and to evaluate the biological safety and biocontrol effect of the antagonistic substances of LF01 strain. In this study, the whole genome of LF01 strain was sequenced based on the third generation Nanopore sequencing platform, and its taxonomic status was identified based on ANI and DDH online analysis, and the genetic evolution at genome level. The gene clusters related to antagonistic substances of LF01 strain was analyzed based on antiSMASH software. The hydrolysis activity of LF01 strain to carbohydrate was analyzed based on CAZyme database. The toxicity of the antagonistic substances of LF01 strain to Oreochromis niloticus and Danio rerio was evaluated by intraperitoneal injection. The biocontrol effect of the antagonistic substances of LF01 strain to O. niloticus was evaluated by artificial infection after 5 d of feeding O. niloticus. The complete genome of LF01 strain had a total length of 3974023 bp and the GC content was 46.56%, and it contains 3843 coding genes. Compared to B. velezensis, the ANI and DDH values of LF01 strain were ≥97.66% and ≥80.10%, respectively. Furthermore, the genomic evolution analysis showed that LF01 strain was clustered with B. velezensis. These results indicated that LF01 strain was identified as B. velezensis at the genomic level. The LF01 strain contains nine gene clusters related to antagonistic substances, including bacillaene, bacillibactin, bacillomycin D, bacilysin, difficidin, fengycin, macrolactin H, plantazolicin and surfactin, which accounts for 8.83% of the total genome sequences. In addition, LF01 strain contains a large number of CAZymes related to the degradation of cellulose, hemicellulose, starch, chitin, pectin, peptidoglycan and glucan. The antagonistic substances of the LF01 strain showed high biological safety to O. niloticus and D. rerio, and they significantly enhanced the resistance of O. niloticus against S. agalactiae infection. In conclusion, LF01 strain contains a large number of gene clusters for biosynthesis of secondary metabolites related to antagonistic substances. The antagonistic substances were found to be safe for O. niloticus and D. rerio, and feeding O. niloticus with them significantly improved the diseases resistance of fish. Therefore, the antagonistic substances of LF01 strain have a broad development and application prospect as biocontrol agents to improve bacteria diseases control in aquaculture.
  • 加载中
  • [1] Ruiz-García C, Béjar V, Martínez-Checa F, et al. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain[J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55(1): 191-195. doi: 10.1099/ijs.0.63310-0
    [2] 陆洋, 郁二蒙, 谢骏, 等. 添加芽孢杆菌对池塘中理化因子和细菌群落结构的影响分析[J]. 水产学报, 2020, 44(1): 130-141.Lu Y, Yu E M, Xie J,et al. Analysis of the effect of adding Bacillus on the physicochemical factors and bacterial community structure in ponds[J]. Journal of Fisheries of China, 2020, 44(1): 130-141 (in Chinese).
    [3] 张德锋, 高艳侠, 王亚军, 等. 贝莱斯芽孢杆菌的分类、拮抗功能及其应用研究进展[J]. 微生物学通报, 2020, 47(11): 3634-3649.Zhang D F, Gao Y X, Wang Y J, et al. Advances in taxonomy, antagonistic function and application of Bacillus velezensis[J]. Microbiology China, 2020, 47(11): 3634-3649 (in Chinese).
    [4] 王金燕, 李彬, 王印庚, 等. 刺参养殖池塘一株贝莱斯芽孢杆菌的分离及其生理特性[J]. 中国水产科学, 2018, 25(3): 567-575. doi: 10.3724/SP.J.1118.2018.17383Wang J Y, Li B, Wang Y G, et al. Screening and characteristic analysis of Bacillus velezensis from sea cucumber (Apostichopus japonicus) ponds[J]. Journal of Fishery Sciences of China, 2018, 25(3): 567-575 (in Chinese). doi: 10.3724/SP.J.1118.2018.17383
    [5] Zhang D F, Gao Y X, Ke X L, et al. Bacillus velezensis LF01: in vitro antimicrobial activity against fish pathogens, growth performance enhancement, and disease resistance against streptococcosis in Nile tilapia (Oreochromis niloticus)[J]. Applied Microbiology and Biotechnology, 2019, 103(21-22): 9023-9035. doi: 10.1007/s00253-019-10176-8
    [6] Yi Y L, Zhang Z H, Zhao F, et al. Probiotic potential of Bacillus velezensis JW: antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus[J]. Fish & Shellfish Immunology, 2018, 78: 322-330.
    [7] 杨可, 郑柯斌, 黄晓慧, 等. 海洋生境贝莱斯芽孢杆菌TCS001的鉴定及抑真菌活性[J]. 农药学学报, 2018, 20(3): 333-339.Yang K, Zheng K B, Huang X H, et al. Identification and antifungal activity of marine Bacillus velezensis strain TCS001[J]. Chinese Journal of Pesticide Science, 2018, 20(3): 333-339 (in Chinese).
    [8] 沙月霞, 隋书婷, 曾庆超, 等. 贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力[J]. 中国农业科学, 2019, 52(11): 1908-1917. doi: 10.3864/j.issn.0578-1752.2019.11.006Sha Y X, Sui S T, Zeng Q C, et al. Biocontrol potential of Bacillus velezensis strain E69 against rice blast and other fungal diseases[J]. Scientia Agricultura Sinica, 2019, 52(11): 1908-1917 (in Chinese). doi: 10.3864/j.issn.0578-1752.2019.11.006
    [9] 杨胜清. 贝莱斯芽孢杆菌S6的鉴定、发酵条件优化及其生防作用研究[D]. 长春: 吉林农业大学, 2017.Yang S Q. Identification, optimization of fermentation conditions of Bacillus velezensis strain S6 and its biocontrol effect[D]. Changchun: Jilin Agricultural University, 2017 (in Chinese).
    [10] 杜淑涛, 李术娜, 朱宝成. 白菜黑斑病拮抗细菌Bacillus velezensis DL-59的筛选鉴定及田间防效实验[J]. 河北农业大学学报, 2010, 33(6): 51-56. doi: 10.3969/j.issn.1000-1573.2010.06.011Du S T, Li S N, Zhu B C. Screening and identification of antagonistic strain DL-59 of Bacillus velezensis against Alternaria brassicae and biocontrol efficiency[J]. Journal of Agricultural University of Hebei, 2010, 33(6): 51-56 (in Chinese). doi: 10.3969/j.issn.1000-1573.2010.06.011
