Whole genome shotgun sequence of Bacillus amyloliquefaciens TF28, a biocontrol entophytic bacterium

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. Electronic supplementary material The online version of this article (doi:10.1186/s40793-016-0182-6) contains supplementary material, which is available to authorized users.


Introduction
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][2][3][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 [5]. 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.

Organism information
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 endosporeforming. 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

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 [8]. 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 FZB42 T (CP000560) as reference-guided assembling. The final assembly yielded 182 contigs and 3 scaffolds representing 3.9 Mbp of sequence information.

Genome properties
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.

Conclusions
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.