黄栌枯萎病拮抗细菌CCBC3-3-1的全基因组测序及比较基因组分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0158

摘要/Abstract

摘要：

【目的】 从黄栌枝干内部分离到的细菌CCBC3-3-1菌株对黄栌枯萎病菌（Verticillium dahliae）表现出较强的拮抗作用，从基因组层面深入研究其拮抗机理，为进一步研发生防菌剂提供理论依据。【方法】 利用PacBio RS测序平台完成CCBC3-3-1的全基因组测序，根据16S rDNA序列信息构建系统发育树，并与同属近缘种菌株进行比较基因组学分析；对其发酵液进行LC-MS非靶标代谢组检测。【结果】 CCBC3-3-1基因组总长度为5.16 Mb，由1条环状双链染色体和3个环状质粒组成，GC含量48.08%，含有5 013个编码基因。经过antiSMASH预测，CCBC3-3-1基因组中有8个抗生素及次生代谢物合成相关的基因簇，其中2个与已知基因簇相似度较低，5个基因簇功能未知，其发酵液中检测出6种已知抗生素类物质。比较基因组学分析结果表明CCBC3-3-1基因组与泛菌属内4个近缘种均有显著差异，而且CCBC3-3-1在16S rDNA系统发育树上形成一个相对独立的分支。【结论】 CCBC3-3-1是泛菌属的一个新变异种Pantoea sp.，能够产生多种抗生素类物质，在黄栌枯萎病生物防治方面具有重要应用潜力。

关键词: 黄栌, 大丽轮枝菌, 泛菌, 拮抗作用, 全基因组测序

Abstract:

【Objective】 A bacterial strain CCBC3-3-1 isolated from the branch of Cotinus coggygria, has strong growth inhibition activity on the causal agent of Verticillium wilt of Cotinus coggygria. Gaining further understanding about the antagonistic mechanism of this strain at the genomic level will provide theoretical basis for developing a new biocontrol agent. 【Method】 PacBio RS sequencing platform was used to have the whole genome sequencing of CCBC3-3-1, a phylogenetic tree was constructed based on 16S rDNA sequences, comparative genomic analysis with genetically closed species was carried out, and LC-MS based untargeted metabolomics analysis were performed. 【Result】 The genome size of CCBC3-3-1 was 5.16 Mb with a GC content of 48.08%, and it contained a circular chromosome and three circular plasmids. A total of 5 013 coding DNA sequences（CDSs）were predicted. Through antiSMASH prediction, it was found that there were eight antibiotic and secondary metabolite gene clusters in the genome of CCBC3-3-1, two of which clusters had low similarity with the known ones, and five others had unknown functions. Six known antibiotics were identified in the fermentation broth of CCBC3-3-1. The results of comparative genomic analysis showed that there were significant differences between the genome of CCBC3-3-1 and other four closely related Pantoea species. CCBC3-3-1 strain formed a relatively independent branch on the 16S rDNA phylogenetic tree. 【Conclusion】 CCBC3-3-1 strain is a new variant species of Pantoea genus, which can produce various antibiotics. It has important application potential in the biological control of the Verticillium wilt of C. coggygria.

Key words: Cotinus coggygria, Verticillium dahlia, Pantoea, antagonism, whole genome sequencing

周江鸿, 夏菲, 仲丽, 仇兰芬, 李广, 刘倩, 张国锋, 邵金丽, 李娜, 车少臣. 黄栌枯萎病拮抗细菌CCBC3-3-1的全基因组测序及比较基因组分析[J]. 生物技术通报, 2024, 40(7): 235-246.

ZHOU Jiang-hong, XIA Fei, ZHONG Li, QIU Lan-fen, LI Guang, LIU Qian, ZHANG Guo-feng, SHAO Jin-li, LI Na, CHE Shao-chen. Whole Genome Sequencing and Comparative Genomic Analysis of Antagonistic Bacterium CCBC3-3-1 against Verticillium dahlia[J]. Biotechnology Bulletin, 2024, 40(7): 235-246.