    [11] Li J, Wu Z B, Zhang Z, et al. Effects of potential probiotic Bacillus velezensis K2 on growth, immunity and resistance to Vibrio harveyi infection of hybrid grouper (Epinephelus lanceolatus ♂×E. fuscoguttatus ♀)[J]. Fish & Shellfish Immunology, 2019, 93: 1047-1055.
    [12] Thurlow C M, Williams M A, Carrias A, et al. Bacillus velezensis AP193 exerts probiotic effects in channel catfish (Ictalurus punctatus) and reduces aquaculture pond eutrophication[J]. Aquaculture, 2019, 503: 347-356. doi: 10.1016/j.aquaculture.2018.11.051
    [13] 熊汉琴, 蔡燕飞, 郭真真, 等. 芽孢杆菌脂肽类抗生素的研究进展[J]. 湖北农业科学, 2015, 54(12): 2817-2821.Xiong H Q, Cai Y F, Guo Z Z, et al. Advances of lipopeptides in Bacillus[J]. Hubei Agricultural Sciences, 2015, 54(12): 2817-2821 (in Chinese).
    [14] 单安山, 田昊天, 邵长轩, 等. 抗菌肽抗细菌机理研究进展[J]. 东北农业大学学报, 2018, 49(3): 84-94. doi: 10.3969/j.issn.1005-9369.2018.03.010Shan A S, Tian H T, Shao C X, et al. Research advance on antibacterial mechanism of antimicrobial peptides[J]. Journal of Northeast Agricultural University, 2018, 49(3): 84-94 (in Chinese). doi: 10.3969/j.issn.1005-9369.2018.03.010
    [15] 曾欣, 张亚惠, 迟惠荣, 等. 温郁金内生拮抗细菌B-11的分离及其抑菌活性[J]. 微生物学通报, 2019, 46(5): 1018-1029.Zeng X, Zhang Y H, Chi H R, et al. Antimicrobial activity of endophytic bacterium strain B-11 isolated from Curcuma wenyujin[J]. Microbiology China, 2019, 46(5): 1018-1029 (in Chinese).
    [16] 陈龙, 吴兴利, 闫晓刚, 等. 贝莱斯芽孢杆菌的分类、次级代谢产物及应用[J]. 家畜生态学报, 2020, 41(1): 1-8. doi: 10.3969/j.issn.1673-1182.2020.01.001Chen L, Wu X L, Yan X G, et al. The classification, secondary metabolites and application of Bacillus velezensis[J]. Acta Ecologiae Animalis Domastici, 2020, 41(1): 1-8 (in Chinese). doi: 10.3969/j.issn.1673-1182.2020.01.001
    [17] 陆洋,郁二蒙,王广军,等. 添加芽孢杆菌对草鱼池塘中真核微生物的影响[J]. 上海海洋大学学报, 2020, 29(2): 218-225.Lu Y, Yu E M, Wang G J, et al. Effect of Bacillus on eukaryotic microorganism in grass carp ponds[J]. Journal of Shanghai Ocean University, 2020, 29(2): 218-225 (in Chinese).
    [18] Koren S, Walenz B P, Berlin K, et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation[J]. Genome Research, 2017, 27(5): 722-736. doi: 10.1101/gr.215087.116
    [19] Walker B J, Abeel T, Shea T, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement[J]. PLoS One, 2014, 9(11): e112963. doi: 10.1371/journal.pone.0112963
    [20] Hyatt D, Chen G L, LoCascio P F, et al. Prodigal: prokaryotic gene recognition and translation initiation site identification[J]. BMC Bioinformatics, 2010, 11(1): 119. doi: 10.1186/1471-2105-11-119
    [21] Nawrocki E P, Eddy S R. Infernal 1.1: 100-fold faster RNA homology searches[J]. Bioinformatics, 2013, 29(22): 2933-2935. doi: 10.1093/bioinformatics/btt509
    [22] Nawrocki E P, Burge S W, Bateman A, et al. Rfam 12.0: updates to the RNA families database[J]. Nucleic Acids Research, 2015, 43(D1): D130-D137. doi: 10.1093/nar/gku1063
    [23] She R, Chu J S C, Wang K, et al. GenBlastA: enabling BLAST to identify homologous gene sequences[J]. Genome Research, 2009, 19(1): 143-149.
    [24] Birney E, Clamp M, Durbin R. GeneWise and genomewise[J]. Genome Research, 2004, 14(5): 988-995. doi: 10.1101/gr.1865504
    [25] 惠文彦, 张和平. 基因组分析方法在微生物分类学中的应用[J]. 微生物学通报, 2016, 43(5): 1136-1142.Hui W Y, Zhang H P. Application of genomic analysis in microbial taxonomy[J]. Microbiology China, 2016, 43(5): 1136-1142 (in Chinese).
    [26] Guindon S, Dufayard J F, Lefort V, et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0[J]. Systematic Biology, 2010, 59(3): 307-321. doi: 10.1093/sysbio/syq010
    [27] Eddy S R. Profile hidden Markov models[J]. Bioinformatics, 1998, 14(9): 755-763. doi: 10.1093/bioinformatics/14.9.755
    [28] 金清, 肖明. 新型抗菌肽——表面活性素、伊枯草菌素和丰原素[J]. 微生物与感染, 2018, 13(1): 56-64. doi: 10.3969/j.issn.1673-6184.2018.01.010Jin Q, Xiao M. Novel antimicrobial peptides: surfactin, iturin and fengycin[J]. Journal of Microbes and Infections, 2018, 13(1): 56-64 (in Chinese). doi: 10.3969/j.issn.1673-6184.2018.01.010
    [29] 张彩文, 程坤, 张欣, 等. 贝莱斯芽胞杆菌(Bacillus velezensis)分类学及功能研究进展[J]. 食品与发酵工业, 2019, 45(17): 258-265.Zhang C W, Cheng K, Zhang X, et al. Taxonomy and functions of Bacillus velezensis: a review[J]. Food and Fermentation Industries, 2019, 45(17): 258-265 (in Chinese).
    [30] Kim S Y, Song H, Sang M K, et al. The complete genome sequence of Bacillus velezensis strain GH1-13 reveals agriculturally beneficial properties and a unique plasmid[J]. Journal of Biotechnology, 2017, 259: 221-227. doi: 10.1016/j.jbiotec.2017.06.1206
    [31] Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008, 16(3): 115-125. doi: 10.1016/j.tim.2007.12.009
    [32] 郅岩. 芽孢杆菌高效合成表面活性素的代谢机制及功能研究[D]. 无锡: 江南大学, 2017.Zhi Y. Metabolic mechanism of highly-efficient biosynthesis of surfactin and its function[D]. Wuxi: Jiangnan University, 2017 (in Chinese).
    [33] 陈志谊. 芽孢杆菌类生物杀菌剂的研发与应用[J]. 中国生物防治学报, 2015, 31(5): 723-732.Chen Z Y. Research and application of bio-fungicide with Bacillus spp.[J]. Chinese Journal of Biological Control, 2015, 31(5): 723-732 (in Chinese).
    [34] Wang T, Liu X H, Wu M B, et al. Molecular insights into the antifungal mechanism of bacilysin[J]. Journal of Molecular Modeling, 2018, 24(5): 118. doi: 10.1007/s00894-018-3645-4
    [35] 吴黎明, 李曦, 伍辉军, 等. 芽胞杆菌抗菌二肽溶杆菌素的研究进展[J]. 南京农业大学学报, 2018, 41(5): 778-783. doi: 10.7685/jnau.201803047Wu L M, Li X, Wu H J, et al. Research advances on bacilysin from Bacillus[J]. Journal of Nanjing Agricultural University, 2018, 41(5): 778-783 (in Chinese). doi: 10.7685/jnau.201803047
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(5) / Tables(2)

