Journal of Biotechnology 207 (2015) 8–9
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of Bacillus pumilus W3: A strain exhibiting high laccase activity Zheng-Bing Guan ∗ , Yu-Jie Cai, Yan-Zhou Zhang, Hong Zhao, Xiang-Ru Liao ∗ The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China
a r t i c l e
i n f o
Article history: Received 18 April 2015 Accepted 27 April 2015 Available online 7 May 2015
a b s t r a c t Here we report the full genome sequence of Bacillus pumilus W3, which was isolated from raw gallnut honey in Nandan County, Guangxi Province of China, showing high CotA-laccase activity. The W3 strain contains 3,745,123 bp with GC content of 41.39%, and contains 3695 protein-coding genes, 21 rRNAs and 70 tRNAs. © 2015 Elsevier B.V. All rights reserved.
Keywords: Bacillus pumilus Genome CotA-laccase
Highly stable bacterial laccases function within a wider pH range and at high temperatures compared to fungal ones, and are also less dependent on metal ions and less susceptible to inhibitory agents (Singh et al., 2011; Sharma et al., 2007). The biological treatment of textile dyeing industrial wastewaters usually requires enzymes to remain active under high pH and temperature conditions or under high concentrations of organic solvents (Klibanov, 2001). Thus bacterial laccases, such as Bacillus CotA-laccase which locates in the outer surface of spore, have significant potential industrial applications in the decolorization of industrial textile dye effluents (Sharma et al., 2007; Koschorreck et al., 2008; Reiss et al., 2011). B. pumilus W3, which was isolated from raw gallnut honey collected in Nandan County, Guangxi Province, China, exhibits high CotA-laccase activity. Our previous work demonstrated that CotAlaccase of B. pumilus W3 was highly stable in alkaline pH and high temperature conditions, also exhibited considerable tolerance to organic solvents and NaCl, and could efficiently decolorize azo and anthraquinonic dyes under alkaline condition (pH 9.0), suggesting this enzyme highly potent for the industrial decolorization of textile dyeing effluents (Guan et al., 2014). B. pumilus is generally non-pathogenic in humans (Sorokulova et al., 2008). In order to elucidate the genetic background of this promising strain and get deep insights into the transcriptional regulation mechanism of CotA-laccase to facilitate the follow-up rational genetic engineering
∗ Corresponding authors. Tel.: +86 510 8532 7725; fax: +86 510 8532 7725. E-mail addresses: [email protected] (Z.-B. Guan), [email protected] (X.-R. Liao). http://dx.doi.org/10.1016/j.jbiotec.2015.04.019 0168-1656/© 2015 Elsevier B.V. All rights reserved.
to this strain, we sequenced the complete genome of B. pumilus W3. The genome of B. pumilus W3 was sequenced with Illumina Hiseq 2000 system. A total of 3,551,566 reads (∼1013 Mb) were generated, reaching a depth of 272-fold genome coverage. All the short reads were assembled using the SPAdes (version 3.1.1) program (Bankevich et al., 2012). GapCloser and GapFiller were used to close gaps where possible after assembly (Boetzer and Pirovano, 2012). Then a draft genome with 15 scaffolds (no gaps in scaffolds) was achieved. The order of scaffolds was determined by a similarity search among published references (Gioia et al., 2007; Tirumalai et al., 2013). Gaps between scaffolds were closed by longrange PCR using TaKaRa LA TaqTM with GC Buffer (Dalian, China) and subsequent Sanger sequencing using an ABI 3730 capillary sequencer. Genome annotation was performed by NCBI Prokaryote Genomes Automatic Annotation Pipeline (PGAAP) software (Pruitt et al., 2009). The complete genome of B. pumilus W3 consists of one 3,745,123 bp circular chromosome with no plasmid. The chromosome has a GC content of 41.39% and contains 3695 coding sequences (CDSs), 70 tRNA genes, and 7 rRNA operons (Table 1). Comparative genome analysis with the strain B. pumilus SAFR-032 (GenBank accession no. CP000813) showed that the sequences of W3 and SARF-32 were colinear, with minor insertions, deletions, and rearrangements (Darling et al., 2010). The available genome information of B. pumilus W3 will facilitate our future rational genetic manipulations, e.g., for increasing the level of CotA-laccase displayed at the outer coat of W3 spore thus it can be used as wholecell biocatalyst in biotechnological applications (bio-bleaching of textile dyes or pulp, etc.) needing massive immobilized alkaline thermo-stable laccase.
