MARGEN-00287; No of Pages 2 Marine Genomics xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/technical resources
Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits Varsha S. Honkalas, Ashwini P. Dabir, Preeti Arora, Dilip R. Ranade, Prashant K. Dhakephalkar ⁎ Microbial Science Division, MACS's Agharkar Research Institute, Pune 411004, India
a r t i c l e
i n f o
Article history: Received 6 January 2015 Received in revised form 28 January 2015 Accepted 28 January 2015 Available online xxxx Keywords: Clostridium celerecrescens Methane hydrate Draft genome
a b s t r a c t Clostridium celerecrescens 152B is an obligate anaerobic, Gram positive rod shaped bacterium isolated from sub-seafloor methane hydrate sediments of Krishna Godavari basin, India. Here, we report the first draft genome sequence of C. celerecrescens 152B, which comprises 5,050,495 bp in 92 contigs with the G + C content of 43.5%. The whole genome of C. celerecrescens 152B was sequenced for further biotechnological exploitation of its genome features especially regarding the production of secondary metabolites as well as for environmental bioremediation and production of industrially valuable enzymes. © 2015 Published by Elsevier B.V.
1. Introduction Since Clostridium celerecrescens was first isolated from methanogenic cellulose enriched culture by Palop et al. (1989), several strains of C. celerecrescens have been widely used for production of hydrogen, ethanol and organic acids from cellulosic biomass (Ren et al., 2007; Blanchard et al., 2009), electrofuel (Zaybak et al., 2013) as well as cinnamic acid derivatives which could act as flavoring agents (Chamkha et al., 2001). C. celerecrescens strains have also been utilized for the reduction of Fe (III) ores to minimize acidification/bio-remediate mining associated environment (Garcia-Balboa et al., 2010). Till the present report, genome sequence of C. celerecrescens was not available in any of the reference databases. Here we announce the draft genome sequence of C. celerecrescens 152B; the first released C. celerecrescens genome sequence. 152B strain, recently isolated from sub-seafloor methane hydrate sediments of Krishna Godavari basin, India, exhibited 99% 16S rRNA gene sequence identity and 90% gyrB gene sequence homology with the type strain of C. celerecrescens 02PIK1. Strain 152B was obtained from MACS Collection of Microorganisms, Agharkar Research Institute, Pune. The whole genome of C. celerecrescens 152B was sequenced to understand the evolutionary lineage of the organism; to gain an insight into the genetic makeup of this organism and to improve biotechnological applications through the production of valuable metabolites and biomolecules. The genome of C. celerecrescens 152B was sequenced using the Ion Torrent PGM sequencer (200-bp library) applying the 316™ sequencing chip according to the manufacturer's instructions (Life Technologies, ⁎ Corresponding author. Tel.: +91 20 25653680; fax: +91 20 25651542. E-mail addresses: [email protected], [email protected] (P.K. Dhakephalkar).
USA). De novo assembly was performed using version 4.0.5 of MIRA Assembler (Chevreux et al., 1999) and SeqMan NGen DNASTAR application (version 11.2.1). The assembly was uploaded for annotation to the Rapid Annotation using Subsystem Technology (RAST) server (Aziz et al., 2008), KEGG database (Kanehisa et al., 2004) and National Centre for Biotechnology Information Prokaryotic Genomes Annotation Pipeline Version 2.6 (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/ static/pipeline.html). The analysis generated 92 contigs featuring a G + C content of 43.5%, an N50 value of 208,566 bp, an N90 value of 35,416 bp and a maximum contig size of 416,014 bp (Table 1). Eight putative gene clusters involved in secondary metabolite production were disclosed using antiSMASH 2.0 (Blin et al., 2013). All the assembled data was deposited in the NCBI genome sequence database. According to the RAST genome analysis, the closest neighbor of 152B was Clostridium saccharolyticum WM1 (Score 521, Genome ID 610130.3). The draft genome of 152B is 5,050,495 bp in size having 83.53 fold genome coverage with 4286 genes annotated using the NCBI prokaryotic genomes annotation pipeline (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/static/pipeline. html), with 27 rRNAs (5S, 16S and 23S subunits) and 72 tRNAs. The genome analysis of 152B revealed the presence of genes encoding resistance to heavy metal ions such as Co2+, Zn2 +, AsO23 −, Cd2 + as well as antibiotics such as fluoroquinolones. Ability of C. celerecrescens to produce hydrogen, an environmentally safe energy source (Calusinska et al., 2010) could be explained on the basis of presence of genes encoding Fe only hydrogenase, Ni–Fe hydrogenases and energy conserving hydrogenases (ferrodoxin). Presence of genes responsible for the production of organic acids including lactate, formate, succinate, and acetate underscored promising biotechnological potential of C. celerecrescens 152B. These pathways/key genes could be modified to qualitatively as well as quantitatively improve the yield.