Article views(1417) PDF downloads(34) Cited by()

Related
Proportional views

Genomic analysis of Bacillus velezensis LF01 strain and the biocontrol effect of its secondary metabolites

    Corresponding author: REN Yan, renyannj@126.com
    Corresponding author: SHI Cunbin, shicunbin2006@163.com
  • 1. Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou    510380, China
  • 2. Key Laboratory of Aquatic Animal Immune Technology, Guangdong Province, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China

Abstract: Bacillus velezensis is a novel species in the Bacillus genus which exhibits a broad spectrum of antimicrobial activities because it produces various secondary metabolites. The current research on B. velezensis mainly focuses on its use for the promotion of the growth of animals and plants, for antagonizing pathogens, and on investigating its gene cluster which plays significant roles in biological control, drug research and development, and food fermentation. The aims of this study are to explore the gene clusters related to antagonistic substances of B. velezensis LF01 strain based on the whole genome sequencing, and to evaluate the biological safety and biocontrol effect of the antagonistic substances of LF01 strain. In this study, the whole genome of LF01 strain was sequenced based on the third generation Nanopore sequencing platform, and its taxonomic status was identified based on ANI and DDH online analysis, and the genetic evolution at genome level. The gene clusters related to antagonistic substances of LF01 strain was analyzed based on antiSMASH software. The hydrolysis activity of LF01 strain to carbohydrate was analyzed based on CAZyme database. The toxicity of the antagonistic substances of LF01 strain to Oreochromis niloticus and Danio rerio was evaluated by intraperitoneal injection. The biocontrol effect of the antagonistic substances of LF01 strain to O. niloticus was evaluated by artificial infection after 5 d of feeding O. niloticus. The complete genome of LF01 strain had a total length of 3974023 bp and the GC content was 46.56%, and it contains 3843 coding genes. Compared to B. velezensis, the ANI and DDH values of LF01 strain were ≥97.66% and ≥80.10%, respectively. Furthermore, the genomic evolution analysis showed that LF01 strain was clustered with B. velezensis. These results indicated that LF01 strain was identified as B. velezensis at the genomic level. The LF01 strain contains nine gene clusters related to antagonistic substances, including bacillaene, bacillibactin, bacillomycin D, bacilysin, difficidin, fengycin, macrolactin H, plantazolicin and surfactin, which accounts for 8.83% of the total genome sequences. In addition, LF01 strain contains a large number of CAZymes related to the degradation of cellulose, hemicellulose, starch, chitin, pectin, peptidoglycan and glucan. The antagonistic substances of the LF01 strain showed high biological safety to O. niloticus and D. rerio, and they significantly enhanced the resistance of O. niloticus against S. agalactiae infection. In conclusion, LF01 strain contains a large number of gene clusters for biosynthesis of secondary metabolites related to antagonistic substances. The antagonistic substances were found to be safe for O. niloticus and D. rerio, and feeding O. niloticus with them significantly improved the diseases resistance of fish. Therefore, the antagonistic substances of LF01 strain have a broad development and application prospect as biocontrol agents to improve bacteria diseases control in aquaculture.

