- Short genome report
- Open Access
Whole genome shotgun sequence of Bacillus amyloliquefaciens TF28, a biocontrol entophytic bacterium
Standards in Genomic Sciencesvolume 11, Article number: 73 (2016)
Bacillus amyloliquefaciens TF28 is a biocontrol endophytic bacterium that is capable of inhibition of a broad range of plant pathogenic fungi. The strain has the potential to be developed into a biocontrol agent for use in agriculture. Here we report the whole-genome shotgun sequence of the strain. The genome size of B. amyloliquefaciens TF28 is 3,987,635 bp which consists of 3754 protein-coding genes, 65 tandem repeat sequences, 47 minisatellite DNA, 2 microsatellite DNA, 63 tRNA, 7rRNA, 6 sRNA, 3 prophage and CRISPR domains.
Bacillus amyloliquefaciens is ubiquitous in nature. Some strains are used as biocontrol agents because of their ability to produce antagonistic metabolites, plant growth promoters and plant health enhancers [1–4]. B. amyloliquefaciens is usually divided into two subspecies by genome comparison and classical bacterial taxonomy. Plant growth-promoting rhizobacterial strains are classified as B. amyloliquefaciens subsp. plantarum , while other strains are regarded as B. amyloliquefaciens subsp. amyloliquefaciens . B. amyloliquefaciens TF28 is an endophytic bacterium that was isolated from soybean root. Previous studies have shown that B. amyloliquefaciens TF28 could inhibit soil borne and air borne plant pathogenic fungi by competition, synthesizing antifungal metabolites and inducing systemic plant resistance [6, 7]. Based on 16S rRNA, DNA gyrase subunit A (gyrA) and RNA polymerase subunit B (rpoB) gene sequence analysis, B. amyloliquefaciens TF28 was classified as B. amyloliquefaciens subsp. plantarum . Here we present a whole-genome shotgun sequence of B. amyloliquefaciens TF28 and its annotation for facilitating its application in the biocontrol of plant diseases.
Classification and features
B. amyloliquefaciens TF28 was isolated from soybean root in China. It exhibited an unusual ability to inhibit a wide range of plant pathogenic fungi. The cell morphology of strain TF28 was determined using scanning electron microscopy (Fig. 1). Cells of B. amyloliquefaciens TF28 are Gram-positive, rod shape, aerobic and endospore- forming. Strain TF28 utilizes glucose and lactose to produce acid and hydrolyzed gelatin and starch. Starin TF28 is positive for Vogues-Proskaur and Methyl red reaction, nitrate reduction and citrate utilization. Current taxonomic classification and general features of B. amyloliquefaciens TF28 are provided in Table 1.
The 16S rRNA gene sequence of strain TF28 and other available 16S rRNA gene sequences of closely related species collected from NCBI database were used to construct a phylogenetic tree (Fig. 2, Additional file 1: Table S1). The evolutionary history was inferred using the Neighbour-joining method with MEGA software version 5.10. BLAST analysis showed strain B. amyloliquefaciens TF28 shared 99.3–99.7 % 16S rRNA gene identities with the other 14 type strains of Bacillus species. Taxonomic analysis showed that 14 type strains were divided into two groups. Strain TF28 together with B. amyloliquefaciens subsp. plantarum FZB42T, B. methylotrophicus CBMB205T, B. amyloliquefaciens subsp. amyloliquefaciens DSM7Tand B. siamensis PD-A10T were clustered into one group. Other strains ( B. atrophaeus NBRC 15539T , B. vallismortis DSM 11031T , B. tequilensis 10bT, B. subtilis 168T, B. subtilis subsp. subtilis DSM 10T , B. subtilis subsp. inaquosorum BGSC 3A28T , B. subtilis subsp. spizizenii NBRC 101239T , B. mojavensis NBRC 15718T , B. malacitensis CR-95T and B. axarquiensis LMG 22476T ) were clustered into another group. Two type stains of B. amyloliquefaciens subspecies, B. amyloliquefaciens B. amyloliquefaciens subsp. plantarum FZB42T and B. amyloliquefaciens subsp. amyloliquefaciens DSM7T were attributed to the different clade. Strain TF28 was most closely related to B. amyloliquefaciens subsp. plantarum FZB42T with 99.7 % 16S rRNA gene sequence identity. Strain TF28 was classified as B. amyloliquefaciens subsp. plantarum .