Z.-B. Guan et al. / Journal of Biotechnology 207 (2015) 8–9 Table 1 General features of B. pumilus W3 genome. Attributes
Value
Genome size (bp) GC content (%) Total predicted CDSs Plasmid rRNAs tRNAs
3,745,123 41.39 3695 0 21 (5S, 16S, 23S) 70
Nucleotide sequence accession number. The genome sequence of B. pumilus W3 has been deposited in NCBI GenBank under accession number CP011150. The strain has been deposited at the China Center for Type Culture Collection (CCTCC No. M2015018). Acknowledgements This work was supported by the National Natural Science Foundation of China (31472003 and 31101331), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program. References Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M., Kulikov, A.S., Lesin, V.M., Nikolenko, S.I., Pham, S., Prjibelski, A.D., Pyshkin, A.V., Sirotkin, A.V., Vyahhi, N., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477.
9
Boetzer, M., Pirovano, W., 2012. Toward almost closed genomes with GapFiller. Genome Biol. 13, R56. Darling, A.C., Mau, B., Blattner, F.R., Perna, N.T., 2010. ProgressiveMauve: multiple alignment with gene gain, loss and rearrangements. PLOS ONE 5, e11147. Gioia, J., Yerrapragada, S., Qin, X., Jiang, H., Igboeli, O.C., Muzny, D., DuganRocha, S., Ding, Y., Hawes, A., Liu, W., Perez, L., Kovar, C., Dinh, H., Lee, S., Nazareth, L., Blyth, P., Holder, M., Buhay, C., Tirumalai, M.R., Liu, Y., Dasgupta, I., Bokhetache, L., Fujita, M., Karouia, F., Eswara Moorthy, P., Siefert, J., Uzman, A., Buzumbo, P., Verma, A., Zwiya, H., McWilliams, B.D., Olowu, A., Clinkenbeard, K.D., Newcombe, D., Golebiewski, L., Petrosino, J.F., Nicholson, W.L., Fox, G.E., Venkateswaran, K., Highlander, S.K., Weinstock, G.M., 2007. Paradoxical DNA repair and peroxide resistance gene conservation in Bacillus pumilus SAFR-032. PLoS ONE 9, e928. Guan, Z.B., Song, C.M., Zhang, N., Zhou, W., Xu, C.W., Zhou, L.X., Zhao, H., Cai, Y.J., Liao, X.R., 2014. Overexpression, characterization, and dye-decolorizing ability of a thermostable, pH-stable, and organic solvent-tolerant laccase from Bacillus pumilus W3. J. Mol. Catal. B: Enzym. 101, 1–6. Klibanov, A., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241–246. Koschorreck, K., Richter, S.M., Ene, A.B., Roduner, E., Schmid, R.D., Urlacher, V.B., 2008. Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids. Appl. Microbiol. Biotechnol. 79, 217–224. Pruitt, K.D., Tatusova, T., Klimke, W., Maglott, D.R., 2009. NCBI reference sequences: current status, policy and new initiatives. Nucleic Acids Res. 37, D32–D36. Reiss, R., Ihssen, J., Linda, T.M., 2011. Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnol. 11, 9. Sharma, P., Goel, R., Capalash, N., 2007. Bacterial laccases. World J. Microbiol. Biotechnol. 23, 823–832. Singh, G., Bhalla, A., Kaur, P., Capalash, N., Sharma, P., 2011. Laccase from prokaryotes: a new source for an old enzyme. Rev. Environ. Sci. Biotechnol. 10, 309–326. Sorokulova, I.B., Pinchuk, I.V., Denayrolles, M., Osipova, I.G., Huang, J.M., Cutting, S.M., Urdaci, M.C., 2008. The safety of two Bacillus probiotic strains for human use digestive diseases and sciences. Digest. Dis. Sci. 53, 954–963. Tirumalai, M.R., Rastogi, R., Zamani, N., O’Bryant Williams, E., Allen, S., Diouf, F., Kwende, S., Weinstock, G.M., Venkateswaran, K.J., Fox, G.E., 2013. Candidate genes that may be responsible for the unusual resistances exhibited by Bacillus pumilus SAFR-032 spores. PLOS ONE 8, e66012.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of Bacillus pumilus W3: A strain exhibiting high laccase activity Zheng-Bing Guan ∗ , Yu-Jie Cai, Yan-Zhou Zhang, Hong Zhao, Xiang-Ru Liao ∗ The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China
a r t i c l e
i n f o
Article history: Received 18 April 2015 Accepted 27 April 2015 Available online 7 May 2015
a b s t r a c t Here we report the full genome sequence of Bacillus pumilus W3, which was isolated from raw gallnut honey in Nandan County, Guangxi Province of China, showing high CotA-laccase activity. The W3 strain contains 3,745,123 bp with GC content of 41.39%, and contains 3695 protein-coding genes, 21 rRNAs and 70 tRNAs. © 2015 Elsevier B.V. All rights reserved.