  • 芽孢杆菌(Bacillus spp.)因其优良的生防作用,在农业生产领域发挥着重要作用。贝莱斯芽孢杆菌(B. velezensis)作为芽孢杆菌属的一个新种[1],该菌具有生长速度快、适应能力强、代谢产物种类丰富、抗菌谱广、生防作用效果好、生物安全性良好等优点受到广泛关注[2-4]。贝莱斯芽孢杆菌不仅对水产动物病原菌如嗜水气单胞菌(Aeromonas hydrophila)、维氏气单胞菌(A. veronii)、迟缓爱德华氏菌(Edwardsiella tarda)、哈维氏弧菌(Vibrio harveyi)、副溶血性弧菌(V. parahaemolyticus)、无乳链球菌(Streptococcus agalactiae)及海豚链球菌(S. iniae)等具有明显的拮抗活性[5-6];对植物病原真菌,如引起黄瓜灰霉病的灰葡萄孢(Botrytis cinerea)、引起黄瓜蔓枯病的西瓜壳二孢菌(Ascochyta citrullina)、引起棉花枯萎病的尖孢镰孢菌(Fusarium oxysporum)、引起香蕉叶斑病的香蕉假尾孢菌(Pseudocercospora musae)、引起番茄灰霉病的灰葡萄孢、引起草莓炭疽病的胶孢炭疽菌(Colletotrichum gloeospoioides)和立枯丝核菌(Rhizoctonia solani)等多种植物真菌性病原也具有较强的抑菌活性[7-8]。此外,贝莱斯芽孢杆菌对农作物病害如番茄早疫病[9]、白菜黑斑病[10]、水稻叶温病[8]等具有良好的田间防控效果;同时,该菌在水产动物病害防控方面也具有广阔的生防应用前景,例如对鲫(Carassius auratus)的嗜水气单胞菌[6]、尼罗罗非鱼(Oreochromis niloticus)的无乳链球菌[5]、杂交石斑鱼(Epinephelus lanceolatus ♂×E. fuscoguttatus ♀)的哈维氏弧菌[11]、斑点叉尾鮰(Ictalurus punctatus)的鮰爱德华氏菌 (E. ictaluri)[12]等病害防控方面均呈现巨大的应用潜力。

    贝莱斯芽孢杆菌能够分泌多种次级代谢产物,其中部分代谢产物具有抑菌和杀菌作用。芽孢杆菌主要通过2种合成途径产生抑菌物质,如由核糖体途径合成的小分子物质以及非核糖体途径合成的多肽类抑菌物质。其中核糖体合成肽类物质如细菌素;非核糖体合成途径产生的抑菌物质主要有脂肽类化合物、聚酮类化合物、多肽类化合物等[3]。细菌素和脂肽类物质因其具有理化性质稳定、抗菌谱广、不易产生耐药性等优点备受医药、农业等领域的青睐[13-14],已成为生物防治研究的重点对象。研究表明,贝莱斯芽孢杆菌分泌产生的蛋白酶、几丁质酶和β-葡聚糖酶等酶类在抑制真菌方面起到重要作用[15]。贝莱斯芽孢杆菌能够分泌纤维素酶、木聚糖酶、果胶酶、淀粉酶和脂肪酶等多种酶类,有望作为动物饲料的酶制剂,可以提高动物饲料消化率,促进动物生长[3, 16]

    前期实验从尼罗罗非鱼肠道中分离到一株贝莱斯芽孢杆菌LF01,证实该菌株具有淀粉酶和蛋白酶活性,并具有广谱拮抗水产常见病原菌的活性[17],饲喂该菌后能够显著提高尼罗罗非鱼对无乳链球菌的抗病力[5]。近年来,随着测序技术革新以及测序费用降低,越来越多的贝莱斯芽孢杆菌基因组序列被公布,这有助于解析贝莱斯芽孢杆菌次级代谢产物的合成途径以及挖掘新的次级代谢产物。本实验为进一步解析LF01菌株次级代谢产物如拮抗物质的种类,实验对该菌进行了全基因组测序,挖掘次级代谢产物基因簇,为揭示其抑菌机制奠定基础。同时,本研究测试了LF01菌株拮抗物质对尼罗罗非鱼的生防作用,为今后开发和利用LF01菌株的代谢产物提供了依据。

1.   材料与方法
  • 实验菌株为贝莱斯芽孢杆菌LF01,保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC 60344。LF01菌株接种至LB培养基中,过夜培养至对数生长期,收集菌体,提取其基因组DNA用于全基因组测序分析。委托北京百迈客生物科技有限公司基于Nanopore测序技术平台进行全基因组测序。

