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ARTICLE IN PRESS
BIOTEC 7053 1–2
Journal of Biotechnology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
1
Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects
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Qiuling Gao, Jinshui Zheng, Lei Zhu, Lifang Ruan, Donghai Peng, Ming Sun ∗ State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
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a r t i c l e
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a b s t r a c t
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Article history: Received 13 March 2015 Accepted 17 March 2015 Available online xxx
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Keywords: Bacillus thuringiensis tenebrionis 4AA1 Bioinsecticide Colepteran insects Genome sequence
Bacillus thuringiensis serovar morrisoni biovar tenebrionis has been developed as bioinsecticide to control Coleopteran insects in agriculture and forestry for a few decades. Its major crystal protein Cry3Aa was also applied to transgenic crops. Here we report the complete genome sequence of strain tenebrionis 4AA1, which has one chromosome of 5,652,292 bp and six plasmids. Two crystal protein genes, cry3Aa and cry15Aa, locate on one single plasmid named pBMB51. This strain also possesses plentiful virulence factors besides crystal proteins. © 2015 Published by Elsevier B.V.
Bacillus thuringiensis is a ubiquitous gram-positive and sporeforming bacterium that produces insecticidal crystal proteins (ICPs) during sporulation. ICPs are found to be highly toxic to larvae of insects, such as in the order of Lepidoptera (moths and butterflies), Diptera (mosquitoes and blackflies), and Coleoptera (beetles), and nematodes. B. thuringiensis has been considered as a useful alternative to synthetic chemical pesticide application in agriculture, forestry, and mosquito control (Schnepf et al., 1998). With the development of transgenic technology, B. thuringiensis also become a key source of genes for transgenic crops to provide resistance to insects (Sanahuja et al., 2011). B. thuringiensis was first found in 1901 and the first commercial production of B. thuringiensis bioinsecticide named Sporeine emerged in 1938 (Beegle and Yamamoto, 1992). In 1977, B. thuringiensis serovar. israelensis was isolated and developed bioinsecticide to control Dipteran insects like mosquito and blackfly (Becker and Margalit, 1993; Goldberg and Margalit, 1977). A few years later, a new strain, named B. thuringiensis var. tenebrionis belonged to serovar morrisoni (H8a8b) was isolated and showed toxicity to Colepteran insects (Krieg et al., 1983), which was then deposited in BGSC as strain 4AA1 (Bacillus Genetic Stock Center, directed by Dr. Dan Zeigler, The Ohio State University). A major insecticidal component of B. thuringiensis tenebrionis is a 68 kD protein named Cry3Aa, which forms crystal with a flat shape and quadrangular in outline (Krieg et al., 1983). Cry3Aa was also the
∗ Corresponding author. Tel.: +86 2787283455; fax: +86 2787280670. E-mail address: [email protected] (M. Sun).
first crystal protein to reveal the tertiary structure (Li et al., 1991), which is served as a model structure for B. thuringiensis crystal proteins (Schnepf et al., 1998). Gene cry3Aa has been introduced into crop to obtain continuous toxicity against Colorado potato beetles (Leptinotarsa decemlineata) (Schnepf et al., 1998). B. thuringiensis tenebrionis was registered as a microbial pesticide to control Coleopteran larvae by U.S. Environmental Protection Agency (EPA) in 1988 and had a good performance on controlling the Colorado potato beetle (Schnepf et al., 1998). Most commercial B. thuringiensis products are derived from three serovar, such as Dipel (serovar kurstaki), Tekar (serovar israelensis), Novodor (tenebrionis, serovar morrisoni), to control Lepidopteran, Dipteran, and Coleopteran insects, respectively (Schnepf et al., 1998). The genome sequences of B. thuringiensis strains developed for commercial bioinsecticides to control Lepidopteran and Dipteran insects have been published, for example, B. thuringiensis serovar kurstaki HD-1 (GenBank accession no. CP004870.1–CP004883.1, JMHW00000000.1), and serovar israelensis ATCC 35646 (GenBank accession no. AAJM00000000.1). However, that for Coleopteran bioinsecticides has not been released. Here we report the complete genome sequence of strain tenebrionis 4AA1. Genomic DNA of the strain 4AA1 was sequenced with a whole genome shotgun strategy using an Illumina HiSeq2000 platform. Two different sequencing libraries (300 bp paired-end and 5000 bp mate-pair) were prepared. We assembled the genome using Abyss v1.3.7 (Simpson et al., 2009). After assembly, we got 43 contigs. Then gaps were filled by GapFiller v1.10 (Boetzer and Pirovano, 2012) and verified by PCR. The chromosome sequence of Bacillus cereus G9842 was used as a reference (GenBank
