Whole Genome Sequencing and Comparative Genomic Analysis of Antagonistic Bacterium CCBC3-3-1 against Verticillium dahlia

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

Abstract

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

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.

Figures/Tables 14

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

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）

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）

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

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

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）

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

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

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

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

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

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

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	-	-	-

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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