  • LF01菌株的测序序列使用Canu v1.5软件[18]进行序列组装,采用Pilon软件[19]基于Illumina Hiseq测序结果进一步对组装的基因组进行纠错,最终得到准确度更高的基因组序列。

  • 编码基因通过Prodigal软件[20]进行预测;非编码基因如转运RNA (tRNA) 使用tRNAscan-SE进行预测,使用Infernal 1.1软件[21]基于Rfam数据库[22]预测基因组中的核糖体RNA (rRNA) 以及除了tRNA和rRNA之外的其他非编码RNA (ncRNA)。通过GenBlastA软件[23]比对,在基因组上搜寻同源基因,再利用GeneWise软件[24]查找基因序列中提前终止密码子和移码突变,得到假基因(pseudogene)。

  • 利用预测得到的基因组序列与COG、KEGG、Swiss-Prot、TrEMBL及Nr等功能数据库进行Blast比对分析,得到基因组功能注释结果。此外,对COG、KEGG代谢通路富集分析、GO功能富集分析。利用预测得到的蛋白序列与转运蛋白分类数据库(TCDB)、抗生素抗性基因数据库(CRDB)、毒力因子数据库(VFDB)等功能数据库进行Blast比对分析,得到相应的注释结果。

  • 平均核苷酸同源性(average nucleotide identity, ANI)是指2个基因组之间同源基因的相似性。通常认为同一种微生物的ANI值需要达到95%以上[25]。为了进一步确定贝莱斯芽孢杆菌LF01的分类学地位,根据其基因组序列,利用在线ANI (https://www.ezbiocloud.net/tools/ani)分析LF01菌株的分类学地位。DNA-DNA杂交(DNA-DAN hybridization, DDH)是指具有互补碱基序列的DNA分子,可以通过碱基对之间形成氢键等稳定的双链区。利用在线DDH (http://ggdc.dsmz.de/ggdc.php)分析LF01菌株的DDH值,进一步确定其分类学地位。

  • 基于LF01基因组,以贝莱斯芽孢杆菌M75基因组为参考序列,对ATCC14580、DSM7、MT45、XH7、KH2、str.168、FZB42、CC09、G341、LS69、S3-1和LF01菌株进行SNP检测。对每一株菌,按照相同的顺序将所有SNP相连,将获得相同长度的fasta格式序列文件作为输入文件。使用PhyML (version 3.0)软件[26],采用最大似然法(maximum likelihood method, ML)构建系统进化树。

  • 使用antiSMASH软件(version 5.1.2) 在线分析预测LF01菌株基因组中的拮抗物质合成相关基因簇。根据在线初步的预测结果,分别下载相应的抗菌物质合成相关基因,并进行逐一比对分析,确定LF01基因组中的拮抗基因是否发生缺失或突变。此外,根据预测的拮抗物质合成相关基因序列,分析拮抗物质合成相关基因簇占基因组序列全长的比例。

  • CAZy按其功能分类主要包括糖苷水解酶 (GH)、糖基转移酶 (GT)、多糖裂解酶 (PL)、碳水化合物酯酶 (CE)、辅助氧化还原酶 (AA) 以及没有催化活性的碳水化合物结合模块 (CBM)。基于碳水化合物活性酶数据库CAZyme,利用hmmer软件[27]进行碳水化合物酶类基因的功能注释与分析。

  • LF01菌株接种至无菌LB培养基中37 °C过夜培养,然后按照1∶10 (体积比) 的比例接种至5 L的LB培养基中,摇床培养48 h。10 000 r/min离心收集上清液,加入1 mol/L的盐酸,调整pH=2.0,4 °C放置过夜。离心收集沉淀,加入40 mL无菌水,调整pH=7.0,分装至无菌西林瓶中(2 mL/瓶),然后进行真空冷冻干燥。冷冻干燥后的拮抗物质粗提物保存于–20 °C冰箱备用。

    称取LF01菌株的拮抗物质粗提物干粉加入无菌磷酸盐缓冲溶液(PBS),制备成1、5和10 mg/mL注射液。分别吸取10 μL拮抗物质注射液点到脱脂绵羊血琼脂平板上,置于37 °C培养24 h后观察溶血活性。LF01菌株拮抗物质注射液(3个浓度组)分别腹腔注射体质量为(24.5±5.8) g的尼罗罗非鱼,每尾注射0.1 mL,每组20尾。对照组(20尾)腹腔注射0.1 mL的无菌PBS。连续观察2周,记录尼罗罗非鱼的临床症状和死亡情况。拮抗物质注射液(3个浓度组)分别腹腔注射健康斑马鱼 (Danio rerio),20 μL/尾,每组20尾,对照组腹腔注射等体积的无菌PBS。连续观察2周,记录斑马鱼的临床症状以及死亡情况。

  • 选取健康尼罗罗非鱼幼鱼[(24.5±5.8) g]随机分成2组,每组90尾(3个重复,每个重复30尾)。称取上述制备的拮抗物质干粉,加入无菌水溶解后,均匀喷洒至尼罗罗非鱼膨化颗粒饲料(20 mg/kg),晾干20 min后开始饲喂。实验组(ET)尼罗罗非鱼饲喂含有拮抗物质的饲料;对照组(CK)的饲料喷洒等体积的无菌水,晾干20 min后开始饲喂。实验组和对照组的尼罗罗非鱼每日饲喂量为鱼体质量的3%,连续饲喂5 d。

