Genome Feature of B.velezensis ES2-4
The genomic structure information showed that the ES2-4 genome is composed of a single circular chromosome with a length of 3,929,792 bp and the GC content is 46.5%(Fig. 1). The whole genome of ES2-4 was predicted to contain 4016 coding sequences (CDSs). The average length is 872bp, and the coding sequence accounts for 89.1% of the entire genome sequence.The chromosome contained 27 rRNA ,86 tRNA ,81 sRNA ,70 tandem repeats and 46 interspersed repeat(Table.1).The COG annotation results showed that 3047 gene annotations were finally obtained (Fig. 2), accounting for 75.9% of the total genes, and the genes with unknown functions accounted for 24.1% of the total gene predictions. There were 20 types of gene annotation results(Table.2). Among them, 289 are amino acid metabolism and transport, accounting for 9.5% of the annotated genes, 232 are transcribed, accounting for 7.6% of the annotated genes, 222 are carbohydrate transport and metabolism, accounting for 7.3% of the annotated genes, and there are 761 more Genes with unknown functions need mining, accounting for 24.9% of the annotated genes. Its function needs further confirmation. The GO annotation results showed that: GO functions were divided into three categories: 1991 genes in biological processes, 1363 genes in cellular components, and 2115 genes in molecular functions. The KEGG annotation results showed that there were a total of 2519 genes, accounting for about 55.3% of the total bacterial genes, with a total of 41 metabolic pathways. The results of carbohydrate activity enzyme analysis showed that: the B.velezensis ES2-4 strain encodes a total of 126 CAZy enzyme gene families, which are divided into six categories of proteins: Glycoside Hydrolases, GlycosylTransferases, Polysaccharide Lyases, Carbohydrate Esterases, Carbohydrate-Binding Modules, and Auxiliary Activities family. Among them, Glycoside hydrolases contain 40 genes related to the hydrolysis of glycosidic bonds for the hydrolysis of sugars and their derivatives mainly including chitinase. Numerous studies have demonstrated that bacterial chitinases display an important role in inhibiting hyphal growth of phytopathogenic fungi. At the same time, some studies have shown that chitinase has been proved to have the activity of inhibiting the random hatching of nematodes(Choub et al. 2021; Tran et al. 2022; Won et al. 2021).
Table 1
General genomic features of Bacillus velezensis ES2-4.
Feature | B.velezensis ES2-4 |
Genome(bp) | 3,929,792 |
G + C(%) | 46.5 |
CDs number | 4016 |
Average gene length(bp) | 872 |
rRNA | 27 |
tRNA | 86 |
sRNA | 81 |
Gene assigned to COG | 3047 |
Prophage | 1 |
Table 2
COG functional classification of 3047 proteins predicted in the genome of strain ES2-4
Category | Type | Type Description | Gene No |
Information storage and processing | B | Chromatin structure and dynamics | 1 |
Metabolism | C | Energy production and conversion | 174 |
Cellular processes and signaling | D | Cell cycle control, cell division, chromosome partitioning | 33 |
Metabolism | E | Amino acid transport and metabolism | 289 |
Metabolism | F | Nucleotide transport and metabolism | 77 |
Metabolism | G | Carbohydrate transport and metabolism | 222 |
Metabolism | H | Coenzyme transport and metabolism | 105 |
Metabolism | I | Lipid transport and metabolism | 102 |
Information storage and processing | J | Translation, ribosomal structure, and biogenesis | 141 |
Information storage and processing | K | Transcription | 232 |
Information storage and processing | L | Replication, recombination, and repair | 121 |
Cellular processes and signaling | M | Cell wall/membrane/envelope biogenesis | 177 |
Cellular processes and signaling | N | Cell motility | 38 |
Cellular processes and signaling | O | Posttranslational modification, protein turnover, chaperones | 98 |
Metabolism | P | Inorganic ion transport and metabolism | 179 |
