Draft Genome Sequences of Three Alkaliphilic Bacillus Strains, Bacillus wakoensis JCM 9140T, Bacillus akibai JCM 9157T, and Bacillus hemicellulosilyticus JCM 9152T Masahiro Yuki,a Kenshiro Oshima,b Wataru Suda,b Yumi Oshida,c Keiko Kitamura,c Toshiya Iida,c Masahira Hattori,b Moriya Ohkumaa,c Biomass Research Platform Team, RIKEN Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, Tsukuba, Ibaragi, Japana; Center for Omics and Bioinformatics, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Chiba, Japanb; Japan Collection of Microorganisms/Microbe Division, RIKEN BioResource Center, Tsukuba, Ibaragi, Japanc
Here, we report the draft genome sequences of the type strains of three cellulolytic or hemicellulolytic alkaliphilic Bacillus species: Bacillus wakoensis, Bacillus akibai, and Bacillus hemicellulosilyticus. The genome information for these three strains will be useful for studies of alkaliphilic Bacillus species, their evolution, and biotechnological applications for their enzymes. Received 24 December 2013 Accepted 6 January 2014 Published 30 January 2014 Citation Yuki M, Oshima K, Suda W, Oshida Y, Kitamura K, Iida T, Hattori M, Ohkuma M. 2014. Draft genome sequences of three alkaliphilic Bacillus strains, Bacillus wakoensis JCM 9140T, Bacillus akibai JCM 9157T, and Bacillus hemicellulosilyticus JCM 9152T. Genome Announc. 2(1):e01258-13. doi:10.1128/genomeA.01258-13. Copyright © 2014 Yuki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Moriya Ohkuma, [email protected].
A
lkaliphilic Bacillus strains have been isolated from various environments and have been well studied for their mechanisms of adaptation to alkaline conditions. These strains produce alkaline enzymes, such as protease, cyclomaltodextrin glucanotransferase, amylases, hemicellulase, and cellulase, which are useful for industry (1). Bacillus wakoensis strain JCM 9140T and Bacillus akibai strain JCM 9157T were isolated from soil as cellulolytic alkaliphiles, and their cellulose-degrading enzymes were characterized (2–5). A 16S rRNA gene sequence analysis indicated that the strains are closely related to each other and to Bacillus krulwichiae (5). Bacillus hemicellulosilyticus strain JCM 9152T, which is related to Bacillus halodurans and Bacillus okuhidensis (5), was isolated as a utilizer of alkaline hemicellulose-rich rayon waste and produces hemicellulases resistant to high pH conditions (6). The genomes of B. wakoensis JCM 9140T, B. akibai JCM 9157T, and B. hemicellulosilyticus JCM 9152T were sequenced using an Ion Torrent PGM system. The sequence reads totaling 621,952 for JCM 9140T, 523,952 for JCM 9157T, and 569,775 for JCM 9152T were assembled using Newbler version 2.8 (Roche) into 187, 142, and 102 contigs, with N50 lengths of 51,263, 79,963, and 128,524 bp, respectively. These assemblies resulted in draft genome sequences of 4,530,938 bp for JCM 9140T, 4,749,369 bp for JCM 9157T, and 4,538,064 bp for JCM 9152T, with 24.6, 23.5, and 27.1⫻ redundancies and G⫹C contents of 38.3, 37.0, and 38.2%, respectively. A total of 4,576, 4,711, and 4,345 protein-encoding genes and 108, 94, and 72 RNA-encoding sequences for JCM 9140T, JCM 9157T, and JCM 9152T, respectively, were identified using the RAST server (7) and the manual inspections detailed below. RAST annotations and the subsequent CAZy database analyses (8) revealed that B. akibai JCM 9157T and B. hemicellulosilyticus JCM 9152T have in common various genes encoding xylanases classified in the glycoside hydrolase 8 (GH8), GH10, and GH43 families and -xylosidases in the GH43 and GH52 families. B. hemicellulosilyticus JCM 9152T has genes encoding cellulases of
January/February 2014 Volume 2 Issue 1 e01258-13
GH5 and GH16, whereas B. akibai JCM 9157T has only that of GH16. Both strains have genes encoding -glucosidase of GH3, and B. hemicellulosilyticus JCM 9152T also has that of GH1. In the genome of B. wakoensis JCM 9140T, genes encoding -xylanase of GH10, -xylosidase of GH43, and -glucosidase of GH1 were found. Although no cellulase gene was found in the RAST annotation, BLASTp analysis using the CAZy database showed that B. wakoensis JCM 9140T has genes encoding cellulases of GH5 and GH9. These cellulolytic and hemicellulolytic enzymes have biotechnological potentials in various industries. Further comparative analyses on a genome scale will facilitate studies on the adaptation and evolution of alkaliphilic Bacillus species and their cellulolytic and hemicellulolytic enzymes. Nucleotide sequence accession numbers. The genome sequences of B. wakoensis JCM 9140T, B. akibai JCM 9157T, and B. hemicellulosilyticus JCM 9152T have been deposited in the DDBJ database under the accession no. BAUT01000001 to BAUT01000187, BAUV01000001 to BAUV01000142, and BAUU01000001 to BAUU01000102, respectively. ACKNOWLEDGMENTS This work was supported by the Genome Information Upgrading Program of the National BioResource Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Hiromi Kuroyanagi for technical support.