图/表 14

图1 黄栌内生细菌CCBC3-3-1无菌发酵液对黄栌枯萎病菌的拮抗作用 a：CCBC3-3-1无菌发酵液对黄栌枯萎病菌（V. dahlia）的抑制作用。b: 对照

Fig. 1 Antagonism of the sterile fermentation broth of endogenous bacterium CCBC3-3-1 in Cotinus cog-gygria on V. dahlia a: The sterile fermentation broth of CCBC3-3-1 effectively inhibited the growth of V. dahlia. b： Control

图2 菌株CCBC3-3-1的染色体基因组图谱从外向内依次为: 1: 基因组的刻度, 大小为M（million bp）; 2: 编码基因; 3: COG数据库注释结果; 4: KEGG数据库注释结果; 5: GO数据库的注释结果; 6:非编码RNA; 7: GC含量（向内的红色部分表示该区域GC含量低于全基因组平均GC含量，向外的绿色部分与之相反，且峰值越高表示与平均GC含量差值越大）; 8: GC偏移（具体算法为G-C/G+C，用来衡量G和C的相对含量，如果G>C，则GC偏移值为正值，用向内的粉色部分表示；G<C，则为负值，用向外的浅绿色部分表示）

Fig. 2 Chromosome genome map of strain CCBC3-3-1 From outer to inner: 1: Genome size（million bp）; 2: coding genes; 3: gene function annotation in the COG database; 4: gene function annotation in the KEGG database; 5: gene function annotation in the GO database; 6: nc RNA; 7: GC content（The inward red part indicates that the GC content in this region is lower than the average GC content of the entire genome, while the outward green part is the opposite. The higher the peak, the greater the difference between the GC content and the average GC content）; 8: GC-skew（The algorithm is G-C/G+C, which is used to measure the relative content of G and C. If G>C, the GC-skew value is positive, represented by the inward pink part; if G<C, the GC-skew value is negative, represented by the outward light green part）

图3 菌株CCBC3-3-1的质粒基因组图谱从外向内依次为: 1: COG数据库注释结果（顺时针箭头表示正链基因）; 2: 基因组的刻度, 大小为K（kilo bp）; 3: GC含量（向内的红色部分表示该区域GC含量低于全基因组平均GC含量，向外的绿色部分与之相反，且峰值越高表示与平均GC含量差值越大）; 4: GC偏移（具体算法为G-C/G+C，用来衡量G和C的相对含量，如果G>C，则GC偏移值为正值，用向内的粉色部分表示；G<C，则为负值，用向外的浅绿色部分表示）

Fig. 3 Plasmid genome map of strain CCBC3-3-1 From outer to inner: 1: Gene function annotation in the COG database（Clockwise arrows indicate positive chain gene）; 2: genome Size（kilo bp）; 3: GC content（The inward red part indicates that the GC content in this region is lower than the average GC content of the entire genome, while the outward green part is the opposite. The higher the peak, the greater the difference between the GC content and the average GC content）; 4: GC-skew（The algorithm is G-C/G+C, which is used to measure the relative content of G and C. If G>C, the GC-skew value is positive, represented by the inward pink part; if G<C, the GC-skew value is negative, represented by the outward light green part）

图4 基于16S rDNA序列的系统发育树

Fig. 4 Neighbour-joining phylogenetic tree based on 16S rDNA sequences

图5 CCBC3-3-1的GO功能注释分类 1：生物黏附；2：生物学调控；3：细胞杀伤；4：细胞成分的组织或生源；5：细胞过程；6：死亡；7：发育过程；8：本地化构建；9：生长；10：免疫系统过程；11：本地化；12：运输；13：代谢过程；14：多生物体过程；15：多细胞机体过程；16：生物过程的负调控；17：氮素利用；18：生物过程的正调控；19：生物过程的调控；20：繁殖；21：繁殖过程；22：刺激反应；23：信号；24：病毒繁殖；25：细胞；26：细胞连接；27：细胞部分；28：细胞外区域；29：细胞外区域部分；30：大分子复合物；31：膜封闭腔；32：细胞器；33:细胞器部分；34：突触；35：突触部分；36：病毒体；37：病毒体部分；38：抗氧化活性；39：结合；40：催化活性；41：电子载体活性；42：酶调节活性；43：分子转导活性；44：核酸结合的转录因子活性；45：蛋白质结合的转录因子活性；46：结构分子活性；47：转运活性

