Journal of Biotechnology 177 (2014) 20–21
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Draft genome sequence of Bacillus firmus DS1 Ce Geng a , Zhichao Tang a , Donghai Peng a , Zongze Shao b , Lei Zhu a , Jinshui Zheng a , Huan Wang a , Lifang Ruan a , Ming Sun a,∗ a State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China b Key Lab of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian 361005, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 13 February 2014 Accepted 14 February 2014 Available online 22 February 2014 Keywords: Bacillus firmus Genome Marine sediment Heavy-metal ion absorption
a b s t r a c t Bacillus firmus DS1, an aerobic, Gram-positive, spore-forming bacterium isolated from marine sediment of the China South Sea coast. Here, the first draft genome sequence of B. firmus DS1 that may help us to clarify the evolutionary status of B. firmus, also will give the opportunity to provide the genetic basis for heavy-metal ion absorption in environmental bioremediation, the enzymes in industrial production and more other active ingredients application. © 2014 Elsevier B.V. All rights reserved.
Since Bacillus firmus was first reported as an isolatable strain different from other Bacillus by Bredemann and Werner (Werner, 1933), hundreds of B. firmus stains were isolated from diverse environmental soil, and were widely used in different areas, which have been used for removal of heavy metals from wastewater in industry (Bachate et al., 2013), also utilized its various extracellular enzymes, cellular endonucleases, such as cyclodextrin glycosyltransferase (CGTase) (Matioli et al., 2001), fibrinolytic enzyme (Seo and Lee, 2004), thermostable xylanases (Chang et al., 2004), thermophilic type II restriction endonucleases (Jutur et al., 2004), even used as efficient adjuvant in respiratory tract immunization (Zanvit et al., 2005) or probiotics in aquaculture. Recently, B. firmus DS1, a newly isolated bacterium from marine sediment of the China South Sea coast, was founded 99% identity with B. firmus standard strain IAM 12464 by 16S rDNA sequence analysis. Though some decades of B. firmus widely used in agricultural and aquacultural disease control, as well as the enzyme usage on extreme condition (Bachate et al., 2013; Seo and Lee, 2004), the exact active ingredients genetic basis which against aquacultural pathogen, also the reason it utilized as candidate for bioremediation of pollution environments is yet unclear. To date, there is no reference
∗ Corresponding author. Tel.: +86 27 87283455; fax: +86 27 87280670. E-mail address: [email protected] (M. Sun). http://dx.doi.org/10.1016/j.jbiotec.2014.02.012 0168-1656/© 2014 Elsevier B.V. All rights reserved.
B. firmus genome sequence available, to get further knowledge and usage of B. firmus, so that we announce the draft genome sequence of B. firmus DS1, the first released B. firmus genome sequence. The draft genome of B. firmus was sequenced with a wholegenome shotgun strategy using an Illumina HiSeq2000 instrument. Quality trimming of 90-nucleotide (nt) paired-end reads, produced from a 500-bp genomic library (1901 Mb; 413-fold coverage), and de novo assembly were performed using the SOAPdenovo2 alignment tool (Luo et al., 2012). The analysis generated 72 large contigs over 689 bp from 808 contigs (4,583,930 bp; G + C content, 41.94%), with an N50 of 294,736 bp, N90 of 76,845 bp and a maximum contig size of 501,602 bp (Table 1). This was the best result obtained with a k-mer size of 15. All the assembled data were deposited in the NCBI nucleotide sequence database. According to the RAST (Rapid Annotation using Subsystem Technology) genome analysis (Aziz et al., 2008), the closest neighbors of B. firmus DS1 are Bacillus sp. 2 A 57 CT2 and Anoxybacillus flavithermus WK1, not the Bacillus pseudofirmus OF4 originally named Bacillus firmus (Takami and Krulwich, 2000). The draft genome of B. firmus DS1 is 4,974,167 bp in size with 5247 RAST-server-annotated ORFs (open reading frames), also with 47 RNAs. 42% of predict coding sequences were assigned to subsystem categories. Compared with Bacillus cereus ATCC14579, several genes clusters were identified in details, which not found the homology genes in B. cereus. Resistance to antibiotics and toxic compounds genes that are responsible for the adaptation or