http://dx.doi.org/10.1016/j.jbiotec.2015.03.009 0168-1656/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Gao, Q., et al., Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.009
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G Model
ARTICLE IN PRESS
BIOTEC 7053 1–2
Q. Gao et al. / Journal of Biotechnology xxx (2015) xxx–xxx
2
Table 1 Sequencing-related and assembly-related statistics of strain 4AA1. Bacillus thuringiensis tenebrionis 4AA1 Sequencing statistics Features Coverage based on G9842 chromosome Depth Assembly statistics Features (accession numbers) Chromosome (CP010577) pBMB232 (CP010578) pBMB92 (CP010579) pBMB76 (CP010580) pBMB68 (CP010581) pBMB51 (CP010582) pBMB48 (CP010583) Annotation statistics rRNA CDS tRNA
75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
Paired-end library 94.7%
Mate-paired library 94.4%
128.6
127.6
Length (bp) 5,652,292 232,994 92,619 76,979 68,444 51,723 4845
G+C content (%) 35.3 33.7 34.4 30.3 32.3 29.3 41.5
49 6419 106
accession no. CP001186.1). Annotation was performed by NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP, http://www.ncbi.nlm.nih.gov/books/NBK174280/). The online tool BtToxin scanner was used to predict B. thuringiensis toxin as we described previously (Ye et al., 2012). The final assembly includes a chromosome with total bases of 5,652,292 bp and six plasmids, named as pBMB232, pBMB92, pBMB76, pBMB68, pBMB51, and pBMB48, respectively. It is predicted that 4AA1 genome contains coding sequences for 49 rRNA, 106 tRNA, and 6419 CDS. The largest plasmid is pBMB232 with a length of 232,994 bp and shares high co-linearity with that plasmid pBMB293 in B. thuringiensis serovar kurstaki YBT-1520 (GenBank accession no. CP004861.1). Plasmid pBMB51 codes two-crystal protein named Cry3Aa and Cry15Aa, which show no general similarity with other published plasmids in B. thuringiensis. Detail information of 4AA1 genome can be seen in Table 1. Cry3Aa is the main crystal protein encoded by strain 4AA1, and has been showed high toxicity to Coleopteran insects (McPherson et al., 1988). Besides that, Cry15Aa shares sequence similarity to mosquitocidal Mtx2 and Mtx3 protein, and shows hemolytic activity and toxicity to some types of Lepidopteran insects (Naimov et al., 2008). Strain 4AA1 genome codes some virulence factors, like two chitinases, metalloproteases InhA1 and InhA3, Bacillus enhancin-like protein Bel as well as non-hemolytic enterotoxin (NHE), enterotoxin FM (EntFM), and Hemolysin BL (HBL) (Raymond et al., 2010). This strain also encodes a newly described virulence factor Bmp1, which shows metalloproteinase activity and serves as a nematicidal virulence factor (Luo et al., 2013). All of these virulence factors what are said above contribute B. thuringiensis an outstanding pathogen. In conclusion, the genome sequence of strain 4AA1 is the first published complete genome of B. thuringiensis serovar morrisoni, especially tenebrionis strain, and of that having toxic to Coleopteran insects. The availability of the genome sequence
of 4AA1 will strongly contribute to a better understanding of Colepteran-specific tenebrionis strain and even serovar morrisoni and provide a reference for this serovar or pathogenic types. 1. Nucleotide sequence accession number This complete genome has been deposited at DDBJ/EMBL/ GenBank under the accession number CP010577–CP010583. Strain 4AA1 is available from Bacillus Genetic Stock Center (Columbus, USA), or Prof. Ming Sun (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China). Acknowledgments This work was supported by grants from the National High Q3 Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), and the National Basic Research Program (973) of China (2009CB118902), the National Natural Science Foundation of China (31170047 and 31171901). References Becker, N., Margalit, J., 1993. Use of Bacillus thuringiensis israelensis against mosquitoes and blackflies. Bacillus thuringiensis, 147–170. Beegle, C.C., Yamamoto, T., 1992. Invitation paper (CP Alexander Fund): history of Bacillus thuringiensis Berliner research and development. Can. Entomol. 124, 587–616. Boetzer, M., Pirovano, W., 2012. Toward almost closed genomes with GapFiller. Genome Biol. 13, R56. Goldberg, L.J., Margalit, J., 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univittatus, Aedes aegypti and Culex pipiens. Mosq. News 37, 355–358. Krieg, A., Huger, A., Langenbruch, G., Schnetter, W., 1983. Bacillus thuringiensis var. tenebrionis, a new pathotype effective against larvae of Coleoptera. Z. Angew. Entomol. 