REFERENCES 1. Horikoshi K. 1999. Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63:735–750. 2. Horikoshi K, Ikeda Y, Nakao M. 1971. Manufacturing method of cellulase. Japan patent no. 46-76685. 3. Fukumori F, Kudo T, Horikoshi K. 1985. Purification and properties of a cellulase from alkalophilic bacillus sp. J. Gen. Microbiol. 1139:131: 3339 –3345. 4. Fukumori F, Kudo T, Narahashi Y, Horikoshi K. 1986. Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139. J. Gen. Microbiol. 132:2329 –2335.
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Yuki et al.
5. Nogi Y, Takami H, Horikoshi K. 2005. Characterization of alkaliphilic bacillus strains used in industry: proposal of five novel species. Int. J. Syst. Evol. Microbiol. 55:2309 –2315. http://dx.doi.org/10.1099/ijs.0.63649-0. 6. Ikura Y, Horikochi K. 1977. Isolation and some properties of alkalophilic bacteria utilizing rayon waste. Agric. Biol. Chem. 41:1373–1377. http://dx .doi.org/10.1271/bbb1961.41.1373. 7. 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
2 genomea.asm.org
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. http://dx.doi.org/10.1186/1471-2164-9-75. 8. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37:D233–D238. http: //dx.doi.org/10.1093/nar/gkn663.
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January/February 2014 Volume 2 Issue 1 e01258-13
Here, we report the draft genome sequences of the type strains of three cellulolytic or hemicellulolytic alkaliphilic Bacillus species: Bacillus wakoensis, Bacillus akibai, and Bacillus hemicellulosilyticus. The genome information for these three strains will be useful for studies of alkaliphilic Bacillus species, their evolution, and biotechnological applications for their enzymes. Received 24 December 2013 Accepted 6 January 2014 Published 30 January 2014 Citation Yuki M, Oshima K, Suda W, Oshida Y, Kitamura K, Iida T, Hattori M, Ohkuma M. 2014. Draft genome sequences of three alkaliphilic Bacillus strains, Bacillus wakoensis JCM 9140T, Bacillus akibai JCM 9157T, and Bacillus hemicellulosilyticus JCM 9152T. Genome Announc. 2(1):e01258-13. doi:10.1128/genomeA.01258-13. Copyright © 2014 Yuki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Moriya Ohkuma, [email protected].
