Antimicrobial Activity of Bacillus velezensis K-9 Against Potato Scab and Its Genome-wide Analysis

doi:10.21203/rs.3.rs-2015769/v1

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Antimicrobial Activity of Bacillus velezensis K-9 Against Potato Scab and Its Genome-wide Analysis

https://doi.org/10.21203/rs.3.rs-2015769/v1

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We isolated a bacterial strain K-9, identified as a species of the genus Bacillus, from a potato tuber sample. The inhibition rate of the strain K-9 against potato scab (Streptomyces scabies) was 44.90%. The whole genome sequence of Bacillus velezensis K-9 was obtained, and genomic analyses were performed. Complete genome sequence of strain K-9 was obtained through PacBio RSII and Illumina platforms. The size of genome is 3891530 bp, with estimated 3915 coding DNA sequences, GC content of 46.45%, 79 tRNA, and 10 rRNA. In addition, functional annotation of the strain K-9 genes was performed by GO, COG, KEGG, and CAZy analyses. There were 12 gene clusters for secondary metabolite synthesis in the genome of Bacillus velezensis K-9. Except for the unknown metabolites of the T3PKS (third polyketone), terpene and lanthipeptide class-II, the other eight gene clusters are associated with the synthesis of secondary metabolites that are common in Bacillus and have antibacterial activity. Based on the functional analysis of the genome, the lipopeptide extract and crude protein extract of the strain were verified as bacteriostatic. The metabolites of its antibacterial activity were analyzed and identified by metabolomics technology. The antibacterial protein-related substances such as polyketide synthetase, plastin synthetase, bacitin-biosynthetic protein and flagellin were isolated and identified from the crude protein extract. In the future, information derived from the thorough genomic analyses of this strain may be helpful in directed genetic modification of bacterial strains for production of antibacterial substances to control potato scab.

Bacillus velezensis K-9

Potato scab

Complete genome sequence

Gene function

Potato (Solanum tuberosum L.) is rich in carbohydrates and a variety of minerals and nutrients needed by humans¹. Potato plays an important role in economy and global food security. However, with the rapid development of potato industry, the incidence and severity of scab have been increasing year by year due to the continuous planting of potatoes, which negatively influenced the development of potato industry. Potato scab, as a disease spread by soil and seed potato, is widespread in the potato-growing areas². Potato scab is widely distributed in the world, and has been found in Argentina and other South American countries, the United States, Germany, Mexico, Finland, Japan, and South Korea, as well as in Yunnan, Heilongjiang and Shandong provinces of China^3–6. Potato scab can invade plants through skin cuts, wounds or young tubers, and can infect also other root crops such as radish, beet, carrot, and parsnip^7–8.

At present, chemical pesticides are difficult to be replaced, but the improper use of chemical drugs not only produces harmful effects on plants, but also leads to human and animal poisoning, killing beneficial microorganisms, leading to drug resistance of pathogens. And in many cases chemical drugs cannot completely cure plant diseases. The use of antagonistic bacteria to reduce or replace the use of pesticides and fertilizers is of great significance for maintaining the biodiversity of plant microecosystems, maintaining ecological balance and realizing sustainable development.

Pathogen and antagonistic bacteria

Streptomyces scabies (S. scabies) was isolated and preserved by plant pathology laboratory of Heilongjiang Bayi Agricultural University. S. scabies cultured in yeast malt agar medium (YME) (yeast extract 4 g, malt extract 10 g, glucose 4 g, sterile water 1000 mL, pH 7.2). The cultures were stored at -80℃.

Strain Bacillus velezensis K-9 was isolated from potato tuber. Tuber homogenate dilution was spread on beef extract medium (NA) plates (beef extract 3 g, peptone 5 g, yeast extract 1 g, sucrose 10 g, agar 17 g, sterile water 1000 mL, pH 7.2). The isolates were purified and cultured at 28℃and stored at -80℃.

Antimicrobial activity of Bacillus velezensis K-9 against potato scab

Antagonistic strains were screened by the dual culture method⁹. Streptomyces scabies was cultured on YME plates for 7 d and then inoculated in YME liquid medium at 30 °C, 180 r/min for 72 h to prepare the S. scabies suspension. The bacterial suspension was filtered through the filter membrane (0.22 μm) into the sterilizing tube to obtain sterile fermentation liquid for use. Then, Bacillus velezensis K-9 strain, bacterial suspension and sterile filtrate were antagonized by plate. The antagonistic bacteria were then inoculated on the YME plates (85 mm) containing 200 μL of S. scabies suspension. The plates containing only Streptomyces scabies were used as the control. The inhibition rate of bacterial strains was determined. The antibacterial activity of antagonistic bacteria was evaluated by measuring the diameter of inhibition zone. Bacteria inhibitive rate (%) = bacteriostatic diameter/plate diameter×100.