96, 500–508. Li, J., Carroll, J., Ellar, D.J., 1991. Crystal structure of insecticidal (-endotoxin from Bacillus thuringiensis at 2.5 A˚ resolution. Nature 353, 815–821. Luo, X., Chen, L., Huang, Q., Zheng, J., Zhou, W., Peng, D., Ruan, L., Sun, M., 2013. Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor. Appl. Environ. Microbiol. 79, 460–468. McPherson, S.A., Perlak, F.J., Fuchs, R.L., Marrone, P.G., Lavrik, P.B., Fischhoff, D.A., 1988. Characterization of the coleopteran-specific protein gene of Bacillus thuringiensis var tenebrionis. Nat. Biotechnol. 6, 61–66. Naimov, S., Boncheva, R., Karlova, R., Dukiandjiev, S., Minkov, I., de Maagd, R.A., 2008. Solubilization, activation, and insecticidal activity of Bacillus thuringiensis serovar thompsoni HD542 crystal proteins. Appl. Environ. Microbiol. 74, 7145–7151. Raymond, B., Johnston, P.R., Nielsen-LeRoux, C., Lereclus, D., Crickmore, N., 2010. Bacillus thuringiensis: an impotent pathogen? Trends Microbiol. 18, 189–194. Sanahuja, G., Banakar, R., Twyman, R.M., Capell, T., Christou, P., 2011. Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol. J. 9, 283–300. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D., Dean, D., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Simpson, J.T., Wong, K., Jackman, S.D., Schein, J.E., Jones, S.J., Birol, I., 2009. ABySS: a parallel assembler for short read sequence data. Genome Res. 19, 1117–1123. Ye, W., Zhu, L., Liu, Y., Crickmore, N., Peng, D., Ruan, L., Sun, M., 2012. Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline. Appl. Environ. Microbiol. 78, 4795–4801.
Please cite this article in press as: Gao, Q., et al., Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.009
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ARTICLE IN PRESS
BIOTEC 7053 1–2
Journal of Biotechnology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
1
Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects
2
3
4
Q1
5
Qiuling Gao, Jinshui Zheng, Lei Zhu, Lifang Ruan, Donghai Peng, Ming Sun ∗ State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
6
7 20
a r t i c l e
i n f o
a b s t r a c t
8 9 10 11 12
Article history: Received 13 March 2015 Accepted 17 March 2015 Available online xxx
13 14 15 16 17 18 19
21Q2 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Keywords: Bacillus thuringiensis tenebrionis 4AA1 Bioinsecticide Colepteran insects Genome sequence
Bacillus thuringiensis serovar morrisoni biovar tenebrionis has been developed as bioinsecticide to control Coleopteran insects in agriculture and forestry for a few decades. Its major crystal protein Cry3Aa was also applied to transgenic crops. Here we report the complete genome sequence of strain tenebrionis 4AA1, which has one chromosome of 5,652,292 bp and six plasmids. Two crystal protein genes, cry3Aa and cry15Aa, locate on one single plasmid named pBMB51. This strain also possesses plentiful virulence factors besides crystal proteins. © 2015 Published by Elsevier B.V.
Bacillus thuringiensis is a ubiquitous gram-positive and sporeforming bacterium that produces insecticidal crystal proteins (ICPs) during sporulation. ICPs are found to be highly toxic to larvae of insects, such as in the order of Lepidoptera (moths and butterflies), Diptera (mosquitoes and blackflies), and Coleoptera (beetles), and nematodes. B. thuringiensis has been considered as a useful alternative to synthetic chemical pesticide application in agriculture, forestry, and mosquito control (Schnepf et al., 1998). With the development of transgenic technology, B. thuringiensis also become a key source of genes for transgenic crops to provide resistance to insects (Sanahuja et al., 2011). B. thuringiensis was first found in 1901 and the first commercial production of B. thuringiensis bioinsecticide named Sporeine emerged in 1938 (Beegle and Yamamoto, 1992). In 1977, B. thuringiensis serovar. israelensis was isolated and developed bioinsecticide to control Dipteran insects like mosquito and blackfly (Becker and Margalit, 1993; Goldberg and Margalit, 1977). A few years later, a new strain, named B. thuringiensis var. tenebrionis belonged to serovar morrisoni (H8a8b) was isolated and showed toxicity to Colepteran insects (Krieg et al., 1983), which was then deposited in BGSC as strain 4AA1 (Bacillus Genetic Stock Center, directed by Dr. Dan Zeigler, The Ohio State University). A major insecticidal component of B. thuringiensis tenebrionis is a 68 kD protein named Cry3Aa, which forms crystal with a flat shape and quadrangular in outline (Krieg et al., 1983). Cry3Aa was also the
∗ Corresponding author. Tel.: +86 2787283455; fax: +86 2787280670. E-mail address: [email protected] (M. Sun).
