- Open access
- Published:
High-quality draft genome sequence of nematocidal Bacillus thuringiensis Sbt003
Standards in Genomic Sciences volume 9, pages 624–631 (2014)
Abstract
Bacillus thuringiensis represents one of the six species of “Bacillus cereus group” in the genus Bacillus within the family Bacillaceae. Strain Sbt003 was isolated from soil and identified as B. thuringiensis. It harbors at least seven plasmids and produces three shapes of parasporal crystals including oval, bipyramidal and rice. SDS-PAGE analysis of spore-crystal suspension of this strain reveals six major protein bands, which implies the presence of multiple parasporal crystal genes. Bioassay of this strain reveals that it shows specific activity against nematodes and human cancer cells. In this study, we report the whole genomic shotgun sequences of Sbt003. The high-quality draft of the genome is 6,175,670 bp long (including chromosome and plasmids) with 6,372 protein-coding and 80 RNA genes.
Introduction
Bacillus thuringiensis, B. cereus, B. anthracis and other three species constitute the “Bacillus cereus group”, a nontaxonomic term, within the genus Bacillus and family Bacillaceae [1]. These species were classified as separate species mainly based on their distinct phenotypes, although extensive genomic studies on strains of these species using different techniques have suggested that they form a single species [2–5]. Strain Sbt003 belongs to the species B. thuringiensis. The type strain of the species produces one or more parasporal crystal proteins showing specific activity against certain larvae from various orders of insects [6]. The specific role and the abundant number of genes encoding of insecticidal crystal proteins of this species have attracted much attention from both academic and industrial researchers. Dozens of B. thuringiensis strains have been sequenced, and dozens more are on their way. In this study, we present a summary classification and a set of features for B. thuringiensis Sbt003, together with the description of the genomic sequencing and annotation.
Classification and features
B. thuringiensis strain Sbt003 harbors at least 7 plasmids and produces three different shapes of parasporal crystals including oval, bipyramidal and rice (Figure 1A, Figure 1B and Table 1). SDS-PAGE analysis of spore-crystal suspension of this strain reveals six major protein bands of 168.8, 148.5, 133.5, 117.2, 107.9 and 103.1 kDa, which implies the presence of multiple parasporal crystal genes (Figure 1C).
A representative genomic 16S rDNA sequence of strain Sbt003 was searched against GenBank database using BLAST [21]. Sequences showing more than 97% identity to the 16S rDNA of Sbt003 were selected for phylogentic analysis, and a 16S rDNA sequence from B. subtilis subsp. subtilis str. 168 was used as the outgroup. Nine sequences were aligned with ClustalW algorithm. The tree was reconstructed using neighbor joining with the Kimura 2-parameter substitution model. The phylogenetic tree was assessed by bootstrapping 1,000 times, and the consensus tree is shown in Figure 2.
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing due to its specific activity against nematodes and human cancer cells. The complete high quality draft genome sequence is deposited in GenBank. The Beijing Genomics Institute (BGI) performed the sequencing and NCBI staff used the Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) to complete the annotation. A summary of the project is given in Table 2.
Growth conditions and DNA isolation
B. thuringiensis Sbt003 was grown in 50 mL Luria broth for 6 hours at 28°C. DNA was isolated by incubating the cells with lysozyme (10 mg/mL) in 2 mL TE (50 mM Tris base, 10 mM EDTA, 20% sucrose, pH8.0) at 4°C for 6 hours. 4 mL 2% SDS was added and the mixture was incubated at 55°C for 30 min; 2 mL 5M NaCl were added, and the mixture was incubated at 4°C for 10 min. DNA was purified by organic extraction and ethanol precipitation.