Metabolism | Q | Secondary metabolites biosynthesis, transport, and catabolism | 75 |
Poorly characterized | S | Function unknown | 761 |
Cellular processes and signaling | T | Signal transduction mechanisms | 127 |
Cellular processes and signaling | U | Intracellular trafficking, secretion, and vesicular transport | 35 |
Cellular processes and signaling | V | Defense mechanisms | 60 |
Comparative Genome Analysis of Strain ES2-4 with Bacillus Strains
In the early stage of the experiment, we observed through morphological observation that ES2-4 is a kind of gram-positive bacteria that can produce resistant endospores, which are rod-shaped and milky-white colonies. In the early stage of the growth of plate culture, the edge of the colony was round and viscous, and in the later stage of growth, the edge was irregular, the middle was slightly raised and folded, and it was easy to dry. When growing statically in a liquid medium, it forms wrinkles and is an aerobic bacteria. So we preliminarily identified ES2-4 as Bacillus. A BLAST comparison with B. velezensis and B. subtilis strains is illustrated in Fig. 5 with annotation of the gene involved in promoting plant growth and triggering plant immunity and secondary metabolite synthesis. In general, it is inaccurate to distinguish B. subtilis, B. amyloliquefaciens, and B. velezensis according to 16S rRNA gene sequences(Dong et al. 2022). As a housekeeping gene, the gyrA gene is highly conserved and has only a single copy of all bacteria, which can avoid the heterogeneity of the 16S rRNA gene due to multiple copies, and its nucleotide sequence length and evolution speed are both high. Much higher than the 16S rRNA gene, showing higher genetic differences(Tayeb et al. 2008). According to gyrA sequencing analysis, ES2-4 had 99% sequence homology with B.velezensis. The phylogenetic analysis of the gyrA sequence showed that strain ES2-4 also clustered with B.velezensis LS69 and B.velezensis SQR9 as Fig. 3. In addition, as Fig. 3 and Fig. 4 show, the ANI and dDDH values show that ES2-4 shares 99.72% and 99.9% with LS69 respectively. According to the generally accepted ANI value of 95% and dDDH value of 70% as a threshold for species classification, this strain can be classified as Bacillus. velezensis, and has a recent evolutionary relationship with LS69. In conclusion, based on cytomorphological features, gyrA, and ANI values, ES2-4 was identified as a new member of B.velezensis.At the same time, it also guides us that when facing Bacillus velezensis and its relatively closely related species, we can use a variety of housekeeping genes such as gyrA, rpoB, cheA, and bioinformatics methods such as ANI and dDDH for comprehensive identification(Kim et al. 2014; Rossello-Mora et al. 2011; Wang et al. 2007).
Table.3 Comparative genomic analysis of Bacillus velezensis ES2-4 with Bacillus genomes
Strains | GeneBank Accession No. | ANI(%) | dDDH(%) | GC(%) | Size(bp) |
Bacillus velezensis ES2-4 | NZ_CP097328.1 | 100 | 100 | 46.5 | 3929792 |
Bacillus velezensis LS69 | NZ_CP015911.1 | 99.72 | 99.9 | 46.5 | 3917761 |
Bacillus velezensis SQR9 | NZ_CP006890.1 | 98.65 | 89.8 | 46.1 | 4117023 |
Bacillus velezensis FZB42 | NC_CP009725.2 | 98.15 | 85.4 | 46.5 | 3918596 |
Bacillus velezensis YYC | NZ_CP076514.1 | 98.14 | 84.7 | 46.5 | 3973236 |
Bacillus velezensis S4 | NZ_CP050424.1 | 98.12 | 84.5 | 46.4 | 4065174 |
Bacillus velezensis W1 | NZ_CP028375.1 | 98.1 | 84.8 | 45.8 | 4237431 |
Bacillus velezensis CC09 | NZ_CP015443.1 | 98.04 | 83.9 | 46.1 | 4167153 |
Bacillus velezensis NST6 | NZ_CP063687.1 | 97.72 | 83.2 | 46 | 4141240 |
Bacillus velezensis JS25R | NZ_CP009679.1 | 97.58 | 79.8 | 46.39 | 4006002 |
Bacillus velezensis WB | NZ_CP076514.1 | 97.58 | 79.8 | 46.7 | 3896799 |
Bacillus Amyloliquefaciens IT-45 | NC_CP020272.1 | 97.53 | 80.1 | 46.6 | 3928857 |
Bacillus velezensis Pm9 | NZ_CP059855.1 | 97.52 | 79.9 | 46.7 | 3890670 |
Bacillu Subtilis 168 | NC_CP000964.3 | 76.38 | 20.9 | 43.5 | 4215606 |