Extraction of genomic DNA from Bacillus velezensis K-9

Bacillus velezensis K-9 single colony was cultured in LB liquid medium on a shaking table at 28 ℃ for 12 h. The antagonistic bacterium was further identified through the analysis of its 16S rRNA gene sequences^10-13. DNA of antagonistic strains was extracted by a Tiangen Bacterial genome kit. The 16S rRNA primers were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification of 16S rDNA was done in a mixture containing PCR MIX 25 μL, Forward and reverse，primers 1 μL each, and bacterial DNA 2 μL. The PCR reaction procedure was: pre-denaturation at 95 ℃ for 5 min; 95 ℃ denaturation for 30 s, 57 ℃ annealing for 30 s, 72 ℃ extension for 45 s, 30 cycles; the final elongation was at 72 ℃ for 7 min. The products were sequenced by Sangon Biotech Co. Ltd (Shanghai, China). The sequencing platform of Bacillus velezensis K-9 genome (including plasmid) was PacBio RS II. After BLAST comparison on NCBI, strains with high similarity in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were selected, and the neighbor-joining method in MEGA 7 software was used to construct a phylogenetic tree. The nucleotide sequences were deposited in GenBank database. The GenBank accession number of the 16S rRNA gene sequence of strain K-9 is OL378201. The GenBank accession number of complete genome sequence of strain K-9 is JAKQYO000000000.

Genomic DNA sequencing and assembly

An Illumina sequencer was used to sequence DNA. The HiSeq raw sequences containing adapter/primers were filtered out using FastQC (V0.11.9). Trimmomatic was used to perform quality shearing on Illumina sequencing data. SPAdes was used to splice second-generation sequencing data, and Gap Filler was used to fill gaps in contigs. PrInSeS-Gis was used for sequence correction to correct editing errors and insertion loss of small fragments in the splicing process.

Gene prediction and annotation

After extracting the coding genes, Interprosca was used to extract the annotation information of GO database. Blastp was used to compare the encoded proteins to KEGG database, and the best result with a comparison coverage greater than 30% was retained as the annotation result. The encoded proteins were annotated to the COG database using rpsblast. The gene protein sequences were compared with CAZy database by HMMER3, and the functional annotation information was obtained. Using the online prediction software antiSMASH6.0.1 (https://antismash.secondarymetabolites.org/#! /start) the secondary metabolite synthesis gene cluster of Bacillus velezensis K-9 was analyzed to find the potential inhibitory metabolite synthesis genes.

Strain K-9 produced ferriophagic activity, lipopeptide crude and protein extracts antibacterial activity

The crude extract was obtained by acid precipitation method, and 100 μL was used for identification of antagonistic activity. Crude protein extract was obtained by ammonium sulfate precipitation method, and 100 μL was used for antagonistic activity identification¹⁴.

Statistical analysis

Statistical analysis was performed using SPSS software(IBM SPSS Statistics, Version 22.0, International Business Machines Corporation, New York, in USA). Mean values were compared using Duncan’s new multiple range test at the 1% (P< 0.01) level of significance between treatments.

Bacillus velezensis K-9 antagonizes S. scabies

The antagonistic activity to S.scabies was screened by the double culture method. The maximum antibacterial rate of strain K-9 was detected for fermentation liquid, and the minimum antibacterial activity was in cell-free filtrate. The antibacterial rate of fermentation broth was 53.92%. The antibacterial rate of strain K-9 was 44.90% and of cell-free filtrate was 27.25% (Fig. 1).

Genomic identification of Bacillus velezensis K-9

The length of 16S rRNA of strain K-9 was 1447 bp. The 16S rRNA gene sequence of strain K-9 was related to Bacillus velezensis, Bacillus amyloliquefaciens and Bacillus subtilis and other strains, sharing more than 97% of the genetic sequences. The results of phylogenetic tree showed that K-9 and Bacillus velezensis SA3 strain branched closely, with 63% similarity rate(Fig. 2).

In the CGView circle of Bacillus velezensis K-9 genome (Fig. 3), corresponding information from outer circle to inner circle is as follows: the first circle (outermost circle), GC content; circle 2, sequencing depth; circle 3, gene category; the fourth circle, CDS information on the positive chain; circle 5, CDS information on the negative chain; the sixth circle, negative chain COG functional classification annotation information; circle 7 (innermost circle), plus link COG functional classification annotation information.