Genome sequencing and assembly
The genome of B. thuringiensis Sbt003 was sequenced using Illumina Hiseq 2,000 platform (with a combination of a 100-bp paired-end reads sequencing from a 500-bp genomic library and a 90-bp mate-paired reads sequencing from a 2-kb genomic library). Reads with average quality scores below Q30 or having more than 3 unidentified nucleotides were eliminated. Using SOAPdenovo 1.05 version, 22,295,588 paired-end reads (achieving ∼325 fold coverage [2.01 Gb]) and 11,166,312 mate-paired reads (achieving ∼ 163 fold coverage [1.00 Gb]) were assembled de novo [22]. The assembly is considered a high-guality draft and consists of 104 contigs arranged in 61 scaffolds with a total size of 6,175,670 bp. According to bioinformatic analysis, we identified two large plasmids belonging to ori44-type and repB-type plasmids, respectively. The former plasmid has two ori44-type replicons. We propose it represents a fusion of two plasmids and its estimated size is about 200 kb. The latter plasmid has an expected size of at least 90 kb, according to the sequence of contig0027, which is typical of repB-type plasmids (80 ∼ 90 kb). In addition, we identified five other plasmids from the plasmid pattern (see Figure 1A). The expected sizes of the smaller three are 13 kb, 8kb and 4kb, respectively, while the sizes of the larger two can’t be deduced either from the plasmid pattern or by bioinformatic analysis.
Genome annotation
Genome annotation was completed using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). Briefly, protein-coding genes were predicted using a combination of GeneMark and Glimmer [23–25]. Ribosomal RNAs were predicted by sequence similarity searching using BLAST against an RNA sequence database and/or using Infernal and Rfam models [26,27]. Transfer RNAs were predicted using tRNAscan-SE [28]. In order to detect missing genes, a complete six-frame translation of the nucleotide sequence was done and predicted proteins (generated above) were masked. All predictions were then searched using BLAST against all proteins from complete microbial genomes. Annotation was based on comparison to protein clusters and on the BLAST results. Conserved domain Database and Cluster of Orthologous Group information were then added to the annotation.
Genome Properties
The high-quality draft assembly of the genome consists of 104 contigs in 61 scaffolds, with an overall 35.21% G+C content. Of the 6,452 genes predicted, 6,372 were protein-coding genes, and 80 RNAs were also identified. The majority of the protein-coding genes (66.67%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins (Table 3). The distribution of genes into COGs functional categories is presented in Table 4.
The whole genomic sequence and the coding sequence of Sbt003 were analyzed by BtToxin_scanner [29], and eight potential crystal protein sequences were identified. Among these, four were considered to be full-length (locus tags: C797_02099, C797_12066, C797_12568 and C797_27783) while the others were considered to be truncated (Locus tags: C797_02094, C797_12046, C797_12061, C797_18417).
Reference
Vilas-Bôas GT, Peruca AP, Arantes OM. Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. Can J Microbiol 2007; 53:673–687. PubMed http://dx.doi.org/10.1139/W07-029
Helgason E, Caugant DA, Lecadet MM, Chen Y, Mahillon J, Lovgren A, Hegna I, Kvaloy K, Kolsto AB. Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources. Curr Microbiol 1998; 37:80–87. PubMed http://dx.doi.org/10.1007/s002849900343
Helgason E, Okstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolsto AB. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl Environ Microbiol 2000; 66:2627–2630. PubMed http://dx.doi.org/10.1128/AEM.66.6.2627-2630.2000
Ticknor LO, Kolsto AB, Hill KK, Keim P, Laker MT, Tonks M, Jackson PJ. Fluorescent amplified fragment length polymorphism analysis of Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol 2001; 67:4863–4873. PubMed http://dx.doi.org/10.1128/AEM.67.10.4863-4873.2001
Helgason E, Tourasse NJ, Meisal R, Caugant DA, Kolsto AB. Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl Environ Microbiol 2004; 70:191–201. PubMed http://dx.doi.org/10.1128/AEM.70.L191-201.2004
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62:775–806. PubMed
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen M, Angiuoli SV, et al. Towards a richer description of our complete collection of genomes and metagenomes “Minimum Information about a Genome Sequence” (MIGS) specification. Nat Biotechnol 2008; 26:541–547. PubMed http://dx.doi.org/10.1038/nbt1360
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 1990; 87:4576–4579. PubMed http://dx.doi.org/10.1073/pnas.87.12.4576
Gibbons NE, Murray RGE. Proposals Concerning the Higher Taxa of Bacteria. Int J Syst Bacteriol 1978; 28:1–6. http://dx.doi.org/10.1099/00207713-28-1-1
Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119–169.