Bacillu Subtilis NCIB 3610 | NZ_CP020102.1 | 76.38 | 20.9 | 43.5 | 4215607 |
Analysis of gene clusters related to secondary metabolite synthesis
Genomic analysis showed that Bacillus velezensis ES2-4 would use no less than 8.3% of the entire genome for the synthesis of secondary metabolites, a capacity that exceeds that of the closely related model Gram-positive bacterium Bacillus subtilis 168 by more than 2-fold (Chen et al. 2009). Through antiSMASH software analysis, we found that ES2-4 contains 14 gene clusters for directing non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) to synthesize bioactive peptides and polyketides with antibiotic properties. Secondary metabolites could be identified (Fig. 5, Table 4). Surfactin, iturin, and fengycin are all lipopeptides, and the cyclic structure formed by fat and polypeptide chains accounts for about 2.6% of the entire genome. Due to their amphiphilic structure, lipopeptides have a variety of biological activities, including antibacterial, antifungal, antiviral, and antitumor activities, and are used in chemical, agricultural, pharmaceutical, and food industries(Crouzet et al. 2020; Jiang et al. 2014; Ongena and Jacques 2008). Not only that, but lipopeptides can also act as inducing factors of plant systemic resistance to enhance the defense ability of plant pathogenic bacteria(J. et al. 2018). Surfactin is synthesized under the guidance of four gene clusters of srfABCD, composed of 7 amino acids and aliphatic segments, and has strong surface activity. In addition to having antibacterial and antiviral activities, it also participates in the formation of biofilms and plays an important role in the chemotaxis of PGPR and the process of colonizing the rhizosphere of plants(Vanittanakom et al. 1986). Iturin is composed of four gene clusters ituABCD. Like surfactin, the peptide chain part is also composed of 7 amino acids, but it has a strong inhibitory ability against a variety of fungi recently, with no obvious antibacterial effect and no antiviral activity(Ongena and Jacques 2008). Fengycin is synthesized by four gene clusters of fenABCDE. Unlike the peptide chain of surfactin and iturin, the peptide chain part of fengycin contains 10 amino acids. Fengycin can significantly inhibit the growth of plant fungi, especially filamentous fungi(Vanittanakom et al. 1986). The siderophore Bacillibactin is synthesized by the dhb gene cluster and has a similar structure to the siderophore Enterobactin produced by Gram-negative bacteria. It incorporates a trilactone ring and three catecholate moieties(Chen et al. 2007; Dertz et al. 2006). Bacillibactin can help host bacteria compete for Fe element to inhibit the growth of pathogenic bacteria, and its chelated Fe element can also be absorbed by plants, thereby promoting plant growth(Qiao et al. 2011). Bacilysin is one of the simplest antifungal dipeptides, which contains an L-Ala residue at the N-terminus and a non-proteinogenic amino acid, L-anticapsin, at the C-terminus. Bacilysin formation is catalyzed by an amino acid Ligase, whose synthesis is directed by the bacABCDE gene cluster (Inaoka et al. 2003; Steinborn et al. 2005). Bacilysin was first discovered in Bacillus subtilis, and it has attracted much attention due to its strong inhibitory properties against various bacteria such as Albicans(Walker and Abraham 1970). About 19kb DNA region of ES2-4 encodes polyketide synthase bae mln and dfn, which are used to synthesize bacillaene, macrolactin, and difficidin, respectively. Polyketides are a large class of secondary metabolites, including numerous bioactive compounds with antibacterial, immunosuppressive, antitumor, or other physiologically relevant biological activities. Amylocyclicin is a novel cyclic bacteriocin first discovered in FZB42 and synthesized by the ribosome under the guidance of the acnBACDE gene cluster. This compound has high antibacterial activity against closely related gram-positive bacteria(Scholz et al. 2014).
