Abstract
The genome sequence of the entomopathogen Bacillus thuringiensis subsp. thuringiensis strain IS5056 was determined. The chromosome is composed of 5,491,935 bp. In addition, IS5056 harbors 14 plasmids ranging from 6,880 to 328,151 bp, four of which contain nine insecticidal protein genes, cry1Aa3, cry1Ab21, cry1Ba1, cry1Ia14, cry2Aa9, cry2Ab1, vip1, vip2, and vip3Aa10.
GENOME ANNOUNCEMENT
Various strains of Bacillus thuringiensis are used worldwide as natural biopesticides to control insect pests. Their entomo-pathogenicity is due to insecticidal proteins produced during vegetative growth (vegetative insecticidal proteins [Vips]) and sporulation (crystal δ-endotoxins [Cry]; cytolytic toxins [Cyt]) (1). B. thuringiensis subsp. thuringiensis strain IS5056 (serotype H1), isolated in 2005 from soil collected in Biebrza National Park (Poland), produces a quasicuboidal bipyramidal crystal composed of Cry toxins highly toxic to Trichoplusia ni larvae (2) and also harbors vip genes (3) encoding toxins known to enhance activity of Cry proteins (4).
The total genomic DNA of IS5056 was used to construct three libraries: (i) a GS FLX+ shotgun library using the GS FLX+ library preparation kit, (ii) an 8-kb-long paired-end library using the GS FLX paired-end kit, and (iii) an Illumina paired-end library using the Illumina TruSeq2.0 kit (Roche Diagnostics GmbH, Mannheim, Germany). The libraries were sequenced using the genome sequencer FLX+ System (Roche), which yielded 178 million nucleotides covering the genome ~18-fold, and the Illumina HiScanSQ genome analyzer (Illumina Inc.), which generated ~400 million nucleotides covering the genome ~40-fold. All high-quality reads were assembled into 161 contigs in 21 scaffolds with the Newbler de novo assembler (454 sequencing system software; Roche). Gaps were filled using the Expand long-template PCR system (Roche), after which the PCR amplicons were sequenced in the ABI3500 genetic analyzer (Applied Biosystems).
The 6.8-Mb genome of IS5056 consisted of a 5,491,935-bp circular chromosome and 14 circular replicons: pIS56-6 (6,880 bp), pIS56-8 (8,251 bp), pIS56-9 (9,671 bp), pIS56-11 (11,331 bp), pIS56-15 (15,185 bp), pIS56-16 (16,206 bp), pIS56-39 (39,749 bp), pIS56-63 (63,864 bp), pIS56-68 (68,616 bp), pIS56-85 (85,134 bp), pIS56-107 (107,431 bp), pIS56-233 (233,730 bp), pIS56-285 (285,459 bp), and pIS56-328 (328,151 bp) (Table 1). The G+C contents of these replicons ranged from 31.0% to 35.7% for pIS56-107 and pIS56-15, respectively, and did not deviate significantly from that of the chromosome (35.4%). Annotation of protein-coding genes (CDSs) was performed using the RAST system (5). The IS5056 chromosome harbors 5,617 CDSs, 85 tRNAs, and 13 rRNA operons. Plasmids represent ~19.0% of the total CDSs. The four megaplasmids, pIS56-328, pIS56-285, pIS56-233, and pIS56-107, contain 302, 294, 186, and 130 CDSs, respectively. The remaining 10 plasmids, pIS56-85, pIS56-68, pIS56-63, pIS56-39, pIS56-16, pIS56-15, pIS56-11, pIS56-9, pIS56-8, and pIS56-6, contain 111, 89, 61, 49, 18, 16, 23, 7, 13, and 6 CDSs, respectively.
Table 1.