Keywords: Bacillus pumilus Genome CotA-laccase
Highly stable bacterial laccases function within a wider pH range and at high temperatures compared to fungal ones, and are also less dependent on metal ions and less susceptible to inhibitory agents (Singh et al., 2011; Sharma et al., 2007). The biological treatment of textile dyeing industrial wastewaters usually requires enzymes to remain active under high pH and temperature conditions or under high concentrations of organic solvents (Klibanov, 2001). Thus bacterial laccases, such as Bacillus CotA-laccase which locates in the outer surface of spore, have significant potential industrial applications in the decolorization of industrial textile dye effluents (Sharma et al., 2007; Koschorreck et al., 2008; Reiss et al., 2011). B. pumilus W3, which was isolated from raw gallnut honey collected in Nandan County, Guangxi Province, China, exhibits high CotA-laccase activity. Our previous work demonstrated that CotAlaccase of B. pumilus W3 was highly stable in alkaline pH and high temperature conditions, also exhibited considerable tolerance to organic solvents and NaCl, and could efficiently decolorize azo and anthraquinonic dyes under alkaline condition (pH 9.0), suggesting this enzyme highly potent for the industrial decolorization of textile dyeing effluents (Guan et al., 2014). B. pumilus is generally non-pathogenic in humans (Sorokulova et al., 2008). In order to elucidate the genetic background of this promising strain and get deep insights into the transcriptional regulation mechanism of CotA-laccase to facilitate the follow-up rational genetic engineering
∗ Corresponding authors. Tel.: +86 510 8532 7725; fax: +86 510 8532 7725. E-mail addresses: [email protected] (Z.-B. Guan), [email protected] (X.-R. Liao). http://dx.doi.org/10.1016/j.jbiotec.2015.04.019 0168-1656/© 2015 Elsevier B.V. All rights reserved.
to this strain, we sequenced the complete genome of B. pumilus W3. The genome of B. pumilus W3 was sequenced with Illumina Hiseq 2000 system. A total of 3,551,566 reads (∼1013 Mb) were generated, reaching a depth of 272-fold genome coverage. All the short reads were assembled using the SPAdes (version 3.1.1) program (Bankevich et al., 2012). GapCloser and GapFiller were used to close gaps where possible after assembly (Boetzer and Pirovano, 2012). Then a draft genome with 15 scaffolds (no gaps in scaffolds) was achieved. The order of scaffolds was determined by a similarity search among published references (Gioia et al., 2007; Tirumalai et al., 2013). Gaps between scaffolds were closed by longrange PCR using TaKaRa LA TaqTM with GC Buffer (Dalian, China) and subsequent Sanger sequencing using an ABI 3730 capillary sequencer. Genome annotation was performed by NCBI Prokaryote Genomes Automatic Annotation Pipeline (PGAAP) software (Pruitt et al., 2009). The complete genome of B. pumilus W3 consists of one 3,745,123 bp circular chromosome with no plasmid. The chromosome has a GC content of 41.39% and contains 3695 coding sequences (CDSs), 70 tRNA genes, and 7 rRNA operons (Table 1). Comparative genome analysis with the strain B. pumilus SAFR-032 (GenBank accession no. CP000813) showed that the sequences of W3 and SARF-32 were colinear, with minor insertions, deletions, and rearrangements (Darling et al., 2010). The available genome information of B. pumilus W3 will facilitate our future rational genetic manipulations, e.g., for increasing the level of CotA-laccase displayed at the outer coat of W3 spore thus it can be used as wholecell biocatalyst in biotechnological applications (bio-bleaching of textile dyes or pulp, etc.) needing massive immobilized alkaline thermo-stable laccase.