In addition, there are 4 secondary metabolite synthesis-related gene clusters in ES2-4, with a total length of not less than 15.6kb, including a polyketide compound whose specific substance is unknown. This compound is synthesized by chalcone synthase and is a potential antibiotic. Two gene clusters are responsible for the synthesis of terpenoids. By prediction, there is another gene cluster that may be responsible for the synthesis of Butirosin, but the similarity of this gene cluster is only 7%. Through gene annotation, it was found that the gene cluster contains a large number of genes related to protein transport and amino acid transport. It can be speculated that this gene cluster is responsible for the synthesis of a similar or novel secondary metabolite to Butirosin. ES2-4 also contains a gene cluster associated with the synthesis of lanthiopeptide compounds. Lanthipep-tides are a large class of natural peptides synthesized by ribosomes and post-translationally modified. These compounds are widely produced from different kinds of bacteria, have a rich structural and biological activity diversity, and provide an important source for active drug research and development. Among them, lanM is responsible for encoding the second-class lantipeptide synthase, which is involved in the translation and modification of lantipeptide compounds, such as dehydrogenation and cyclization. In addition, through gene annotation, we found that ES2-4 also contains lcnC, lcnDR2, nisFEGRK, nukEFG, and other gene clusters responsible for the transport, modification, and regulation of lantipeptide compounds(Repka et al. 2017; Zhang et al. 2012). The above results show that ES2-4 can synthesize various kinds of antibiotic substances, and has great potential for biological control.
Table 4
Predicted secondary metabolite clusters in genomes of Bacillus velezensis ES2-4
Cluster | Compound | enzyme | gene clusters | Size(kb) | Identity(%) |
Cluster1 | Surfactin | NRPS | srfAA/AB/AC/AD,sfp | 26.8 | 100 |
Cluster2 | BacillomycinD/Iturin | NRPS/PKS | ituABCD | 36.9 | 100 |
Cluster3 | Fengycin | NRPS | fenABCDE | 37.6 | 100 |
Cluster4 | Bacillibatcin | NRPS | dhbACEB | 11.7 | 100 |
Cluster5 | Bacilysin | NRPS | bacABCDEFG | 6.7 | 100 |
Cluster6 | Macrolactin | PKS | mlnABCDEFGHI | 53.1 | 100 |
Cluster7 | Bacillaene | PKS/NRPS | baeBCDE,acpK,baeGHIJKLMNRS | 71.6 | 100 |
Cluster8 | Difficidin | PKS | dfnAYXBCDEFGHIJKLMN | 69.1 | 100 |
Cluster9 | lanthipeptide | RSP | lanM,lcnC, lcnDR2, nisFEGRK, nukEFG | 8.4 | - |
Cluster10 | amylocyclicin | RSP | acnBACDE | 4.4 | |
Cluster11 | unkown | t3pks | bpsAB | 1.6 | - |
Cluster12 | unkown | terpene | - | - | - |
Cluster13 | unkown | terpene | - | - | - |
Cluster14 | butirosin | otherks | - | - | 7 |
Genes involved in promoting plant growth and triggering plant immunity |
In addition to producing a variety of secondary metabolites with antibacterial and antifungal activities, ES2-4 also contains a series of interactions with colonizing plants, synthesizing phytohormones, inducing systemic responses, etc. to enhance plant nutrition and trigger plant defense responses(Table.5). ES2-4 contains genes required for swarming movement, including genes involved in chemotaxis and biofilm formation. ES2-4 contains the protein encoded by the swrA gene, which is necessary for bacterial colony movement, and its hydrophilic and surfactant properties facilitate colonization of plant cell surfaces and uptake of nutrients(Compant et al. 2005), in addition, including fliD, hag, and other