http://dx.doi.org/10.1016/j.margen.2015.01.008 1874-7787/© 2015 Published by Elsevier B.V.
Please cite this article as: Honkalas, V.S., et al., Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.01.008
2
V.S. Honkalas et al. / Marine Genomics xxx (2015) xxx–xxx
Acknowledgments
Table 1 Genome features of Clostridium celerecrescens 152B. Attributes
Values
Genome size Total no. of contigs G + C content (%) Total no. of subsystems Total no. of coding sequences Genes with predicted functions Plasmids tRNAs rRNAs
5,050,495 bp 92 43.5 362 4286 4036 0 72 27
We found key genes responsible for the production of several enzymes such as α-amylase, β-glucosidase, chitinase, and β-galactosidase. Presence of extracellular polysaccharide genes, osmotic and oxidative stress genes, and heat shock and cold shock genes could explain the ability of this organism to thrive under extreme environmental conditions. Availability of the C. celerecrescens genome sequence has provided us with an opportunity for further biotechnological exploitation of its genome features especially regarding the production of secondary metabolites as well as for environmental bioremediation and production of industrially valuable enzymes. Genome sequence analysis will also help to further understand the evolutionary relationship between Clostridium strains of marine as well as terrestrial origin. 2. Nucleotide sequence accession number This whole-genome shotgun project has been deposited at DDBJ/ EMBL/GenBank under the accession number JPME00000000. The first version (JPME00000000.1) is described in this paper. C. celerecrescens 152B is available in MACS Collection of Microorganisms (Registration No. WDCM 561) under the name MCM B-936, Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India.
Methane hydrate samples were provided by National Gas Hydrate Repository, Oil and Natural Gas Corporation (ONGC), Panvel, Mumbai, India. CSIR is gratefully acknowledged for providing fellowship to Preeti Arora. 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. Blanchard, J., Leschine, S., Petit, E., Fabel, J., 2009. Generic methods and compositions for improving the production of fuels in microorganisms. https://www.google.com/ patents/US20090286294. 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. Calusinska, M., Happe, T., Joris, B., Wilmotte, A., 2010. The surprising diversity of clostridial hydrogenases: a comparative genomic perspective. Microbiology 156, 1575–1588. Chamkha, M., Garcia, J.L., Labat, M., 2001. Metabolism of cinnamic acids by some Clostridiales and emendation of the descriptions of Clostridium aerotolerans, Clostridium celerecrescens and Clostridium xylanolyticum. Int. J. Syst. Evol. Microbiol. 51, 2105–2111. Chevreux, B., Wetter, T., Suhai, S., 1999. Genome sequence assembly using trace signals and additional sequence information. Computer Science and Biology: Proceedings of the German Conference on Bioinformatics (GCB) 99, pp. 45–56. Garcia-Balboa, C., Bedoya, I.C., Gonzalez, F., Blazquez, M.L., Munoz, J.A., Ballester, A., 2010. Bio-reduction of Fe (III) ores using three pure strains of Aeromonas hydrophila, Serratia fonticola and Clostridium celerecrescens and a natural consortium. Bioresour. Technol. 101, 7864–7871. Kanehisa, M., Goto, S., Kawashima, S., Okuno, Y., Hattori, M., 2004. The KEGG resource for deciphering the genome. Nucleic Acids Res. 32, 277–280. Palop, M.L., Valles, S., Pinaga, F., Flors, A., 1989. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int. J. Syst. Evol. Microbiol. 39, 68–71. Ren, Z., Ward, T.E., Logan, B.E., Regan, J.M., 2007. Characterization of the cellulolytic and hydrogen‐producing activities of six mesophilic Clostridium species. J. Appl. Microbiol. 103, 2258–2266. Zaybak, Z., Pisciotta, J.M., Tokash, J.C., Logan, B.E., 2013. Enhanced start-up of anaerobic facultatively autotrophic biocathodes in bioelectrochemical systems. J. Biotechnol. 168, 478–485.