Z.-B. Guan et al. / Journal of Biotechnology 207 (2015) 8–9 Table 1 General features of B. pumilus W3 genome. Attributes
Value
Genome size (bp) GC content (%) Total predicted CDSs Plasmid rRNAs tRNAs
3,745,123 41.39 3695 0 21 (5S, 16S, 23S) 70
Nucleotide sequence accession number. The genome sequence of B. pumilus W3 has been deposited in NCBI GenBank under accession number CP011150. The strain has been deposited at the China Center for Type Culture Collection (CCTCC No. M2015018). Acknowledgements This work was supported by the National Natural Science Foundation of China (31472003 and 31101331), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project (No. 111-2-06), and the Jiangsu Province “Collaborative Innovation Center for Advanced Industrial Fermentation” industry development program. References Bankevich, A., Nurk, S., Antipov, D., Gurevich, A.A., Dvorkin, M., Kulikov, A.S., Lesin, V.M., Nikolenko, S.I., Pham, S., Prjibelski, A.D., Pyshkin, A.V., Sirotkin, A.V., Vyahhi, N., Tesler, G., Alekseyev, M.A., Pevzner, P.A., 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477.
9
Boetzer, M., Pirovano, W., 2012. Toward almost closed genomes with GapFiller. Genome Biol. 13, R56. Darling, A.C., Mau, B., Blattner, F.R., Perna, N.T., 2010. ProgressiveMauve: multiple alignment with gene gain, loss and rearrangements. PLOS ONE 5, e11147. Gioia, J., Yerrapragada, S., Qin, X., Jiang, H., Igboeli, O.C., Muzny, D., DuganRocha, S., Ding, Y., Hawes, A., Liu, W., Perez, L., Kovar, C., Dinh, H., Lee, S., Nazareth, L., Blyth, P., Holder, M., Buhay, C., Tirumalai, M.R., Liu, Y., Dasgupta, I., Bokhetache, L., Fujita, M., Karouia, F., Eswara Moorthy, P., Siefert, J., Uzman, A., Buzumbo, P., Verma, A., Zwiya, H., McWilliams, B.D., Olowu, A., Clinkenbeard, K.D., Newcombe, D., Golebiewski, L., Petrosino, J.F., Nicholson, W.L., Fox, G.E., Venkateswaran, K., Highlander, S.K., Weinstock, G.M., 2007. Paradoxical DNA repair and peroxide resistance gene conservation in Bacillus pumilus SAFR-032. PLoS ONE 9, e928. Guan, Z.B., Song, C.M., Zhang, N., Zhou, W., Xu, C.W., Zhou, L.X., Zhao, H., Cai, Y.J., Liao, X.R., 2014. Overexpression, characterization, and dye-decolorizing ability of a thermostable, pH-stable, and organic solvent-tolerant laccase from Bacillus pumilus W3. J. Mol. Catal. B: Enzym. 101, 1–6. Klibanov, A., 2001. Improving enzymes by using them in organic solvents. Nature 409, 241–246. Koschorreck, K., Richter, S.M., Ene, A.B., Roduner, E., Schmid, R.D., Urlacher, V.B., 2008. Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids. Appl. Microbiol. Biotechnol. 79, 217–224. Pruitt, K.D., Tatusova, T., Klimke, W., Maglott, D.R., 2009. NCBI reference sequences: current status, policy and new initiatives. Nucleic Acids Res. 37, D32–D36. Reiss, R., Ihssen, J., Linda, T.M., 2011. Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnol. 11, 9. Sharma, P., Goel, R., Capalash, N., 2007. Bacterial laccases. World J. Microbiol. Biotechnol. 23, 823–832. Singh, G., Bhalla, A., Kaur, P., Capalash, N., Sharma, P., 2011. Laccase from prokaryotes: a new source for an old enzyme. Rev. Environ. Sci. Biotechnol. 10, 309–326. Sorokulova, I.B., Pinchuk, I.V., Denayrolles, M., Osipova, I.G., Huang, J.M., Cutting, S.M., Urdaci, M.C., 2008. The safety of two Bacillus probiotic strains for human use digestive diseases and sciences. Digest. Dis. Sci. 53, 954–963. Tirumalai, M.R., Rastogi, R., Zamani, N., O’Bryant Williams, E., Allen, S., Diouf, F., Kwende, S., Weinstock, G.M., Venkateswaran, K.J., Fox, G.E., 2013. Candidate genes that may be responsible for the unusual resistances exhibited by Bacillus pumilus SAFR-032 spores. PLOS ONE 8, e66012.