genes. srfABCD, sinR, resE, espA-O, etc are involved in the formation of biofilm, which is beneficial to reducing the surface tension of ES2-4 during chemotaxis(Kearns et al. 2005). Recently, it was found that L-sucrase synthesized by sacB can convert sucrose to levan, which stimulates strong production of surfactin and hyperflagellation, and promote solid surface motility (SSM) and Bacillus subtilis Root colonization(Idris et al. 2007). The direct role of PGPR in the mechanism of promoting plant growth is the secretion of plant hormones such as IAA. Through genome analysis, it was found that ES2-4 also contains genes yhcX, dhaS, and ysnE related to the synthesis of indole triacetic acid. These three genes have been confirmed to be involved in the biosynthesis of IAA through experiments(Idris et al. 2007). Interestingly, we found that ES2-4 contains the gene encoding the alkaline serine protease aprE, which is highly similar (98%) to bace16 of Bacillus sp. B16 encodes an alkaline serine protease which can degrade nematode cuticle and kill it (Qiuhong et al. 2006). We speculate that ES2-4 also has the effect of killing nematodes, but it needs experimental verification. In addition, ES2-4 is capable of synthesizing acetoin and 2,3-butanediol, which have been shown to promote plant growth and induce systemic resistance(He et al. 2012). In summary, ES2-4 has a relatively abundant genetic material basis in promoting plant growth and enhancing sensitivity and immunity and shows great potential in promoting plant growth.
Table 5
Representative genes of B.velezensis ES2-4 probably involved in plant bacterium interactions
Gene | Position | Protein | Description |
sinR | 2435910–2436251 | Transcriptional regulator | Biofilm formation |
epsA-O | 3275462–3291175 | - | Biofilm formation |
abrB | 45830 − 45546 | Transcriptional regulator abrb family | Biofilm formation |
resE | 2231683 − 2229902 | Histidine kinase | Biofilm formation |
lytS | 2739520 − 2737739 | Histidine kinase | Biofilm formation |
Spo0A | 2401748–2402548 | Sporulation transcription factor | defenseBiofilm formation |
ycbA | 255268–256560 | Histidine kinase | Biofilm formation |
sacB | 3884893–3886374 | Levansucrase | Root adhesion |
efp | 2422362 − 2421805 | Elongation factor P | Essential for swarming motility |
SwrA | 3370510 − 3370175 | Swarming motility protein | Essential for swarming motility |
comP | 1196189–1197439 | Histidine kinase | Regulator of surfactin production |
acpT | 370500 − 369826 | 4'-phosphopantetheinyl transferase | Necessary for surface motility and biofilm formation |
fliD | 3384406 − 3382886 | Flagellar capping protein | Elicitation of plant basal defense |
flgK | 3389587 − 3388070 | Flagellar hook-associated protein flgk | Elicitation of plant basal defense |
xynD | 1885217 − 1883814 | Glucuronoxylanase | Extracellular degradation of plant cell walls |
xynC | 1883762 − 1882491 | Glucuronoxylanase | Extracellular degradation of plant cell walls |
xynB | 1811124–1812539 | Xylan 1,4-beta-xylosidase | Carbohydrate metabolism |
lacR | 1167187–1167948 | Lactose phosphotransferase system repressor | Lactose metabolism |
lacE | 1165145 − 1163448 | Pts system | Cellobiose degradation |
lacF | 1165471 − 1165157 | Iia component | Cellobiose degradation |
lacG | 1166934 − 1165534 | Beta-glucosidase | Hydrolyzation of phospholactose |
bglA | 2070530 − 2069088 | Aryl-phospho-beta-D-glucosidase | Glucan degradation |
alsD | 3455837 − 3455070 | Alpha-acetolactate decarboxylase | Synthesis of 2,3-butanediol |