Please cite this article as: Honkalas, V.S., et al., Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.01.008
Contents lists available at ScienceDirect
Marine Genomics journal homepage: www.elsevier.com/locate/margen
Genomics/technical resources
Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits Varsha S. Honkalas, Ashwini P. Dabir, Preeti Arora, Dilip R. Ranade, Prashant K. Dhakephalkar ⁎ Microbial Science Division, MACS's Agharkar Research Institute, Pune 411004, India
a r t i c l e
i n f o
Article history: Received 6 January 2015 Received in revised form 28 January 2015 Accepted 28 January 2015 Available online xxxx Keywords: Clostridium celerecrescens Methane hydrate Draft genome
a b s t r a c t Clostridium celerecrescens 152B is an obligate anaerobic, Gram positive rod shaped bacterium isolated from sub-seafloor methane hydrate sediments of Krishna Godavari basin, India. Here, we report the first draft genome sequence of C. celerecrescens 152B, which comprises 5,050,495 bp in 92 contigs with the G + C content of 43.5%. The whole genome of C. celerecrescens 152B was sequenced for further biotechnological exploitation of its genome features especially regarding the production of secondary metabolites as well as for environmental bioremediation and production of industrially valuable enzymes. © 2015 Published by Elsevier B.V.
1. Introduction Since Clostridium celerecrescens was first isolated from methanogenic cellulose enriched culture by Palop et al. (1989), several strains of C. celerecrescens have been widely used for production of hydrogen, ethanol and organic acids from cellulosic biomass (Ren et al., 2007; Blanchard et al., 2009), electrofuel (Zaybak et al., 2013) as well as cinnamic acid derivatives which could act as flavoring agents (Chamkha et al., 2001). C. celerecrescens strains have also been utilized for the reduction of Fe (III) ores to minimize acidification/bio-remediate mining associated environment (Garcia-Balboa et al., 2010). Till the present report, genome sequence of C. celerecrescens was not available in any of the reference databases. Here we announce the draft genome sequence of C. celerecrescens 152B; the first released C. celerecrescens genome sequence. 152B strain, recently isolated from sub-seafloor methane hydrate sediments of Krishna Godavari basin, India, exhibited 99% 16S rRNA gene sequence identity and 90% gyrB gene sequence homology with the type strain of C. celerecrescens 02PIK1. Strain 152B was obtained from MACS Collection of Microorganisms, Agharkar Research Institute, Pune. The whole genome of C. celerecrescens 152B was sequenced to understand the evolutionary lineage of the organism; to gain an insight into the genetic makeup of this organism and to improve biotechnological applications through the production of valuable metabolites and biomolecules. The genome of C. celerecrescens 152B was sequenced using the Ion Torrent PGM sequencer (200-bp library) applying the 316™ sequencing chip according to the manufacturer's instructions (Life Technologies, ⁎ Corresponding author. Tel.: +91 20 25653680; fax: +91 20 25651542. E-mail addresses: [email protected], [email protected] (P.K. Dhakephalkar).