C. Geng et al. / Journal of Biotechnology 177 (2014) 20–21 Table 1 Bacillus firmus DS1 genome features.
21
Acknowledgements
Attributes
Value
Genome size Total number of contigs GC content (%) Plasmid rRNA operons tRNA operons Total predicted ORFs Genes with predicted function
4,974,167 bp 72 41.94 0 6 41 5247 2180
utilization of a wide various heavy metal contamination, such as Arsenic, Cobalt, Zinc, Cadmium, Mercuric tolerance related genes we located, which could explain its novel function in bioremediation. Scanned by antiSMASH 2.0 (Blin et al., 2013), moreover we also found a type III polyketides biosynthetic gene cluster in the contig 45 of draft B. firmus DS1 genome, so that, to some degree the existence of antibiotic gene cluster in B. firmus accounted for its widely utilization on aquacultural pathogen control. More importance, a plenty of capsular and extracellular polysaccharides genes, osmotic and oxidative stress response genes were required for its growth in extreme condition. Further comparative genome analyses are now under way to better elucidate the active ingredients of B. firmus. What’s more, availability of first time B. firmus genome sequence provides us opportunity to further understand the evolution relationship between Bacillus strains of marine origin and that isolated from soil, to explain the genetic reasons for its growth in the extreme condition, to get more knowledge about the basic genetics of widely used B. firmus, to exploit more secondary metabolites and virulence products applied in agriculture and aquaculture. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number APVL00000000. The first version (accession number APVL01000000) is described in this paper. The strain of B. firmus DS1 is available from Prof. Ming Sun (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, PR China).
This work was supported by grants from the National Basic Research Program (973) of China (2013CB127504), the National High Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), and the National Natural Science Foundation of China (30970037, 31171901). References Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K., Gerdes, S., Glass, E.M., Kubal, M., Meyer, F., Olsen, G.J., Olson, R., Osterman, A.L., Overbeek, R.A., McNeil, L.K., Paarmann, D., Paczian, T., Parrello, B., Pusch, G.D., 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. Bachate, S.P., Nandre, V.S., Ghatpande, N.S., Kodam, K.M., 2013. Simultaneous reduction of Cr(VI) and oxidation of As(III) by Bacillus firmus TE7 isolated from tannery effluent. Chemosphere 90, 2273–2278. Blin, K., Medema, M.H., Kazempour, D., Fischbach, M.A., Breitling, R., Takano, E., Weber, T., 2013. antiSMASH 2.0—a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41, W204–W212. Chang, P., Tsai, W.S., Tsai, C.L., Tseng, M.J., 2004. Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus. Biochem. Biophys. Res. Commun. 319, 1017–1025. Jutur, P.P., Hoti, S.L., Reddy, A.R., 2004. Bsu2413I and Bfi2411I, two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification. Thermophilic endonucleases from two Bacillus species. Mol. Biol. Rep. 31, 139–142. Luo, R., Liu, B., Xie, Y., Li, Z., Huang, W., Yuan, J., He, G., Chen, Y., Pan, Q., Liu, Y., Tang, J., Wu, G., Zhang, H., Shi, Y., Liu, Y., Yu, C., Wang, B., Lu, Y., Han, C., Cheung, D.W., Yiu, S.M., Peng, S., Xiaoqian, Z., Liu, G., Liao, X., Li, Y., Yang, H., Wang, J., Lam, T.W., Wang, J., 2012. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience 1, 18. Matioli, G., Zanin, G.M., De Moraes, F.F., 2001. Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain no. 37. Appl. Biochem. Biotechnol. 91–93, 643–654. Seo, J.H., Lee, S.P., 2004. Production of fibrinolytic enzyme from soybean grits fermented by Bacillus firmus NA-1. J. Med. Food 7, 442–449. Takami, H., Krulwich, T.A., 2000. Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4. Extremophiles 4, 19–22. Werner, W.E.G., 1933. Botanische Beschreibung häufiger am Buttersäureabbau beteiligter sporenbildender Bakterienspezies. Zentralbl Bakteriol Parasitenkd Abt II 87, 446–475. Zanvit, P., Havlickova, M., Tacner, J., Jirkovska, M., Petraskova, P., Novotna, O., Cechova, D., Julak, J., Sterzl, I., Prokesova, L., 2005. Immune response after adjuvant mucosal immunization of mice with inactivated influenza virus. Immunol. Lett. 97, 251–259.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Draft genome sequence of Bacillus firmus DS1 Ce Geng a , Zhichao Tang a , Donghai Peng a , Zongze Shao b , Lei Zhu a , Jinshui Zheng a , Huan Wang a , Lifang Ruan a , Ming Sun a,∗ a State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China b Key Lab of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian 361005, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 13 February 2014 Accepted 14 February 2014 Available online 22 February 2014 Keywords: Bacillus firmus Genome Marine sediment Heavy-metal ion absorption
a b s t r a c t Bacillus firmus DS1, an aerobic, Gram-positive, spore-forming bacterium isolated from marine sediment of the China South Sea coast. Here, the first draft genome sequence of B. firmus DS1 that may help us to clarify the evolutionary status of B. firmus, also will give the opportunity to provide the genetic basis for heavy-metal ion absorption in environmental bioremediation, the enzymes in industrial production and more other active ingredients application. © 2014 Elsevier B.V. All rights reserved.