Fig. 5 GO function classification of strain CCBC3-3-1 1: Biological adhesion. 2: Biological regulation. 3: Cell killing. 4: Cellular component organization or biogenesis. 5: Cellular process. 6: Death. 7: Developmental process. 8: Establishment of localization. 9: Growth. 10: Immune system process. 11: Localization. 12: Locomotion. 13: Metabolic process. 14: Multi-organism process. 15: Multi cellular organismal process. 16: Negative regulation of biological process. 17: Nitrogen utilization. 18: Positive regulation of biological process. 19: Regulation of biological process. 20: Reproduction. 21: Reproductive process. 22: Response to stimulus. 23: Signaling. 24: Viral reproduction. 25: Cell. 26: Cell junction. 27: Cell part. 28: Extracellular region. 29: Extracellular region part. 30: Macromolecular complex. 31: Membrane-enclosed lumen. 32: Organelle. 33: Organelle part. 34: Synapse. 35: Synapse part. 36: Virion. 37: Virion part. 38: Antioxidant activity. 39: Binding. 40: Catalytic activity. 41: Electron carrier activity. 42: Enzyme regulator activity. 43: Molecular transducer activity. 44: Nucleic acid binding transcription factor activity. 45: Protein binding transcription factor activity. 46: Structural molecule activity. 47: Transporter activity

图6 CCBC3-3-1的KEEG功能注释分类 A：细胞过程（1：运输和分解代谢；2：细胞群落-原核生物；3：细胞的运动性；4：细胞生长与死亡）；B：环境信息处理（5：信号转导；6：膜运输）；C：遗传信息处理（7：翻译；8：转录；9：复制和修复；10：折叠、分类和降解）；D：人类疾病（11：神经退行性疾病；12：侵染性疾病；13：免疫性疾病；14：内分泌和代谢疾病；15：抗药性；16：心血管疾病；17：癌症）；E：新陈代谢（18：外源物质生物降解和代谢；19:核苷酸代谢；20：萜类和聚酮类代谢; 21：其他氨基酸代谢；22：辅助因子和维生素代谢；23：脂类代谢；24：多糖的生物合成与代谢；25：能量代谢；26：糖代谢；27：其他次生代谢物的生物合成；28：氨基酸代谢）；F：有机体系统（29：神经系统；30：免疫系统；31：排泄系统；32：环境适应；33：内分泌系统；34：消化系统；35：衰老）

Fig. 6 KEEG function classification of strain CCBC3-3-1 A: Cellular processes（1: Transport and catabolism. 2: Cellular community-prokaryotes. 3: Cell motility. 4: Cell growth and death）. B: Environmental information processing（5: Signal transduction. 6: Membrane transport）. C: Genetic information processing（7: Translation. 8: Transcription. 9: Replication and repair. 10: Folding, sorting and degradation）. D: Human diseases（11: Neurodegenerative diseases. 12: Infectious diseases. 13: Immune diseases. 14: Endocrine and metabolic diseases. 15: Drug resistance. 16: Cardiovascular diseases. 17: Cancers）. E: Metabolism（18: Xenobiotics biodegradation and metabolism. 19: Nucleotide metabolism. 20: Metabolism of terpenoids and polyketides. 21: Metabolism of other amino acids. 22: Metabolism of cofactors and vitamins. 23: Lipid metabolism. 24: Glycan biosynthesis and metabolism. 25: Energy metabolism. 26: Carbohydrate metabolism. 27: Biosynthesis of other secondary metabolites. 28: Amino acid metabolism）.F: Organismal systems（29: Nervous system. 30: Immune system. 31: Excretory system. 32: Environmental adaptation. 33: Endocrine system. 34: Digestive system. 35: Aging）