Genome sequencing information
Genome project history
Genome of B. amyloliquefaciens TF28 was sequenced by Huada Gene Technology Co., Ltd, Shenzhen, China. The Whole Genome Shotgun sequence has been deposited in GenBank database under the accession number JUDU00000000. The summary of the project information is shown in Table 2.
Growth conditions and genomic DNA preparation
B. amyloliquefaciens TF28 was grown in LB medium at 30 °C for 16 h. One liter cultures at the exponential growth phase was taken and centrifuged at 4 °C, 5000 rpm for 10 min. The pellet was collected and about 5 g cell pellet was used to extract genomic DNA by CTAB method . The quality of DNA was assessed using a Qubit Fluorometer. Total DNA (280.6 μg) was obtained to do genome sequencing.
Genome sequencing and assembly
Genomic DNA was sheared randomly. The required length DNA fragments were retained by electrophoresis and used for construction of a 500 bp and 6000 bp long paired-end library. Sequencing was performed by Illumina HiSeq 2000 sequencing platform. Sequencing of the 500 bp library generated 6,649,820 reads (representing 554 Mbp of sequence information), while sequencing of the 6,000 bp paired-end library generated 3,633,388 reads (290 Mbp). Both libraries achieved a genome coverage of 190× for an estimated genome size of 4.4 Mbp. All generated reads were quality trimmed to obtain clear reads. The trimmed reads were assembled by SOAPdenovo software 2.04 using the available genome sequence of B. amyloliquefaciens subsp. plantarum FZB42T(CP000560) as reference-guided assembling. The final assembly yielded 182 contigs and 3 scaffolds representing 3.9 Mbp of sequence information.
The genome sequence was annotated by a combination of several annotation tools. Genes were identified by Glimmer 3.02 . DNA tandem repeat sequences, minisatellite DNA and microsatellite DNA were found with the Tandem Repeats Finder 4.04 . Prediction of non-coding RNA was performed using rRNA database blasting or rRNAmmer 1.2 for rRNA , tRNAscan-SE 1.23 for tRNA and their secondary structure , and infernal software and Rfam database for sRNA . Prophage was predicted using PHAST software 2013.03.20 . CRISPR domains were found using CRISPR Finder 0.4 . Functional annotation of protein coding genes was based on gene comparisons with GO database (version 1.419) , KEGG database (version 59) , Cluster of Orthologous Groups of proteins(COG)(version 20090331) , NR database(version 20121005), SwissProt (version 201206)  and Pfam databases (version 25) .
The genome statistics are provided in Table 3 and Fig. 3. The high quality draft genome of B. amyloliquefaciens TF28 was distributed in 182 contigs with a total size of 3,987,635 bp and an average G + C content of 46.38 %. Genome analysis showed that the genome of strain TF28 contained 3,754 protein coding genes, 65 tandem repeat sequences, 47 minisatellite DNA, 2 microsatellite DNA, 63 tRNA, 7 rRNA, 6 sRNA, 3 prophage and 3 CRISPR domains. The predicted protein coding genes represented 89.57 % of the total genome sequence, with a total length of 3,571,596 bp. The majority of protein coding genes (76.13 %) were assigned to putative functions. The distribution of genes into COG functional categories is presented in Table 4.