first crystal protein to reveal the tertiary structure (Li et al., 1991), which is served as a model structure for B. thuringiensis crystal proteins (Schnepf et al., 1998). Gene cry3Aa has been introduced into crop to obtain continuous toxicity against Colorado potato beetles (Leptinotarsa decemlineata) (Schnepf et al., 1998). B. thuringiensis tenebrionis was registered as a microbial pesticide to control Coleopteran larvae by U.S. Environmental Protection Agency (EPA) in 1988 and had a good performance on controlling the Colorado potato beetle (Schnepf et al., 1998). Most commercial B. thuringiensis products are derived from three serovar, such as Dipel (serovar kurstaki), Tekar (serovar israelensis), Novodor (tenebrionis, serovar morrisoni), to control Lepidopteran, Dipteran, and Coleopteran insects, respectively (Schnepf et al., 1998). The genome sequences of B. thuringiensis strains developed for commercial bioinsecticides to control Lepidopteran and Dipteran insects have been published, for example, B. thuringiensis serovar kurstaki HD-1 (GenBank accession no. CP004870.1–CP004883.1, JMHW00000000.1), and serovar israelensis ATCC 35646 (GenBank accession no. AAJM00000000.1). However, that for Coleopteran bioinsecticides has not been released. Here we report the complete genome sequence of strain tenebrionis 4AA1. Genomic DNA of the strain 4AA1 was sequenced with a whole genome shotgun strategy using an Illumina HiSeq2000 platform. Two different sequencing libraries (300 bp paired-end and 5000 bp mate-pair) were prepared. We assembled the genome using Abyss v1.3.7 (Simpson et al., 2009). After assembly, we got 43 contigs. Then gaps were filled by GapFiller v1.10 (Boetzer and Pirovano, 2012) and verified by PCR. The chromosome sequence of Bacillus cereus G9842 was used as a reference (GenBank
http://dx.doi.org/10.1016/j.jbiotec.2015.03.009 0168-1656/© 2015 Published by Elsevier B.V.
Please cite this article in press as: Gao, Q., et al., Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.009
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74
G Model
ARTICLE IN PRESS
BIOTEC 7053 1–2
Q. Gao et al. / Journal of Biotechnology xxx (2015) xxx–xxx
2
Table 1 Sequencing-related and assembly-related statistics of strain 4AA1. Bacillus thuringiensis tenebrionis 4AA1 Sequencing statistics Features Coverage based on G9842 chromosome Depth Assembly statistics Features (accession numbers) Chromosome (CP010577) pBMB232 (CP010578) pBMB92 (CP010579) pBMB76 (CP010580) pBMB68 (CP010581) pBMB51 (CP010582) pBMB48 (CP010583) Annotation statistics rRNA CDS tRNA
75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
Paired-end library 94.7%
Mate-paired library 94.4%
128.6
127.6
Length (bp) 5,652,292 232,994 92,619 76,979 68,444 51,723 4845
G+C content (%) 35.3 33.7 34.4 30.3 32.3 29.3 41.5
49 6419 106
accession no. CP001186.1). Annotation was performed by NCBI Prokaryotic Genome Automatic Annotation Pipeline (PGAAP, http://www.ncbi.nlm.nih.gov/books/NBK174280/). The online tool BtToxin scanner was used to predict B. thuringiensis toxin as we described previously (Ye et al., 2012). The final assembly includes a chromosome with total bases of 5,652,292 bp and six plasmids, named as pBMB232, pBMB92, pBMB76, pBMB68, pBMB51, and pBMB48, respectively. It is predicted that 4AA1 genome contains coding sequences for 49 rRNA, 106 tRNA, and 6419 CDS. The largest plasmid is pBMB232 with a length of 232,994 bp and shares high co-linearity with that plasmid pBMB293 in B. thuringiensis serovar kurstaki YBT-1520 (GenBank accession no. CP004861.1). Plasmid pBMB51 codes two-crystal protein named Cry3Aa and Cry15Aa, which show no general similarity with other published plasmids in B. thuringiensis. Detail information of 4AA1 genome can be seen in Table 1. Cry3Aa is the main crystal protein encoded by strain 4AA1, and has been showed high toxicity to Coleopteran insects (McPherson et al., 1988). Besides that, Cry15Aa shares sequence similarity to mosquitocidal Mtx2 and Mtx3 protein, and shows hemolytic activity and toxicity to some types of Lepidopteran insects (Naimov et al., 2008). Strain 4AA1 genome codes some virulence factors, like two chitinases, metalloproteases