A
lkaliphilic Bacillus strains have been isolated from various environments and have been well studied for their mechanisms of adaptation to alkaline conditions. These strains produce alkaline enzymes, such as protease, cyclomaltodextrin glucanotransferase, amylases, hemicellulase, and cellulase, which are useful for industry (1). Bacillus wakoensis strain JCM 9140T and Bacillus akibai strain JCM 9157T were isolated from soil as cellulolytic alkaliphiles, and their cellulose-degrading enzymes were characterized (2–5). A 16S rRNA gene sequence analysis indicated that the strains are closely related to each other and to Bacillus krulwichiae (5). Bacillus hemicellulosilyticus strain JCM 9152T, which is related to Bacillus halodurans and Bacillus okuhidensis (5), was isolated as a utilizer of alkaline hemicellulose-rich rayon waste and produces hemicellulases resistant to high pH conditions (6). The genomes of B. wakoensis JCM 9140T, B. akibai JCM 9157T, and B. hemicellulosilyticus JCM 9152T were sequenced using an Ion Torrent PGM system. The sequence reads totaling 621,952 for JCM 9140T, 523,952 for JCM 9157T, and 569,775 for JCM 9152T were assembled using Newbler version 2.8 (Roche) into 187, 142, and 102 contigs, with N50 lengths of 51,263, 79,963, and 128,524 bp, respectively. These assemblies resulted in draft genome sequences of 4,530,938 bp for JCM 9140T, 4,749,369 bp for JCM 9157T, and 4,538,064 bp for JCM 9152T, with 24.6, 23.5, and 27.1⫻ redundancies and G⫹C contents of 38.3, 37.0, and 38.2%, respectively. A total of 4,576, 4,711, and 4,345 protein-encoding genes and 108, 94, and 72 RNA-encoding sequences for JCM 9140T, JCM 9157T, and JCM 9152T, respectively, were identified using the RAST server (7) and the manual inspections detailed below. RAST annotations and the subsequent CAZy database analyses (8) revealed that B. akibai JCM 9157T and B. hemicellulosilyticus JCM 9152T have in common various genes encoding xylanases classified in the glycoside hydrolase 8 (GH8), GH10, and GH43 families and -xylosidases in the GH43 and GH52 families. B. hemicellulosilyticus JCM 9152T has genes encoding cellulases of
January/February 2014 Volume 2 Issue 1 e01258-13
GH5 and GH16, whereas B. akibai JCM 9157T has only that of GH16. Both strains have genes encoding -glucosidase of GH3, and B. hemicellulosilyticus JCM 9152T also has that of GH1. In the genome of B. wakoensis JCM 9140T, genes encoding -xylanase of GH10, -xylosidase of GH43, and -glucosidase of GH1 were found. Although no cellulase gene was found in the RAST annotation, BLASTp analysis using the CAZy database showed that B. wakoensis JCM 9140T has genes encoding cellulases of GH5 and GH9. These cellulolytic and hemicellulolytic enzymes have biotechnological potentials in various industries. Further comparative analyses on a genome scale will facilitate studies on the adaptation and evolution of alkaliphilic Bacillus species and their cellulolytic and hemicellulolytic enzymes. Nucleotide sequence accession numbers. The genome sequences of B. wakoensis JCM 9140T, B. akibai JCM 9157T, and B. hemicellulosilyticus JCM 9152T have been deposited in the DDBJ database under the accession no. BAUT01000001 to BAUT01000187, BAUV01000001 to BAUV01000142, and BAUU01000001 to BAUU01000102, respectively. ACKNOWLEDGMENTS This work was supported by the Genome Information Upgrading Program of the National BioResource Project from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Hiromi Kuroyanagi for technical support.
REFERENCES 1. Horikoshi K. 1999. Alkaliphiles: some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63:735–750. 2. Horikoshi K, Ikeda Y, Nakao M. 1971. Manufacturing method of cellulase. Japan patent no. 46-76685. 3. Fukumori F, Kudo T, Horikoshi K. 1985. Purification and properties of a cellulase from alkalophilic bacillus sp. J. Gen. Microbiol. 1139:131: 3339 –3345. 4. Fukumori F, Kudo T, Narahashi Y, Horikoshi K. 1986. Molecular cloning and nucleotide sequence of the alkaline cellulase gene from the alkalophilic Bacillus sp. strain 1139. J. Gen. Microbiol. 132:2329 –2335.
Genome Announcements
genomea.asm.org 1
Yuki et al.
5. Nogi Y, Takami H, Horikoshi K. 2005. Characterization of alkaliphilic bacillus strains used in industry: proposal of five novel species. Int. J. Syst. Evol. Microbiol. 55:2309 –2315. http://dx.doi.org/10.1099/ijs.0.63649-0. 6. Ikura Y, Horikochi K. 1977. Isolation and some properties of alkalophilic bacteria utilizing rayon waste. Agric. Biol. Chem. 41:1373–1377. http://dx .doi.org/10.1271/bbb1961.41.1373. 7. 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
2 genomea.asm.org
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. http://dx.doi.org/10.1186/1471-2164-9-75. 8. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2009. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37:D233–D238. http: //dx.doi.org/10.1093/nar/gkn663.
Genome Announcements
January/February 2014 Volume 2 Issue 1 e01258-13