Insights from the genome sequence
Protein coding genes were mainly classified into 3 parts based on their functions by GO analysis (Fig. 4). 1901, 2993 and 4309 genes participated in cellular component, molecular function and biological process, respectively. The metabolic pathway analysis using KEGG annotation showed that the majority of protein coding genes participated in metabolism, genetic information processing, environmental information processing and cellular processes (Fig. 5). 154 metabolic pathways were found using KEGG orthology, including glycolysis, TCA cycle and pentose phosphate pathways, fructose, mannose and galactose metabolisms pathways, fatty acid biosynthesis and metabolism pathways, ubiquinone and other terpenoid-uquinoid synthesis pathways, bacterial chemotaxis, biosynthsis of siderophore group nonribosomal peptides, antibiotic biosynthesis (tetracycline, penicillin and cephalosporin, streptomycin, novobiocin and vancomycin) as well as noxious substance degradation pathways (caprolactam, atrazine, ethylbenzene, toluene, polycyclic aromatic hydrocarbon, chloroalkane and chloroalkene, bisphenol, naphthalene, aminobenzoate, limonene and pinene), and so on.
Genome similarity was detected based on Mummer blast by comparing the genome sequence of strain TF28 with the type strain B. amyloliquefaciens subsp. plantarum FZB42Tat amino acid level . The results showed that genome similarity of B. amyloliquefaciens TF28 and B. amyloliquefaciens subsp. plantarum FZB42T reached 98.69 %. Core-pan gene was also determined based on NCBI blast and Muscle analysis . 201 strain-specific genes for B. amyloliquefaciens TF28 was observed, which may contribute to species-specific features of this bacterium. Among them, 83 genes are classified into 17 COG functional categories major belonging to carbohydrate transport and metabolism (6.97 %), general function prediction only (4.48 %), defense mechanisms (4.48 %), signal transduction mechanisms (3.48 %), amino acid transport and metabolism (3.98 %). The remaining 116 unique genes (57.71 %) are not classified into any COG categories (Table 5). Comparative genome analysis revealed that B. amyloliquefaciens TF28 possessed the giant gene clusters for non-ribosomal synthesis of the polyketides difficidin (TH57_02955-TH57_03045) and bacillaene (TH57_05575-TH57_05655), the antifungal lipopetides surfactin (TH57_12375-TH57_12430), plipastatin (TH57_04780-TH57_04835), mycosubtilin (TH57-04955-TH57-04980), bacilysin (TH57_15685-TH57_15710) and bacillibactin (TH57_05755-TH57_05800) (Additional file 2: Table S2). The size of these gene clusters accounted for 6.8 % of genome, which was smaller than that of strain FZB42T(8.9 %) . Mycosubtilin and plipastatin synthesis gene clusters were only observed in strain TF28. These gene clusters produce the secondary metabolites like NRPSs, PKS, and peptide antibiotics usually displaying antifungal and antibacterial activities [23–25]. The finding of these gene clusters revealed that strain TF28 possessed a high potential to biocontrol. In addition, sporulation genes, spo0ABFJ(TH57_02695,01435,16015and14190),spoVABCDEFKSMRT(TH57_03250,03255,03260,03265,03270,03275,03280),SpoIIBPMERDQSASB(TH57_05470,00570,06315,09520,13850),spoIIIABCDEFGH(TH57_01370,02065,03200,07810,07815,13800,16095,16205and16305),coaX(TH57_01485),YtrIH(TH57_00725,009730),ylbJB(TH57_06685),ydcC(TH57_11735),ydhD(TH57_07580),cse15(TH57_07135), yunB(TH57_18555) and motility genes, motAB (TH57_074157, 07420) and swrABC (TH57_16980,05970 and 10760), were found in the genome.