图7 CCBC3-3-1的COG功能注释分类 A：RNA加工和修饰；B：能量产生与转换；C：细胞周期控制, 细胞分裂和染色体分裂；D：氨基酸转运与代谢；E：核苷酸转运与代谢；F：碳水化合物转运与代谢；G：辅酶转运与代谢；H：脂质转运与代谢；I：翻译、核糖体结构和生物合成；J：转录；K：复制, 重组与修复；L：细胞壁/膜/包膜的生物合成；M：细胞运动：N：翻译后修饰，蛋白质转化与分子伴侣；O：无机离子的转运与代谢；P：次生代谢产物的生物合成、运输与分解代谢；Q：一般功能预测；R：功能未知；S：信号转导机制；T：细胞内运输，分泌和囊泡；U：防御机制；V：细胞外结构；W：移动基因组：原噬菌体、转座子

Fig. 7 COG function classification of strain CCBC3-3-1 A: RNA processing and modification. B: Energy production and conversion. C: Cell cycle control, cell division, chromosome partitioning. D: Amino acid transport and metabolism. E: Nucleotide transport and metabolism. F: Carbohydrate transport and metabolism. G: Coenzyme transport and metabolism. H: Lipid transport and metabolism. I: Translation, ribosomal structure and biogenesis. J: Transcription. K: Replication, recombination and repair. L: Cell wall/membrane/envelope biogenesis. M: Cell motility. N: Posttranslational modification, protein turnover, chaperones. O: Inorganic ion transport and metabolism. P: Secondary metabolites biosynthesis, transport and catabolism. Q: General function prediction only. R: Function unknown. S: Signal transduction mechanisms. T: Intracellular trafficking, secretion, and vesicular. U: Defense mechanisms. V: Extracellular structures. W: Mobilome: prophages, transposons

图8 CCBC3-3-1的CAZy功能注释分类 AAs：辅助功能的酶基因；CBMs：碳水化合物结合模块基因；CEs：糖酯酶基因；GHs：糖苷水解酶基因；GTs：糖基转移酶基因；PLs：多糖裂解酶基因

Fig. 8 CAZy function classification of strain CCBC3-3-1 AAs: Auxiliary activities. CBMs: Carbohydrate-binding modules. CEs: Carbohydrate esterases. GHs: Glycoside hydrolases.GTs: Glycosyl transferases. PLs: Polysaccharide lyases

表1 菌株CCBC3-3-1与Pantoea属其他4个种的染色体序列特征比较

Table 1 Comparison of chromosome sequence feature between strain CCBC3-3-1 with other 4 species of Pantoea

项目Item	CCBC3-3-1	P. vagans C9-1	P. agglomerans TH81	P. ananatis PA13	P. eucalypti LMG24197
GeneBank number	CP034363.1	CP002206.1	CP031649.1	CP003085.1	CP045720.1
Length of chromosome /bp	5 008 525	4 024 986	4 128 817	4 586 378	4 035 995
GC content/%	52.9	55.5	55.2	53.7	54.6
Number of CDSs	4 610	3 707	3 766	4 254	3 692
Number of rRNAs	22	22	21	22	21
Number of tRNAs	87	78	72	86	76
Number of CRISPRS	1	0	0	0	0

表2 菌株CCBC3-3-1与Pantoea属其他4个种的质粒序列特征比较

Table 2 Comparison of strain CCBC3-3-1 plasmid sequence with other 4 species of Pantoea