pta | 3604646 − 3603675 | Phosphate acetyltransferase | Strongly up-regulated by root exudates |
butB | 625234 − 624194 | Butanediol dehydrogenase | Promote plant growth |
ilvH | 2678411 − 2677893 | Acetolactate synthase | Promote plant growth |
ilvB | 2680225 − 2678408 | Acetolactate synthase | Promote plant growth |
acuA | 2826837–2827469 | Acetoin utilization protein | Induced systemic resistance |
yhcX | 910595–912133 | GNAT family N-acetyltransferase | IAA synthesis |
ysnE | 3654901–3655359 | GNAT family N-acetyltransferase | IAA synthesis |
dhaS | 2037121–2038608 | Aldehyde dehydrogenase family protein | IAA synthesis |
aprE | 1013755–1014903 | Alkaline protease | Kill cuticle |
cpbD | 1832911 − 1832291 | Chitin-binding protein | Chitin degradation |
csn | 3097663–3098499 | Chitosanase | Chitin degradation |
Plant Pathogens’ Growth Inhibition Assays |
To explore the control potential of strain ES2-4 against plant pathogens, the antagonistic activity was determined in PDA media. As is shown in Fig. 6 and Fig. 7, the strain ES2-4 was cultured with R. Solani, B. dothidea, Gibberella zeae, and F. oxysporum and the results showed that the strain ES2-4 could significantly inhibit the growth of these fungal pathogens. Among them, the inhibitory efficiency against R. solani reached 74.4%, showing a strong inhibitory ability. This result indicated that strain ES2-4 exhibited a broad spectrum against plant pathogens. To further study the inhibitory mechanism of ES2-4 against these four pathogens, we collected some samples from the area of the fungus closest to ES2-4 colonies and observed the morphological characteristics of the hyphae by scanning electron microscopy. As shown in Fig. 8, A-B represents the morphological characteristics of the four fungal hyphae under normal culture conditions, and the hyphae surface is smooth and regular. Compared with the control group, C-D represented the morphological characteristics of the four fungal hyphae in the inhibited state. The hyphae of the four fungi were atrophied, deformed, or twisted to varying degrees. Fig. H The apical atrophy of the hyphae affects the growth of the hyphae. Therefore, we can infer that ES2-4 can change the morphological structure of hyphae to affect the growth activity of fungi.
Detached Leaf Bioassays of Crude Lipopeptides Against A. solani in Tomato
In order to determine the inhibitory ability of strain ES2-4 to plant pathogenic infecting plant leaves, we first prepared a sterile fermentation broth of strain ES2-4 with different dilution ratios and treated in vitro tomato leaves, and then selected the strongest antibacterial ability in the plate confrontation experiment. Rhizoctonia solani was inoculated on tomato leaves, and the diameter of the lesions on the leaves was observed. As shown in Fig. 9 and Fig. 10, after 7 days of culture, the leaf necrosis area of the control group without fermentation broth basically occupied the entire leaf. However, the leaves treated with different dilution times of fermentation broth showed lesions with different diameters, which were significantly different from those of the control group. Among them, when the dilution ratio is 0, the diameter of the lesions is the smallest, which may be the most antibacterial substances and the strongest inhibitory ability against Rhizoctonia solani. Therefore, we can infer that ES2-4 can synthesize and secrete secondary metabolites that can inhibit the further infection of tomato leaves by Rhizoctonia solani, and has a strong inhibitory effect.