Since Bacillus firmus was first reported as an isolatable strain different from other Bacillus by Bredemann and Werner (Werner, 1933), hundreds of B. firmus stains were isolated from diverse environmental soil, and were widely used in different areas, which have been used for removal of heavy metals from wastewater in industry (Bachate et al., 2013), also utilized its various extracellular enzymes, cellular endonucleases, such as cyclodextrin glycosyltransferase (CGTase) (Matioli et al., 2001), fibrinolytic enzyme (Seo and Lee, 2004), thermostable xylanases (Chang et al., 2004), thermophilic type II restriction endonucleases (Jutur et al., 2004), even used as efficient adjuvant in respiratory tract immunization (Zanvit et al., 2005) or probiotics in aquaculture. Recently, B. firmus DS1, a newly isolated bacterium from marine sediment of the China South Sea coast, was founded 99% identity with B. firmus standard strain IAM 12464 by 16S rDNA sequence analysis. Though some decades of B. firmus widely used in agricultural and aquacultural disease control, as well as the enzyme usage on extreme condition (Bachate et al., 2013; Seo and Lee, 2004), the exact active ingredients genetic basis which against aquacultural pathogen, also the reason it utilized as candidate for bioremediation of pollution environments is yet unclear. To date, there is no reference
∗ Corresponding author. Tel.: +86 27 87283455; fax: +86 27 87280670. E-mail address: [email protected] (M. Sun). http://dx.doi.org/10.1016/j.jbiotec.2014.02.012 0168-1656/© 2014 Elsevier B.V. All rights reserved.
B. firmus genome sequence available, to get further knowledge and usage of B. firmus, so that we announce the draft genome sequence of B. firmus DS1, the first released B. firmus genome sequence. The draft genome of B. firmus was sequenced with a wholegenome shotgun strategy using an Illumina HiSeq2000 instrument. Quality trimming of 90-nucleotide (nt) paired-end reads, produced from a 500-bp genomic library (1901 Mb; 413-fold coverage), and de novo assembly were performed using the SOAPdenovo2 alignment tool (Luo et al., 2012). The analysis generated 72 large contigs over 689 bp from 808 contigs (4,583,930 bp; G + C content, 41.94%), with an N50 of 294,736 bp, N90 of 76,845 bp and a maximum contig size of 501,602 bp (Table 1). This was the best result obtained with a k-mer size of 15. All the assembled data were deposited in the NCBI nucleotide sequence database. According to the RAST (Rapid Annotation using Subsystem Technology) genome analysis (Aziz et al., 2008), the closest neighbors of B. firmus DS1 are Bacillus sp. 2 A 57 CT2 and Anoxybacillus flavithermus WK1, not the Bacillus pseudofirmus OF4 originally named Bacillus firmus (Takami and Krulwich, 2000). The draft genome of B. firmus DS1 is 4,974,167 bp in size with 5247 RAST-server-annotated ORFs (open reading frames), also with 47 RNAs. 42% of predict coding sequences were assigned to subsystem categories. Compared with Bacillus cereus ATCC14579, several genes clusters were identified in details, which not found the homology genes in B. cereus. Resistance to antibiotics and toxic compounds genes that are responsible for the adaptation or