The genome size was 3 891 530 bp. The GC content was 46.45%, and there were 11 contigs. The number of tRNA and rRNA was 79 and 10, respectively. There were 3915 protein-coding genes in Bacillus velezensis K-9, and the proportion of protein -coding genes was 89.87%(Table 1).

Table 1

Complete genome features of *Bacillus velezensis*K-9
Property	Value
Sequencing platform	PacBio RS II
Genome size (bp)	3891530
Contig numbers	11
DNA G + C (%)	46.45
Number of tRNA genes	79
Number of rRNA genes	10
Number of protein coding genes	3915
Ratio of protein coding genes	89.87

Annotation of the Clusters of Orthologous Groups (COG) of proteins has been developed based on protein sequence alignment based on gene sequences, with each COG representing a gene family^15–16. The COG classification number of Bacillus velezensis K-9 genome was 4, and the number of COG types was 20. The number of genes with COG annotation was 1238, accounting for 33.05% of all genes. As can be seen from COG annotation information (Fig. 4), apart from the predicted gene function represented by "R" and the unknown function represented by "S", "K" represents the transcription gene family with the largest number of members (239), followed by "E" representing the amino acid transfer and metabolism family with 238 members.

Through genomic GO (Gene Ontology) annotations, biological terms can be standardized for easy communication. Statistical results of GO annotation of Bacillus velezensis K-9 genome are shown in Fig. 5. In Bacillus velezensis K-9 genome, the number of GO classifications was 3, and the number of genes involved in annotation was 3004, accounting for 80.19% of all genes.

In the KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations of Bacillus velezensis K-9 genome, the gene functions were divided into six major categories. Among them, 82 genes were annotated to cellular processes, 374 genes to environmental information processing, 240 to genetic information processing, 2066 to metabolism, 68 to human diseases, and 28 genes to organismal systems. Detailed breakdown of each category is shown in Fig. 6.

As shown in Fig. 7, among the genes coding for fermentation activity, the glycoside hydrolase genes were the most abundant in Bacillus velezensis K-9, accounting for 29.65%, followed by 48 genes of glycosyl transferases, accounting for 27.90%. The gene numbers for carbohydrate esterases, carbohydrate-binding modules, auxiliary oxidoreductases, and polysaccharide lyases were 39 (22.67%), 18 (10.47%), 11 (6.40%), and 5 (2.91%), respectively. The main components of plant diseases and residues in soil are sugars and proteins, and the strain K-9 contains β-glucosidase (EC3.2.1.21), chitin (β-1,6-glucanosyltransferase, EC 2.4.1.-), α-amylase (EC 3.2.1.1), cellulase (EC 3.2.1.4) and other enzymes. The biocontrol mechanism of strain K-9 was further analyzed from the perspective of gene.

AntiSMASH6.0.1 software was used to predict the secondary metabolites of Bacillus velezensis K-9. The results showed that there were 12 secondary metabolite synthesis gene clusters in the genome of Bacillus velezensis K-9. Except for the unknown metabolites of the third polyketone (T3PKS), terpene and lanthipeptide-class-II, the other eight gene clusters are associated with biosynthesis of secondary metabolites that have antibacterial activity and are common in Bacillus: Clusters 1 and 5, polyketone synthesis genes; Cluster 4, synthesis gene cluster of Camelina; Cluster 6, macrolide synthesis; Cluster 9 butamectin synthesis; Cluster 10, lysobacterin synthesis; Cluster 11, siderophores synthesis; and Cluster 12, surfactant synthesis. Gene clusters 4, 11 and 12 are associated with synthesis of secondary metabolites through non-ribosomal pathways (Table 2, Fig. 8-A).

Table 2

Prediction of secondary metabolites of *Bacillus velezensis*K-9 strain
Gene cluster^a	Type^ག	Location^གྷ	Secondary metabolites^ང	Function^e	Similarity^f
1	TransAT-PKS	546640–638999	Difficidin	Anti-bacterial	100%
2	T3PKS	767634–808360	—	—	—
3	Terpene	873700–893826	—	—	—
4	NRPS	922563–1056873	Fengycin	Anti-fungal	100%
5	TransAT-PKS	1130615–1231180	Bacillaene	Anti-fungal, anti-bacterial	100%
6	TransAT-PKS	1450368–1538601	Macrolactin	Anti-fungal, anti-bacterial, anti-virus	100%
7	Lanthipeptide-class-ii	1705164–1734052	—	—	—
8	Terpene	1854545–1875285	—	—	—
9	PKS-like	1957329–1998573	Butirosin	Anti-bacterial	7%
10	Other	83195–124613	Bacilysin	Anti-bacterial	100%
11	NRPS	660923–712714	Bacillibactin	Accumulate and take up iron ions	100%
12	NRPS	199691–265098	Surfactin	Anti-virus, anti-mycoplasma,anti-tumour	82%

a : the secondary metabolite synthesis gene cluster annotated with antiSMASH6.0.1, b : the gene cluster type,c : the location of gene clusters in the strain genome, d : the secondary metabolite that may be produced based on the gene cluster, e : the bioactive function of metabolites synthesized by gene cluster, f : similarity to known gene clusters,“—”: indicates unknown.