Murray RGE. The Higher Taxa, or, a Place for Everything…? In: Holt JG (ed), Bergey’s Manual of Systematic Bacteriology, First Edition, Volume 1, The Williams and Wilkins Co., Baltimore, 1984, p. 31–34.
List of new names and new combinations previously effectively, but not validly, published. List no. 132. Int J Syst Evol Microbiol 2010; 60:469–472. http://dx.doi.org/10.1099/ijs.0.022855-0
Ludwig W, Schleifer KH, Whitman WB. Class I. Bacilli class nov. In: De Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds), Bergey’s Manual of Systematic Bacteriology, Second Edition, Volume 3, Springer-Verlag, New York, 2009, p. 19–20.
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of Bacterial Names. Int J Syst Bacteriol 1980; 30:225–420. http://dx.doi.org/10.1099/00207713-30-1-225
Prévot AR. In: Hauderoy P, Ehringer G, Guillot G, Magrou. J., Prévot AR, Rosset D, Urbain A (eds), Dictionnaire des Bactéries Pathogènes, Second Edition, Masson et Cie, Paris, 1953, p. 1–692.
Fischer A. Untersuchungen über bakterien. Jahrbücher für Wissenschaftliche Botanik 1895; 27:1–163.
Cohn F. Untersuchungen über Bakterien. Beitr Biol Pflanz 1872; 1:127–224.
Gibson T, Gordon RE. Genus I. Bacillus Cohn 1872, 174; Nom. gen. cons. Nomencl. Comm. Intern. Soc. Microbiol. 1937, 28; Opin. A. Jud. Comm. 1955, 39. In: Buchanan RE, Gibbons NE (eds), Bergey’s Manual of Determinative Bacteriology, Eighth Edition, The Williams and Wilkins Co., Baltimore, 1974, p. 529–550.
Berliner E. Über die Schlaffsucht der Mehlmottenraupe (Ephestia kuhniella Zell) und ihren Erreger Bacillus thuringiensis n. sp. Zeitschrift für angewandte Entomologie Berlin 1915; 2:29–56.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 2000; 25:25–29. PubMed http://dx.doi.org/10.1038/75556
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403–410. PubMed
Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K, et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 2010; 20:265–272. PubMed http://dx.doi.org/10.1101/gr.097261.109
Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 2001; 29:2607–2618. PubMed http://dx.doi.org/10.1093/nar/29.12.2607
Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. Improved microbial gene identification with GLIMMER. Nucleic Acids Res 1999; 27:4636–4641. PubMed http://dx.doi.org/10.1093/nar/27.23.4636
Lukashin AV, Borodovsky M. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 1998; 26:1107–1115. PubMed http://dx.doi.org/10.1093/nar/26.4.1107
Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR. Rfam: an RNA family database. Nucleic Acids Res 2003; 31:439–441. PubMed http://dx.doi.org/10.1093/nar/gkg006
Eddy SR. A memory-efficient dynamic programming algorithm for optimal alignment of a sequence to an RNA secondary structure. BMC Bioinformatics 2002; 3:18. PubMed http://dx.doi.org/10.1186/1471-2105-3-18
Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997; 25:955–964. PubMed http://dx.doi.org/10.1093/nar/25.5.0955
Ye W, Zhu L, Liu Y, Crickmore N, Peng D, Ruan L, Sun M. Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline. Appl Environ Microbiol 2012; 78:4795–4801. PubMed http://dx.doi.org/10.1128/AEM.00340-12
Acknowledgements
This work was supported by grants from the National High Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), the National Basic Research Program (973) of China (2009CB118902), the National Natural Science Foundation of China (31170047 and 31171901), and the Genetically Modified Organisms Breeding Major Projects of China (2009ZX08009-032B).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
About this article
Cite this article
Liu, Y., Ye, W., Zheng, J. et al. High-quality draft genome sequence of nematocidal Bacillus thuringiensis Sbt003. Stand in Genomic Sci 9, 624–631 (2014). https://doi.org/10.4056/sigs.4738557
Published:
Issue Date:
DOI: https://doi.org/10.4056/sigs.4738557