  • 饲喂5 d后的第8 小时进行无乳链球菌人工感染实验。无乳链球菌WC1535株接种至BHI液体培养至对数生长期,收集菌体悬浮于无菌PBS中,并调整其浓度为3.0×108 CFU/mL。实验组和对照组尼罗罗非鱼分别人工腹腔注射无乳链球菌菌液0.1 mL/尾。空白对照组仅注射等体积的无菌PBS。观察并记录尼罗罗非鱼的发病症状以及死亡数、死亡时间,通过SPSS 22.0软件绘制尼罗罗非鱼人工感染后的生存曲线,并采用ONE-ANOVA方法分析2组之间累积死亡率的差异。

2.   结果
  • LF01菌株测序获得128 288 有效序列数(clean reads),其中N50长度为13 822 bp,基因组大小为3 974 023 bp,G+C含量为46.56%。LF01基因组包含3 843个编码基因、19个假基因(pesudogene)、27个rRNA、86个tRNA和79个ncRNA,其基因组圈图如图1所示。LF01菌株基因组的GenBank登录号为CP058216。

    Figure 1.  Circle map of the genome of B. velezensis LF01 strain

    COG数据库分析结果显示,共有2 887个基因获得功能注释,其中氨基酸转运及代谢、转录、碳水化合物转运及代谢、无机离子转运及代谢、细胞壁/膜和被膜生物合成与翻译、核糖体结构和生物合成等相关的基因丰度较高,注释上的基因数分别为350、303、254、212、179和160 (图1)。预测基因注释到TCDB数据库的基因有247个;注释到VFDB数据库的基因为495个。此外,对致病性芽孢杆菌常见的毒力相关基因如溶血素BL基因(hblA/B/C/D)、非溶血性的肠毒素NHE基因(nheA/B/C)、肠毒素T基因(bceT)、细胞毒素K基因(cytK)及呕吐毒素(cereulide)基因(cesAcesHcesPcesTcesBcesCcesD)的比对分析结果显示,LF01菌株的基因组中均不含这些毒力基因,表明该菌不具有表达相关毒力因子的能力,暗示在基因组水平上该菌的致病力非常弱或不具有致病力。LF01菌株基因组注释到抗性基因数据库(ARDB)数据库的基因有5个,分别为杆菌肽耐药基因(bacA)、磷霉素耐药基因 (fosB)、林克霉素耐药基因(lmrB)、四环素耐药基因 (tetL)和多重耐药基因(mdr)。然而,LF01菌株除了对林可霉素中度敏感以及对杆菌肽耐药外,对多种药物均敏感[17],可能是由于这些耐药基因不是关键的耐药基因,对菌株耐药表型的影响有限。

  • 基于ANI在线分析结果显示,LF01菌株与贝莱斯芽孢杆菌的ANI值介于97.66%~99.73%,但是与解淀粉芽孢杆菌(B. amyloliquefaciens)、枯草芽孢杆菌(B. subtilis)、地衣芽孢杆菌(B. licheniformis)和蜡样芽孢杆菌(B. cereus)的ANI值≤94.01% (表1),低于种的分类阈值95%,表明LF01菌株与贝莱斯芽孢杆菌的亲缘关系更近。通过比较DDH值结果显示,LF01菌株与贝莱斯芽孢杆菌的DDH值介于80.10%~98.30%,高于种的分类阈值70%,然而LF01菌株与解淀粉芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌、蜡样芽孢杆菌的DDH值介于19.60%~55.40%,均低于70%,进一步表明LF01菌株与贝莱斯芽孢杆菌的亲缘关系最近。

    菌株 (GenBank序列号)
    strain (GenBank accession no.)
    LF01菌株
    LF01 strain
    ANIDDH
    贝莱斯芽孢杆菌 B. velezensis M75 (CP016395) 99.73 98.30
    贝莱斯芽孢杆菌 B. velezensis G341 (CP011686) 97.66 79.90
    贝莱斯芽孢杆菌 B. velezensis LS69 (CP015911) 97.74 80.10
    贝莱斯芽孢杆菌 B. velezensis FZB42 (CP000560) 97.73 80.10
    解淀粉芽孢杆菌 B. amyloliquefaciens ATCC23350 (NC_014551) 93.96 55.40
    解淀粉芽孢杆菌 B. amyloliquefaciens MT45 (CP011252) 94.01 55.20
    枯草芽孢杆菌 B. subtilis str. 168 (NC_000964) 77.17 20.70
    地衣芽孢杆菌 B. licheniformis ATCC 14580 (NC_006270) 72.60 19.60
    蜡样芽孢杆菌 B. cereus ATCC 14579 (CP034551) 68.17 34.20

    Table 1.  The values of ANI and DDH between the LF01 strain and its related species %

    基于LF01菌株和参考菌株的SNP构建系统发育树,结果显示,LF01菌株与贝莱斯芽孢杆菌M75聚为一支,与贝莱斯芽孢杆菌FZB42、CC09、G341、S3-1、LS69等菌株聚为一个大的分支,与解淀粉芽孢杆菌、枯草芽孢杆菌和地衣芽孢杆菌聚为不同的分支,表明LF01菌株在基因组水平上与贝莱斯芽孢杆菌亲缘关系最近 (图2)。综上可知,基于分子水平LF01菌株被归类为贝莱斯芽孢杆菌。