The sequence features of 14 plasmids from Bacillus thuringiensis subsp. thuringiensis strain IS5056
| Plasmid | Length (bp) |
G+C content (%) |
No. of CDSs (forward/reverse) |
Total length of CDSs (bp) |
Coding sequences (%)a |
Average length of CDSs (range) (bp) |
GenBank accession no. |
|---|---|---|---|---|---|---|---|
| pIS56-328 | 328,151 | 32.6 | 302 (144/158) | 237,030 | 72.2 | 784.9 (114-8,583) | CP004137 |
| pIS56-285 | 285,459 | 33.0 | 294 (163/131) | 206,469 | 72.3 | 702.3 (114-4,020) | CP004136 |
| pIS56-233 | 233,730 | 32.7 | 186 (71/115) | 179,304 | 76.7 | 964.0 (114-10,011) | CP004135 |
| pIS56-107 | 107,431 | 31.0 | 130 (93/37) | 89,415 | 83.2 | 687.8 (114–3,687) | CP004134 |
| pIS56-85 | 85,134 | 33.2 | 111 (35/76) | 65,664 | 77.1 | 591.6 (117–3,435) | CP004133 |
| pIS56-68 | 68,616 | 31.8 | 89 (17/72) | 56,721 | 82.7 | 637.3 (114–2,655) | CP004132 |
| pIS56-63 | 63,864 | 34.7 | 61 (12/49) | 55,212 | 86.5 | 905.1 (132–3,594) | CP004131 |
| pIS56-39 | 39,749 | 34.9 | 49 (17/32) | 33,015 | 83.1 | 673.8 (120–3,024) | CP004130 |
| pIS56-16 | 16,206 | 33.3 | 18 (11/7) | 10,497 | 64.8 | 583.2 (141–2,139) | CP004129 |
| pIS56-15 | 15,185 | 35.7 | 6 (3/13) | 10,557 | 69.5 | 659.8 (123–2,964) | CP004128 |
| pIS56-11 | 11,331 | 31.6 | 23 (16/7) | 8,850 | 78.1 | 384.8 (114–1,380) | CP004127 |
| pIS56-9 | 9,671 | 33.0 | 7 (0/7) | 5,559 | 57.5 | 794.1 (294–1,338) | CP004126 |
| pIS56-8 | 8,251 | 32.4 | 13 (5/8) | 5,292 | 64.1 | 407.1 (123–1,230) | CP004125 |
| pIS56-6 | 6,880 | 31.8 | 6 (2/4) | 3,189 | 46.4 | 531.5 (141–1,095) | CP004124 |
Ratio of total gene length to plasmid length.
Altogether, IS5056 harbors nine genes encoding insecticidal proteins, which reside on four plasmids. The vip1 and vip2 genes occur in pIS56-328. The cry1Aa3, cry1Ia14, cry2Aa9, and cry2Ab1 homologues, together with the vip3Aa10 gene, create a pathogenicity island in pIS56-285. The cry1Ba1 and cry1Ab21 homologues are present in pIS56-107 and pIS56-63, respectively. The availability of the IS5056 genome should facilitate the study of plasmid and insecticidal protein gene diversity and the regulation of insecticidal protein expression and will contribute to deeper understanding of the evolution of entomopathogenicity among B. thuringiensis strains.
Nucleotide sequence accession numbers.
The complete genome sequence of B. thuringiensis IS5056 has been deposited in GenBank under accession numbers CP004123 (chromosome) and CP004124 to CP004137 (plasmids).
ACKNOWLEDGMENTS
The work was partly funded by grant N N302 656640 and by funds allocated to “Specific scientific equipment—2012,” both from the Ministry of Science and Higher Education in Poland (to I.S.). E.M. expresses her gratitude to the Podlaskie Province Marshal’s Office, Poland, and WOTT University of Bialystok for the scholarship designated for the project “Podlasie Scholarship Fund,” Priority of VIII Operation Program Human Capital, financed by the European Social Fund (ESF) and the Polish government.
Footnotes
Citation Murawska E, Fiedoruk K, Bideshi DK, Swiecicka I. 2013. Complete genome sequence of Bacillus thuringiensis subsp. thuringiensis strain IS5056, an isolate highly toxic to Trichoplusia ni. Genome Announc. 1(2):e00108-13. doi:10.1128/genomeA.00108-13.
REFERENCES
- 1. Sanahuja G, Banakar R, Twyman RM, Capell T, Christou P. 2011. Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol. J. 9:283–300 [DOI] [PubMed] [Google Scholar]
- 2. Swiecicka I, Bideshi DK, Federici BA. 2008. Novel isolate of Bacillus thuringiensis subsp. thuringiensis that produces a quasicuboidal crystal of Cry1Ab21 toxic to larvae of Trichoplusia ni. Appl. Environ. Microbiol. 74:923–930 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Swiecicka I, Sztachelska M, Czajkowska M, Bideshi DK, Federici BA. 2011. Characterization of Bacillus thuringiensis isolates from soil and small mammals that harbour vip3A gene homologues. Biocontrol Sci. Technol. 21:461–473 [Google Scholar]
- 4. Thamthiankul Chankhamhaengdecha S, Tantichodok A, Panbangred W. 2008. Spore stage expression of a vegetative insecticidal gene increase toxicity of Bacillus thuringiensis subsp. aizawai SP41 against Spodoptera exigua. J. Biotechnol. 136:122–128 [DOI] [PubMed] [Google Scholar]
- 5. 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, Zagnitko O. 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75 [DOI] [PMC free article] [PubMed] [Google Scholar]