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30 October 2014

Genome Sequence of Bacillus amyloliquefaciens GB03, an Active Ingredient of the First Commercial Biological Control Product

ABSTRACT

Bacillus amyloliquefaciens GB03 has been used as a representative commercialized strain of the bacilli for biological control against a broad spectrum of plant pathogens and as a bio-fertilizer to promote growth and yield of field crops for more than two decades. Herein, we present the genome sequence and a brief analysis of strain GB03.

GENOME ANNOUNCEMENT

Bacillus spp. offer many benefits over Gram-negative bacteria for use as biological control agents, including a long shelf life under starvation or unfavorable conditions due to their ability to form endospores (1). Accordingly, several commercial products, such as Quantum, Kodiak, BioYield, Epic, Rhizo-Plus, Serenade, Subtilex, and System 3, that originated from Bacillus spp. are available in the U.S. and in other countries (1). Bacillus amyloliquefaciens strain GB03, originally described as Bacillus subtilis strain A13, was originally isolated from the healthy foliage of a Douglas fir in Australia (2). In an early study, the strain demonstrated plant growth promotion capacity in the field (3). A comprehensive mechanism study indicated that B. amyloliquefaciens GB03 was an efficient root colonizer that directly competes with potential root pathogens for nutrients and space at the surface of roots and produces antifungal agents such as iturins (4). B. amyloliquefaciens GB03 also reduces disease by inducing the plant's innate immunity, a process referred to as induced systemic resistance (4). Bacillus amyloliquefaciens GB03, the active ingredient of Kodiak and Quantum, is a representative strain of the bacilli developed as a biological control agent against soilborne fungal pathogens (4).
Genome sequencing of Bacillus amyloliquefaciens GB03 was carried out at the National Instrumentation Center for Environmental Management (Seoul, Korea) using an Illumina Genome Analyzer IIx. A total of 27,492,038 reads (2.17 Gb, 101 nucleotides) were produced from paired-end sequencing of a genomic library with an average insert size of 393 bp. Sequence Read Archive (SRA) data are available under the accession no. SRS505354. De novo assembly using CLC Genomics Workbench version 4.8, after quality trimming and filtering (about 564-fold coverage after pretreatment of the reads), resulted in 37 contigs with a total length of 3,849,547 bp. The N50 and maximum contig length were 387,471 bp and 784,095 bp, respectively. Automatic genome annotation, performed using the RAST server (5), predicted 87 RNA genes and 3,892 coding sequences, 48% of which were categorized into subsystems. The G+C content was 46.6%. While the RAST analysis result suggested Bacillus subtilis subsp. subtilis 168 was the closest neighbor to the strain GB03, average nucleotide identity (ANI) analysis by the JSpecies program (6) revealed that B. amyloliquefaciens FZB42 and B. amyloliquefaciens Y2 are most similar to GB03 (>98.3% ANI values) among the completely sequenced Bacillus genomes.
Genome analysis revealed that nonribosomal peptide synthetase and polyketide synthase gene clusters for the production of surfactin, bacillomycinD, fengycin, bacillibactin, bacilysin, macrolactin, bacillaene, and difficidin are well conserved between strains GB03 and FZB42. However, the nrsABCDEF gene cluster of FZB42 was not found in the GB03 genome. Although the amino acid sequence of ComP has 68% identity between GB03 and FZB42, other genes related to bacterial traits for plant interactions such as root colonization, swarming, biofilm formation, biofertilization, and production of phytohormones like indole acetic acid and 2,3-butanediol are well conserved (7). In conclusion, the genome information of the GB03 strain will be helpful for strain improvement and for understanding the interaction between microorganisms and plants.

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. AYTJ00000000. The version described in this paper is version AYTJ01000000.

ACKNOWLEDGMENTS

This work was supported by the Industrial Source Technology Development Program of the Ministry of Knowledge Economy (10044909) of Korea, the Next-Generation BioGreen 21 Program (SSAC grant # PJ009524), Rural Development Administration, and the KRIBB Research Initiative Program, Ministry of Science, ICT and Future Planning, Republic of Korea.

REFERENCES

1.
McSpadden Gardener BB. 2004. Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathology 94:1252–1258.
2.
Broadbent P, Baker KF, and Waterworth Y. 1971. Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. Aust. J. Biol. Sci. 24:925–944.
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Kloepper JW, Reddy MS, Rodriguez-Kabana R, Kenney DS, Kokalis-Burelle N, and Martinez-Ochoa N. 2004. Application for rhizobacteria in transplant production and yield enhancement. Acta Hort. 631:219–229.
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Kloepper JW, Ryu CM, and Zhang S. 2004. Induced systemic resistance and promotion of plant growth by bacillus spp. Phytopathology 94:1259–1266.
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Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, and Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75.
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Richter M and Rosselló-Móra R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. U. S. A. 106:19126–19131.
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Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I, Morgenstern B, Voss B, Hess WR, Reva O, Junge H, Voigt B, Jungblut PR, Vater J, Süssmuth R, Liesegang H, Strittmatter A, Gottschalk G, and Borriss R. 2007. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat. Biotechnol. 25:1007–1014.

Information & Contributors

Information

Published In

cover image Genome Announcements
Genome Announcements
Volume 2Number 530 October 2014
eLocator: e01092-14
PubMed: 25359911

History

Received: 13 September 2014
Accepted: 16 September 2014
Published online: 30 October 2014

Contributors

Authors

Soo-Keun Choi
Super-Bacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea
Haeyoung Jeong
Korean Bioinformation Center, KRIBB, Daejeon, Republic of Korea
Joseph W. Kloepper
Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama, USA
Choong-Min Ryu
Super-Bacteria Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea

Notes

Address correspondence to Choong-Min Ryu, [email protected].
S.-K.C. and H.J. contributed equally to this work.

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