According to the bacteriostatic activity test results of crude protein extract of Bacillus velezensis K-9 against Scab and protein gene analysis (Figure 8-B), It is presumed to be the main antibacterial substance of the K-9 against Scab.

Subsequently, the crude protein extract was detected by SDS-PAGE. As can be seen from Fig. 9, the protein subunits were distributed below 100kDa, and there were many bands. The gel electrophoresis pattern is clear and the bands are rich, which can be used for subsequent LC-MS/MS metabolic test.

Each protein was enzymatically digested to produce peptides of different lengths, which were then identified by LC-MS/MS(Table 3). Based on Uniprot-Bacillus Velezensis database, 4001 peptides and 500 proteins were found. The protein information related to antimicrobial activity is shown in Table 3. Polyketide synthetase (gene name: PksN、PksL), plastin synthetase, subtilisin, flagellar biosynthetic protein (gene name: FliP) and so on were identified. Polyketide synthetase, coptigenin synthetase and non-ribosomal peptide synthetase have been identified from crude proteins to catalyze the synthesis of antibacterial substances. The genes of these three enzymes are often used as indicator genes to screen for the production of novel structural natural active compounds^17–18. Subtilisin, an antimicrobial substance synthesized by the ribosome pathway, has the characteristics of good stability and strong acid-base adaptability, and has antibacterial effect on most Gram-positive bacteria¹⁹. FliP genes play a role in the flagellum-specific transport system. Flagellin is a typical virulence factor, which helps bacterial infection and colonization and plays an important role in bacteriostatic activity²⁰. Chitin binding protein can help improve plant defense system, thereby enhancing plant disease resistance²¹. Chitosanase is a hydrolytic enzyme that exclusively degrades chitosan. However, it is also known as Pathogenesis related protein, which can increase the disease resistance of plants. Bacillus Velezensis K−9 inhibited the activity of potato scab pathogen by producing these five antibacterial substances.

Table 3

*Bacillus velezensis* K-9 results of qualitative analysis of protein substance by LS-MS
Login number	Protein information	The name of the protein
A0A7W4QCN4	Polyketide synthase PksN OS = Bacillus velezensis OX = 492670 GN = pksN_2 PE = 4 SV = 1	Polyketide synthase PksN
A0A7W4LRG1	Polyketide synthase PksL OS = Bacillus velezensis OX = 492670 GN = pksL_5 PE = 4 SV = 1	Polyketide synthase PksL
A0A1D9PPV3	Bacilysin biosynthesis protein BacB OS = Bacillus velezensis OX = 492670 GN = bacB PE = 4 SV = 1	Bacilysin biosynthesis protein BacB
L7WLS3	Subtilisin OS = Bacillus velezensis OX = 492670 GN = kerBPN PE = 3 SV = 1	Subtilisin
A0A8G1PD45	Flagellar biosynthetic protein FliP OS = Bacillus velezensis OX = 492670 GN = fliP PE = 3 SV = 1	Flagellar biosynthetic protein FliP
A0A7S8F0Q0	Fengycin synthetase B (Fragment) OS = Bacillus velezensis OX = 492670 GN = fenB PE = 4 SV = 1	Fengycin synthetase B
A0A6H3B0Z0	Non-ribosomal peptide synthetase OS = Bacillus velezensis OX = 492670 GN = CHR37_17550 PE = 3 SV = 1	Non-ribosomal peptide synthetase NRPS
A0A8G1LH67	Flagellin OS = Bacillus velezensis OX = 492670 GN = K4L72_17430 PE = 3 SV = 1	Flagellin
A0A8F0EXT8	Chitosanase OS = Bacillus velezensis OX = 492670 GN = csn PE = 3 SV = 1	Chitosanase
A0A1D9PQS1	Chitin-binding protein OS = Bacillus velezensis OX = 492670 GN = gbpA PE = 4 SV = 1	Chitin-binding protein
Note: OS is the Latin name of the species, GN is the gene name, PE is the protein existence certificate (PE = 1: experimental evidence at protein level, PE = 2: experimental evidence at tranlevel, PE = 3: protein inferred from homology, PE = 4: protein predicted, PE = 5: protein uncertain), SV is the protein sequence version.