质粒 Plasmid	数量 Number	GenBank登录号 GenBank number	长度 Length/bp	GC含量 GC Content /%	CDSs数量 CDSs number	rRNA数量 rRNA number	tRNA数量 tRNA number	CRISPRS数量 CRISPRS number
CCBC3-3-1	3	CP034364.1 CP034365.1 CP034366.1	25 471 29 467 96 304	46.7 42.7 49.9	38 37 114	0 0 0	0 0 0	0 0 0
P. vagans C9-1	3	CP001893.1 CP001894.1 CP001895.1	167 983 165 693 529 676	53.0 51.1 53.9	144 183 508	0 0 0	0 0 0	0 0 0
P. agglomerans TH81	3	CP031650.1 CP031651.1 CP031652.1	520 959 182 169 152 523	53.7 52.3 48.9	517 161 160	0 0 0	0 0 0	0 0 0
P. ananatis PA13	1	CP003086.1	280 753	52.3	257	0	0	0
P. eucalypti LMG24197	3	CP045721.1 CP045722.1 CP045723.1	529 303 138 725 94 967	52.2 50.9 52.2	528 114 99	0 0 0	0 0 0	0 0 0

图9 CCBC3-3-1与P. vagans C9-1、P. ananatis PA13、P. agglomerans TH81和P. eucalypti LMG24197的基因组共线性分析同种颜色区域部分代表基因组间共线性部分

Fig. 9 Genomic collinearity analysis of CCBC3-3-1 with P. vagans C9-1, P. ananatis PA13, P. agglomerans TH81 and P. eu-calypti LMG24197 The same color modules joined by linear indicate collinear region

表3 菌株CCBC3-3-1与Pantoea 属12个菌株的OrthoANIu和dDDH值

Table 3 OrthoANIu and dDDH scores between strain CCBC3-3-1 and 12 strains of Pantoea

物种 Species	CCBC3-3-1
物种 Species	OrthoANIu/%	dDDH/%
P. agglomerans TH81	75.95	20.90
P. agglomerans C410P1	75.66	21.00
P. agglomerans CHTF15	75.93	20.80
P. agglomerans CQ10	76.03	20.80
P. ananatis PA13	74.99	20.60
P. ananatis VY148	74.99	20.60
P. ananatis LMG20103	74.96	20.70
P. eucalypti LMG24197	75.89	20.90
P. jilinensis D25	75.49	20.50
P. vagans C9-1	75.91	20.90
P. vagans FAAARGOS160	75.67	20.60
P. vagans LMG24199	76.02	20.90

表4 菌株CCBC3-3-1抗生素及次生代谢产物合成相关基因簇分析

Table 4 Analysis of antibiotic and secondary metabolite gene clusters of strain CCBC3-3-1

编号 Cluster ID	类型 Cluster type	起始-终止位点 Start-stop site	已知基因簇 Known cluster	相似度 Similarity/%	来源 Resources
基因簇1 Cluster 1	其他（磷酸盐） Other（Phosphonate）	1 233863 - 1 251863	-	-	-
基因簇2 Cluster 2	糖化物 Saccharide	1 593 081 - 1 618 729	O-抗原 O-antigen	14	Pseudomonas aeruginosa
基因簇3 Cluster 3	核糖体肽类似酶 RiPP-like	1 674 964 - 1 735 767	-	-	-
基因簇4 Cluster 4	萜烯 Terpene	2 149 356 - 2 241 512	类胡萝卜素 Carotenoid	100	Enterobacteriaceae bacterium DC413
基因簇5 Cluster 5	非核糖体多肽合成酶 NRPS	2 335 269 - 2 383 545	-	-	-
基因簇6 Cluster 6	非核糖体多肽合成酶 NRPS	3 112 137 - 3 156 045	Amonabactin	57	Aeromonas hydrophila subsp. hydrophila ATCC 7966
基因簇7 Cluster 7	核糖体肽类似酶 RiPP-like	3 734 150 - 3 762 973	-	-	-
基因簇8 Cluster 8	核糖体肽类似酶 RiPP-like	4 182 791 - 4 203 075	-	-	-

表5 CCBC3-3-1发酵液中鉴定出的抗生素类物质

Table 5 Antibiotics in the fermentation broth of CCBC3-3-1

序号No.	名称Name	MS2分值MS2 score
1	依诺沙星Enoxacin	0.99
2	新生霉素Novobiocin	0.94
3	链脲霉素Streptozocin	0.85
4	手霉素A Manumycin A	0.68
5	司帕沙星Sparfloxacin	0.67
6	阿克拉霉素Aclarubicin	0.65

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