    Figure 2.  Phylogenetic tree of LF01 and the reference strains base on the SNP

  • 通过antiSMARSH (version 5.1.2)软件在线分析,结果发现LF01基因组含有bacillaene、bacillibactin、杆菌霉素D(bacillomycin D)、溶杆菌素(bacilysin)、difficidin、泛革素(fengycin)、macrolactin H、plantazolicin和表面活性素(surfactin)等抗菌物质合成基因簇,进一步对这些拮抗物质合成基因进行比对分析发现,LF01基因组包含了上述9种抗菌物质合成基因簇(图3),其中表面活性素合成相关基因簇中缺失了yckEycxCycxD,但是有研究表明,这3个基因并非是surfactin合成相关的必须基因[28]。以上这些基因簇核心基因的片段大小为350.1 kb,约占基因组全长的8.83%。

    Figure 3.  The gene clusters of LF01 strain for the biosynthesis of nine secondary metabolites related to antimicrobial substances

  • LF01菌株编码的CAZy酶类基因家族有160个,分别为GH (44)、GT (39)、CE (32)、PL (4)、AA (8)和CBM (33)基因家族,其中GH家族最多,占整个基因家族的27.5%,GT家族次之,约占24.4%。在这些基因家族中,与纤维素和半纤维素降解有关的主要有GH5、GH11、GH26、GH43、GH51和GH53等;与淀粉水解相关的主要有GH13和GH126;与几丁质降解相关的有GH18、GH23、CE9和CBM50等;与木聚糖降解相关的有GH43、CE1、CE3、CE4、CE6和CE7等;与果胶降解酶类相关的主要有GH43、CE1、PL1、PL9等;与肽聚糖降解相关的有GH23和GH73等;与葡聚糖酶类相关的有GH3、GH16和GH30等 (表2)。这表明LF01菌株具有降解纤维素、半纤维素、淀粉、几丁质、果胶、肽聚糖和葡聚糖等物质的潜力。

    CAZy酶类家族       
    CAZymes families       
    基因亚族数量/个
    no. of gene subfamilies
    糖苷水解酶 glycoside hydrolase, GH GH1 (3)、GH3 (1)、GH4 (4)、GH5 (1)、GH11 (1)、GH13 (4)、GH16 (1)、GH18 (2)、GH23 (3)、GH26 (1)、GH30 (2)、GH32 (3)、GH43 (4)、GH46 (1)、GH51 (2)、GH53 (1)、GH68 (1)、GH73 (3)、GH76 (1)、GH109 (4)、GH126 (1)
    糖基转移酶 glycosyl transferase, GT GT1 (3)、GT2 (15)、GT4 (7)、GT8 (1)、GT19 (1)、GT26 (1)、GT28 (3)、GT46 (2)、GT51 (4)、GT83 (2)
    碳水化合物酯酶 carbohydrate esterase, CE CE1 (8)、CE3 (2)、CE4 (7)、CE6 (1)、CE7 (2)、CE9 (2)、CE10 (4)、CE12 (2)、CE14 (4)
    多糖裂解酶 polysaccharide lyase, PL PL1 (2)、PL9 (1)、PL22 (1)
    辅助氧化还原酶 auxiliary activitie, AA AA4 (1)、AA6 (2)、AA7 (4)、AA10 (1)
    碳水化合物结合模块 carbohydrate-binding module, CBM CBM2 (1)、CBM3 (1)、CBM6 (1)、CBM12 (1)、CBM16 (1)、CBM26 (1)、CBM37 (1)、CBM50 (26)

    Table 2.  CAZymes families in the genome of B. velezensis LF01

  • LF01菌株拮抗物质的溶血活性实验结果显示,当拮抗物质的浓度为1和5 mg/mL时,无明显的溶血活性,但是当拮抗物质浓度为10 mg/mL时,出现了溶血现象(图4),这表明LF01拮抗物质的溶血活性属于剂量依赖型。

    Figure 4.  Hemolytic activity of the antimicrobial substances of LF01 strain

    LF01菌株拮抗物质对尼罗罗非鱼和斑马鱼的生物安全性实验结果显示,在1、5和10 mg/mL剂量浓度下,腹腔注射的尼罗罗非鱼和斑马鱼在观察期内均未出现死亡,而且实验鱼均未表现出明显的临床异常症状,表明LF01菌株拮抗物质在低于10 mg/mL的剂量条件下对尼罗罗非鱼和斑马鱼均不致病,具有较高的生物安全性。进一步计算可知LF01菌株拮抗物质对尼罗罗非鱼的安全剂量为≤40.81 mg/kg (拮抗物质干粉/尼罗罗非鱼体质量)。

  • LF01拮抗物质饲喂尼罗罗非鱼后的人工感染实验结果显示,实验组(ET-sub1/2/3)的尼罗罗非鱼累积死亡率平均为36.67%;而对照组(CK-sub1/2/3)尼罗罗非鱼的累积死亡率平均为90.00%。这表明饲喂拮抗物质后能够显著提高尼罗罗非鱼对无乳链球菌的抗病能力(P<0.01)。进一步分析发现对照组的尼罗罗非鱼在2 d之内的死亡率远高于实验组(图5),而且主要表现为急性死亡现象,具有身体发黑、眼球突出、打转、狂游、脾脏和肾脏肿大等链球菌病典型症状。

    Figure 5.  Survival curve of O. niloticus in experimental and control groups after being infected with S. agalactiae