InhA1 and InhA3, Bacillus enhancin-like protein Bel as well as non-hemolytic enterotoxin (NHE), enterotoxin FM (EntFM), and Hemolysin BL (HBL) (Raymond et al., 2010). This strain also encodes a newly described virulence factor Bmp1, which shows metalloproteinase activity and serves as a nematicidal virulence factor (Luo et al., 2013). All of these virulence factors what are said above contribute B. thuringiensis an outstanding pathogen. In conclusion, the genome sequence of strain 4AA1 is the first published complete genome of B. thuringiensis serovar morrisoni, especially tenebrionis strain, and of that having toxic to Coleopteran insects. The availability of the genome sequence
of 4AA1 will strongly contribute to a better understanding of Colepteran-specific tenebrionis strain and even serovar morrisoni and provide a reference for this serovar or pathogenic types. 1. Nucleotide sequence accession number This complete genome has been deposited at DDBJ/EMBL/ GenBank under the accession number CP010577–CP010583. Strain 4AA1 is available from Bacillus Genetic Stock Center (Columbus, USA), or Prof. Ming Sun (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China). Acknowledgments This work was supported by grants from the National High Q3 Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), and the National Basic Research Program (973) of China (2009CB118902), the National Natural Science Foundation of China (31170047 and 31171901). References Becker, N., Margalit, J., 1993. Use of Bacillus thuringiensis israelensis against mosquitoes and blackflies. Bacillus thuringiensis, 147–170. Beegle, C.C., Yamamoto, T., 1992. Invitation paper (CP Alexander Fund): history of Bacillus thuringiensis Berliner research and development. Can. Entomol. 124, 587–616. Boetzer, M., Pirovano, W., 2012. Toward almost closed genomes with GapFiller. Genome Biol. 13, R56. Goldberg, L.J., Margalit, J., 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univittatus, Aedes aegypti and Culex pipiens. Mosq. News 37, 355–358. Krieg, A., Huger, A., Langenbruch, G., Schnetter, W., 1983. Bacillus thuringiensis var. tenebrionis, a new pathotype effective against larvae of Coleoptera. Z. Angew. Entomol. 96, 500–508. Li, J., Carroll, J., Ellar, D.J., 1991. Crystal structure of insecticidal (-endotoxin from Bacillus thuringiensis at 2.5 A˚ resolution. Nature 353, 815–821. Luo, X., Chen, L., Huang, Q., Zheng, J., Zhou, W., Peng, D., Ruan, L., Sun, M., 2013. Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor. Appl. Environ. Microbiol. 79, 460–468. McPherson, S.A., Perlak, F.J., Fuchs, R.L., Marrone, P.G., Lavrik, P.B., Fischhoff, D.A., 1988. Characterization of the coleopteran-specific protein gene of Bacillus thuringiensis var tenebrionis. Nat. Biotechnol. 6, 61–66. Naimov, S., Boncheva, R., Karlova, R., Dukiandjiev, S., Minkov, I., de Maagd, R.A., 2008. Solubilization, activation, and insecticidal activity of Bacillus thuringiensis serovar thompsoni HD542 crystal proteins. Appl. Environ. Microbiol. 74, 7145–7151. Raymond, B., Johnston, P.R., Nielsen-LeRoux, C., Lereclus, D., Crickmore, N., 2010. Bacillus thuringiensis: an impotent pathogen? Trends Microbiol. 18, 189–194. Sanahuja, G., Banakar, R., Twyman, R.M., Capell, T., Christou, P., 2011. Bacillus thuringiensis: a century of research, development and commercial applications. Plant Biotechnol. J. 9, 283–300. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D., Dean, D., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775–806. Simpson, J.T., Wong, K., Jackman, S.D., Schein, J.E., Jones, S.J., Birol, I., 2009. ABySS: a parallel assembler for short read sequence data. Genome Res. 19, 1117–1123. Ye, W., Zhu, L., Liu, Y., Crickmore, N., Peng, D., Ruan, L., Sun, M., 2012. Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a computational pipeline. Appl. Environ. Microbiol. 78, 4795–4801.
Please cite this article in press as: Gao, Q., et al., Complete genome sequence of Bacillus thuringiensis tenebrionis 4AA1, a typical strain with toxicity to Coleopteran insects. J. Biotechnol. (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.03.009
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