The problems of high residue, high pollution and harm to human and livestock caused by the use of chemical pesticides have become increasingly prominent.Green prevention and control technology has become a research hotspot, among which the application of biological antagonism to control various diseases has been proved to be a safe and effective new method. Bacillus has good control effect on crop diseases in biological control field. Traditional experimental and identification methods are difficult to fully analyze the antimicrobial substances of Bacillus, and can not fully explore all its antimicrobial genes. In order to further study this strain, the whole genome sequencing and bioinformatics analysis techniques were used to obtain the genome sequence of strain K-9. The genome size of strain K-9 was determined to be 3 870 130 bp, and a total of 4 161 genes were encoded. Meanwhile, the genome data were compared and analyzed with GO, COG, KEGG and other databases. The functional annotation and data statistics of K-9 genome were completed, and the classification status of K-9 was determined by 16S rRNA gene, and the gene clusters of secondary metabolites and antibacterial proteins that might play a role in its antibacterial action were explored from the perspective of molecular biology. The CAZy family of Bacillus velezensis K-9 contains genes related to β-1, 3-glucanase, chitinase and other cell wall degradation enzymes. It is speculated that the cell wall degradation of pathogenic bacteria is one of the antibacterial mechanisms of strain K-9.

Bacillus is an excellent resource of biocontrol bacteria. Its secondary metabolites are rich, including antibacterial proteins, lipopeptides, polyketones and other antibacterial substances. The gene clusters of secondary metabolites of strain K-9 were predicted, and 12 gene clusters of lipopeptide and polyketone antibiotics were obtained. There were 4 unknown gene clusters, 7 antibiotic synthesis gene clusters (Difficidin,Fengycin, Bacillaene, Macrolactin,Bacilysin, Bacillibactin, and Surfactin) with high similarity, and 1 gene cluster with low similarity 7% antibiotic gene cluster (Butirosin). After homology comparison of functional types, Bacillus velezensis K-9 contained multiple gene clusters related to antibacterial active substances such as PKS (polyketone synthase), terpene, NRPS (non-ribosomal peptide synthase), and bacilysin. These gene clusters may be involved in the anti-pathogen activity. Freitas et al²². conducted a whole-genome analysis of Bacillus 64 − 1 and found that polyketo synthase (PKS), non-ribosomal peptide synthase (NRPS) and bacteriolysin may be related to antibacterial activity. Zeng et al²³. predicted the function of Bacillus pumilus GLB197 gene. Six gene clusters were related to the antibacterial activity of Bacillus pumilus GLB197, and NRPS gene clusters were predicted to be used in the production of antibiotics. Xu et al²⁴. sequenced the genome of Bacillus amylolitica HAB-2 and found that 13 gene clusters (NRPS and PKS, etc.) were involved in antibacterial action. In summary, the related gene clusters (NRPS, PKS and bacteriocin, etc.) that control the synthesis of active substances that are important to the anti-pathogenic activity of Bacillus. The similarity of Butirosin antibiotic gene cluster found in this study was only 7%, indicating that this gene cluster may synthesize new antibacterial substances, which needs further study. It was predicted that strain K-9 could prevent scab by producing cell wall hydrolases, antagonistic active compounds and bacteriostatic proteins, which had great application potential in agriculture. Bacillus velezensis K-9 can produce protein antibacterial substances, and lipopeptides, as a new type of bioactive agent, not only have good emulsifying properties, but also have antibacterial effects. This provides a theoretical basis for the further development of biocontrol agents for Bacillus velezensis and its metabolites.

Funding This research was supported by The Integrated Research and Demonstration of Potato Fertilizer and Pesticide Reduction Technology of the National Researchand Development Program of China(2018YFD020080706).

Author contributions

MaShuang wrote the main manuscript text and WangTeng give modification suggestions and data processing , Jiarong Ru prepared figures 2, Lili Jiang prepared table 1,Wang Cong prepared figures 1 and Yanjie Wang prepared language change . All authors reviewed the manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The GenBank accession number of the 16S rRNA gene sequence of strain K-9 is OL378201. The GenBank accession number of complete genome sequence of strain K-9 is JAKQYO000000000.

Credit author statement

All of the authors have read and approved the manuscript. This work has not been published previously, nor is it being considered by any other peer-reviewed journal.

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Antimicrobial Activity of Bacillus velezensis K-9 Against Potato Scab and Its Genome-wide Analysis

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