3.   讨论
  • 贝莱斯芽孢杆菌因其能够拮抗多种动植物病原菌,而且具有促生长作用,在农业、工业、医药、环境保护、养殖业等领域都有广泛研究。目前,贝莱斯芽孢杆菌作为植物病害的生防制剂已经开展了较为深入的研究,有些菌株甚至进行到示范应用阶段[3, 29]。然而,在水生动物病害防控方面,贝莱斯芽孢杆菌的相关研究较为薄弱,亟待深入研究。本研究为了进一步解析贝莱斯芽孢杆菌LF01的拮抗和生防作用,通过细菌的全基因组测序分析,在分子水平对该菌株进行种水平上的鉴定,并解析其拮抗物质合成相关基因簇;通过拮抗物质的生防作用实验,分析拮抗物质作为绿色防控药物应用的潜力;通过LF01菌株拮抗物质的生物安全性评价,评估其代谢产物对尼罗罗非鱼和斑马鱼的毒性,为今后LF01菌株及其代谢产物资源开发利用奠定基础。

    贝莱斯芽孢杆菌LF01的基因组测序结果显示,该菌的基因组序列全长为3 974 023 bp,GC含量为46.56%。通过ANI和DDH值分析,结果显示LF01归属为贝莱斯芽孢杆菌;根据核基因SNP进化分析,进一步表明LF01菌株与贝莱斯芽孢杆菌同属一个分支,并且LF01菌株与贝莱斯芽孢杆菌M75的亲缘关系最近。M75菌株对植物病原真菌具有较强的拮抗活性,其基因组序列分析发现,该菌含有大量的拮抗活性物质合成相关基因簇,如bacillibactin、泛革素、表面活性素、bacillaene、difficidin、macrolactin H、溶杆菌素和mersacidin[30]。相似地,LF01菌株的基因组含有bacillaene、bacillibactin、bacillomycin D、溶杆菌素、difficidin、泛革素、macrolactin H、plantazolicin和表面活性素等抗菌物质合成相关基因簇。这表明与M75菌株相比,LF01菌株产生的拮抗物质种类可能更加丰富。

    脂肽类化合物根据其结构特征主要分为表面活性素、伊枯草菌素(iturin)和泛革素等3大类,这些物质的理化性质相对稳定,能够耐受氯仿等有机溶剂,耐受紫外线、蛋白酶等处理。表面活性素具有抗病毒、抗细菌活性,但是没有明显的抗真菌活性[31],值得注意的是表面活性素在高浓度下会现出溶血活性[32]。本研究发现LF01拮抗物质在高浓度下具有一定的溶血活性,该现象可能与表面活性素的产生有关。泛革素具有强烈的抗真菌活性和较弱的抑制细菌活性[31, 33]。聚酮类化合物如difficidin、macrolactin和bacillaene等对细菌具有强烈的抑菌活性[33]。溶杆菌素是一种二肽,具有广泛抑制真菌和细菌的活性[34-35]。Bacillibactin和plantazolicin对细菌具有抑制活性,而且bacillomycin还具有抑制真菌的活性[3]。由于LF01菌株的基因组中含有以上多种拮抗物质合成基因簇,表明该菌不仅具有广泛的抑制细菌活性,而且具有抑制病原真菌的潜力,其真菌抑菌活性需要进一步验证。此外,体外抑菌实验结果显示,LF01菌株拮抗物质冻干粉对嗜水气单胞菌、维氏气单胞菌、无乳链球菌和海豚链球菌的MIC分别为64、64、64和32 μg/mL,表现出较强的抑菌活性,暗示该菌拮抗物质在水产动物细菌性病害防控方面具有替代传统抗生素的潜力。

    CAZy酶类分析结果显示,LF01菌株基因组中含有降解真菌细胞壁成分几丁质、肽聚糖和葡聚糖等物质的相关酶类,表明该菌对病原真菌具有一定的拮抗作用。同时,LF01基因组中含有大量与纤维素、半纤维素、淀粉、几丁质、木聚糖、肽聚糖等降解相关的基因簇,表明LF01菌株具有降解水产动物饲料中纤维素、淀粉、多糖类物质的能力,这暗示该菌株作为水产动物饲料添加剂,在提高动物饲料消化率和生长性能方面具有广阔的应用前景。

    LF01菌株基因组中不含有致病性芽孢杆菌常见的毒素基因,因此该菌作为致病菌的可能性极小。该菌拮抗物质在一定浓度范围内不具有溶血活性,而且对尼罗罗非鱼和斑马鱼是安全的,这为今后该菌代谢产物的商业化应用提供了参考依据。虽然LF01菌株基因组中含有部分耐药基因,但是这些耐药相关基因存在其基因组上,与质粒上的耐药基因相比,不易扩散和传播。而且这些耐药基因可能是非关键的耐药基因(无明显的耐药表型特征),其基因的上下游缺少可移动遗传元件,表明LF01菌株传播耐药基因的风险较小。LF01菌株拮抗物质添加到饲料中饲喂尼罗罗非鱼,能够显著提高尼罗罗非鱼对无乳链球菌的抗病力,表明该拮抗物质具有作为尼罗罗非鱼链球菌病新型药物研发的潜力,为水产动物疾病的药物筛选提供了新方向。本研究表明,贝莱斯芽孢杆菌的拮抗物质是水产动物细菌性病原重要的药物资源库,值得深入挖掘。由于本研究未能将LF01菌株的拮抗物质进行逐一提纯,只是将粗提物视为一个整体进行相关研究,后续研究需要将粗提物进一步纯化后分析其抑菌效果、生物安全性以及生防作用机制。

Reference (35)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return