C. Geng et al. / Journal of Biotechnology 177 (2014) 20–21 Table 1 Bacillus firmus DS1 genome features.
21
Acknowledgements
Attributes
Value
Genome size Total number of contigs GC content (%) Plasmid rRNA operons tRNA operons Total predicted ORFs Genes with predicted function
4,974,167 bp 72 41.94 0 6 41 5247 2180
utilization of a wide various heavy metal contamination, such as Arsenic, Cobalt, Zinc, Cadmium, Mercuric tolerance related genes we located, which could explain its novel function in bioremediation. Scanned by antiSMASH 2.0 (Blin et al., 2013), moreover we also found a type III polyketides biosynthetic gene cluster in the contig 45 of draft B. firmus DS1 genome, so that, to some degree the existence of antibiotic gene cluster in B. firmus accounted for its widely utilization on aquacultural pathogen control. More importance, a plenty of capsular and extracellular polysaccharides genes, osmotic and oxidative stress response genes were required for its growth in extreme condition. Further comparative genome analyses are now under way to better elucidate the active ingredients of B. firmus. What’s more, availability of first time B. firmus genome sequence provides us opportunity to further understand the evolution relationship between Bacillus strains of marine origin and that isolated from soil, to explain the genetic reasons for its growth in the extreme condition, to get more knowledge about the basic genetics of widely used B. firmus, to exploit more secondary metabolites and virulence products applied in agriculture and aquaculture. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number APVL00000000. The first version (accession number APVL01000000) is described in this paper. The strain of B. firmus DS1 is available from Prof. Ming Sun (State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, PR China).
This work was supported by grants from the National Basic Research Program (973) of China (2013CB127504), the National High Technology Research and Development Program (863) of China (2011AA10A203), China 948 Program of Ministry of Agriculture (2011-G25), and the National Natural Science Foundation of China (30970037, 31171901). References Aziz, R.K., Bartels, D., Best, A.A., DeJongh, M., Disz, T., Edwards, R.A., Formsma, K., Gerdes, S., Glass, E.M., Kubal, M., Meyer, F., Olsen, G.J., Olson, R., Osterman, A.L., Overbeek, R.A., McNeil, L.K., Paarmann, D., Paczian, T., Parrello, B., Pusch, G.D., 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. Bachate, S.P., Nandre, V.S., Ghatpande, N.S., Kodam, K.M., 2013. Simultaneous reduction of Cr(VI) and oxidation of As(III) by Bacillus firmus TE7 isolated from tannery effluent. Chemosphere 90, 2273–2278. Blin, K., Medema, M.H., Kazempour, D., Fischbach, M.A., Breitling, R., Takano, E., Weber, T., 2013. antiSMASH 2.0—a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41, W204–W212. Chang, P., Tsai, W.S., Tsai, C.L., Tseng, M.J., 2004. Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus. Biochem. Biophys. Res. Commun. 319, 1017–1025. Jutur, P.P., Hoti, S.L., Reddy, A.R., 2004. Bsu2413I and Bfi2411I, two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification. Thermophilic endonucleases from two Bacillus species. Mol. Biol. Rep. 31, 139–142. Luo, R., Liu, B., Xie, Y., Li, Z., Huang, W., Yuan, J., He, G., Chen, Y., Pan, Q., Liu, Y., Tang, J., Wu, G., Zhang, H., Shi, Y., Liu, Y., Yu, C., Wang, B., Lu, Y., Han, C., Cheung, D.W., Yiu, S.M., Peng, S., Xiaoqian, Z., Liu, G., Liao, X., Li, Y., Yang, H., Wang, J., Lam, T.W., Wang, J., 2012. SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience 1, 18. Matioli, G., Zanin, G.M., De Moraes, F.F., 2001. Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain no. 37. Appl. Biochem. Biotechnol. 91–93, 643–654. Seo, J.H., Lee, S.P., 2004. Production of fibrinolytic enzyme from soybean grits fermented by Bacillus firmus NA-1. J. Med. Food 7, 442–449. Takami, H., Krulwich, T.A., 2000. Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4. Extremophiles 4, 19–22. Werner, W.E.G., 1933. Botanische Beschreibung häufiger am Buttersäureabbau beteiligter sporenbildender Bakterienspezies. Zentralbl Bakteriol Parasitenkd Abt II 87, 446–475. Zanvit, P., Havlickova, M., Tacner, J., Jirkovska, M., Petraskova, P., Novotna, O., Cechova, D., Julak, J., Sterzl, I., Prokesova, L., 2005. Immune response after adjuvant mucosal immunization of mice with inactivated influenza virus. Immunol. Lett. 97, 251–259.
