Bacillus sp. 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 sp. K-9
The length of 16S rDNA of strain K-9 was 1447 bp. The 16S rDNA 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 sp. SA3 strain branched closely, with 63% similarity rate.
In the CGView circle of Bacillus sp. K-9 genome (Fig. 1), 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 sp.K-9, and the proportion of protein -coding genes was 89.87%.
Table 1
Complete genome features of Bacillus sp.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 (Radisic et al. 2021; Paolo et al. 2020). The COG classification number of Bacillus sp. 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 sp. K-9 genome are shown in Fig. 5. In Bacillus sp. 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 sp. 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 sp. 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 sp. K-9. The results showed that there were 12 secondary metabolite synthesis gene clusters in the genome of Bacillus sp. 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(Fig. 8-A); 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-B).
Table 2
Prediction of secondary metabolites of Bacillus sp.K-9 strain
Gene clustera
|
Typeག
|
Locationགྷ
|
Secondary metabolitesང
|
Functione
|
Similarityf
|
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. |
Antibacterial peptide LcI gene exists in strain K-9, and it has been reported (Chen,1996) that antibacterial peptide LcI protein in Bacillus can effectively inhibit the growth of pathogens, and it is speculated that secretion of antibacterial substances in strain K-9 is one of its antibacterial mechanisms. The specific gene sequence of LcI in K-9 genome is:ATGAACTTCAAAAAAGTGTTAACCGGTTCTGCGCTGTCTC TTGCCTTACTGATGTCTGCCGCTCCTGCCTTTGCCGCATCACCAACAGCATCCGCATCCATGGAAAATAGCCCAATCTCAACGAAAGCAGATGCCGGCATTAATGCCATCAAACTCGTTCAAAGCCCTAACGGAAACTTCGCTGCTTCTTTCGTTTTAGATGGAACAAAGTGGATTTTCAAAAGCAAATACTATGACAGCAGCAAAGGGTATTGGGTAGGGATTTATGAAAGTGTAGATAAATAA.
The specific protein sequence of LCI in k-9 is:MNFKKVLTGSALSLALLMS AAPAFAASPTASASMENSPISTKADAGINAIKLVQSPNGNFAASFVLDGTKWIFKSKYYDSSKGYWVGIYESVDK. Antimicrobial peptide LCI protein sequences in K-9 were compared online in NCBI database. The results showed that the similarity of antimicrobial peptide LCI protein with Bacillus (WP_032872774.1) was 100%. It has been reported that LCI protein of Bacillus subtilis has significant effects on Xanthomonas campestris PV Oryzea, Pseudomonas Solanacearum PE1 is one of the main effective ingredients with strong antibacterial activity(Gong 2011). According to the bacteriostatic activity test results of crude protein extract of K-9 against Scab and protein gene analysis (Figure 8-C), it was speculated that the protein bacteriostatic activity substance of K-9 against Scab was LCI protein and the bacteriostatic gene was LcI gene.