Comparative genomic analysis of B. amyloliquefaciens TF28 and other 22 strains of B. amyloliquefaciens possessing complete genomic sequences indicated that the genome size of the strain TF28 was somewhat bigger than that of B. amyloliquefaciens subsp. plantarum FZB42T and B. amyloliquefaciens subsp. amyloliquefaciens DSM7T . Three strains, B. amyloliquefaciens IT-45, B. amyloliquefaciens NAU-B3 and B. amyloliquefaciens TF28, possessed CRISPR domains by CRISPR Finder on line (Additional file 3: Table S3, Additional file 4: Table S4, Additional file 5: Table S5, Additional file 6: Table S6). B. amyloliquefaciens TF28 possessed 3 CRISPR domains. The CRISPR length is 422 bp with 81 bp direct repeat (DR) sequences be separated by 5 spacers. No CRISPR associated gene was observed due to the incomplete genome sequence. B. amyloliquefaciens NAU-B3 had 1 CRISPR domains. The CRISPR length is 67 bp with 26 bp DR sequences be separated by 1 spacer. B. amyloliquefaciens IT-45 had 2 CRISPR domains. The CRISPR length is 129 bp with 37 bp DR sequences be separated by 1 spacer. The full-length sequence of protein-coding gene, DNA gyrase subunit A (gyrA) and RNA polymerase subunit B (rpoB) derived from 22 strains of B. amyloliquefaciens , were chosen to phylogenetic analysis. The neighbor-joining (NJ) phylogenetic tree revealed that strain TF28 with most of B. amyloliquefaciens subsp. plantarum clustered into the same group, which is distinct from the type strain B. amyloliquefaciens subsp. amyloliquefaciens DSM 7T (Fig. 6).
In this study, we characterized the genome of B. amyloliquefaciens TF28 isolated from soybean root. Strain TF28 was classified as B. amyloliquefaciens subsp. plantarum on comparative analysis of 16S rRNA sequence, DNA gyrase subunit A (gyrA) and RNA polymerase subunit B (rpoB) gene sequences. The genome of strain TF28 has the giant gene clusters that are linked with biocontrol, including non-ribosomal synthesis of the polyketides difficidin and bacillaene, the antifungal lipopetides surfactin, plipastatin, mycosubtilin, bacilysin and bacillibactin. Mycosubtilin and plipastatin synthesis gene clusters were only observed in strain TF28. Ubiquinone and other terpenoid-uquinoid synthesis, bacterial chemotaxis, biosynthsis of siderophore group nonribosomal peptides, antibiotic biosynthesis and noxious substance degradation pathways were found which reflected a high capacity of strain TF28 to promote plant growth, inhibit pathogens and support environment fitness. 201 specific genes are found in strain TF28 which provides information for further analysis of the strain function. The availability of the genome provides insights to better understand the biocontrol mechanisms and facilitate the utilization of the strain in the future.
- gyrA :
DNA gyrase subunit A
- rpoB :
RNA polymerase subunit B
Tricarboxylic acid cycle
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We appreciate Lu Gao, Shenzhen Huada Gene Technology Co., Ltd. very much for her assistance in bioinformatics analysis. We are also grateful to Xiaoyong Wang, Institute of Microbiology, Heilongjiang Academy of Sciences, for her help in the paper writing.
This work was supported by the grants from Heilongjiang Academy of Sciences for subject team planning (2014sw09) and subject special planning (YXK14ZSM15).
SZ prepared and wrote the manuscript, JL and CS designed research, WJ characterized strain TF28, LQ performed bioinformatics analysis, XC and JH participated in the statistical analysis, YL and JC participated in the sequence alignment. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Strain ID Summary (DOCX 16 kb)
Gene clusters of secondary metabolites synthesis in B.amyloliquefaciens TF28 and B.amyloliquefaciens subsp. plantarum FZB42T (DOCX 13 kb)
General feature of genome from 22 strains of B. amyloliquefaciens (DOCX 17 kb)
GenBank Accession Summary (DOCX 15 kb)
Scientific Name Summary (DOCX 12 kb)
Reference Search Summary (DOCX 12 kb)
Annotation Summary (DOCX 16 kb)