USA). De novo assembly was performed using version 4.0.5 of MIRA Assembler (Chevreux et al., 1999) and SeqMan NGen DNASTAR application (version 11.2.1). The assembly was uploaded for annotation to the Rapid Annotation using Subsystem Technology (RAST) server (Aziz et al., 2008), KEGG database (Kanehisa et al., 2004) and National Centre for Biotechnology Information Prokaryotic Genomes Annotation Pipeline Version 2.6 (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/ static/pipeline.html). The analysis generated 92 contigs featuring a G + C content of 43.5%, an N50 value of 208,566 bp, an N90 value of 35,416 bp and a maximum contig size of 416,014 bp (Table 1). Eight putative gene clusters involved in secondary metabolite production were disclosed using antiSMASH 2.0 (Blin et al., 2013). All the assembled data was deposited in the NCBI genome sequence database. According to the RAST genome analysis, the closest neighbor of 152B was Clostridium saccharolyticum WM1 (Score 521, Genome ID 610130.3). The draft genome of 152B is 5,050,495 bp in size having 83.53 fold genome coverage with 4286 genes annotated using the NCBI prokaryotic genomes annotation pipeline (PGAAP) (http://www.ncbi.nlm.nih.gov/genomes/static/pipeline. html), with 27 rRNAs (5S, 16S and 23S subunits) and 72 tRNAs. The genome analysis of 152B revealed the presence of genes encoding resistance to heavy metal ions such as Co2+, Zn2 +, AsO23 −, Cd2 + as well as antibiotics such as fluoroquinolones. Ability of C. celerecrescens to produce hydrogen, an environmentally safe energy source (Calusinska et al., 2010) could be explained on the basis of presence of genes encoding Fe only hydrogenase, Ni–Fe hydrogenases and energy conserving hydrogenases (ferrodoxin). Presence of genes responsible for the production of organic acids including lactate, formate, succinate, and acetate underscored promising biotechnological potential of C. celerecrescens 152B. These pathways/key genes could be modified to qualitatively as well as quantitatively improve the yield.
http://dx.doi.org/10.1016/j.margen.2015.01.008 1874-7787/© 2015 Published by Elsevier B.V.
Please cite this article as: Honkalas, V.S., et al., Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.01.008
2
V.S. Honkalas et al. / Marine Genomics xxx (2015) xxx–xxx
Acknowledgments
Table 1 Genome features of Clostridium celerecrescens 152B. Attributes
Values
Genome size Total no. of contigs G + C content (%) Total no. of subsystems Total no. of coding sequences Genes with predicted functions Plasmids tRNAs rRNAs
5,050,495 bp 92 43.5 362 4286 4036 0 72 27
We found key genes responsible for the production of several enzymes such as α-amylase, β-glucosidase, chitinase, and β-galactosidase. Presence of extracellular polysaccharide genes, osmotic and oxidative stress genes, and heat shock and cold shock genes could explain the ability of this organism to thrive under extreme environmental conditions. Availability of the C. celerecrescens genome sequence has provided us with an opportunity for further biotechnological exploitation of its genome features especially regarding the production of secondary metabolites as well as for environmental bioremediation and production of industrially valuable enzymes. Genome sequence analysis will also help to further understand the evolutionary relationship between Clostridium strains of marine as well as terrestrial origin. 2. Nucleotide sequence accession number This whole-genome shotgun project has been deposited at DDBJ/ EMBL/GenBank under the accession number JPME00000000. The first version (JPME00000000.1) is described in this paper. C. celerecrescens 152B is available in MACS Collection of Microorganisms (Registration No. WDCM 561) under the name MCM B-936, Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India.
Methane hydrate samples were provided by National Gas Hydrate Repository, Oil and Natural Gas Corporation (ONGC), Panvel, Mumbai, India. CSIR is gratefully acknowledged for providing fellowship to Preeti Arora. 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. Blanchard, J., Leschine, S., Petit, E., Fabel, J., 2009. Generic methods and compositions for improving the production of fuels in microorganisms. https://www.google.com/ patents/US20090286294. 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. Calusinska, M., Happe, T., Joris, B., Wilmotte, A., 2010. The surprising diversity of clostridial hydrogenases: a comparative genomic perspective. Microbiology 156, 1575–1588. Chamkha, M., Garcia, J.L., Labat, M., 2001. Metabolism of cinnamic acids by some Clostridiales and emendation of the descriptions of Clostridium aerotolerans, Clostridium celerecrescens and Clostridium xylanolyticum. Int. J. Syst. Evol. Microbiol. 51, 2105–2111. Chevreux, B., Wetter, T., Suhai, S., 1999. Genome sequence assembly using trace signals and additional sequence information. Computer Science and Biology: Proceedings of the German Conference on Bioinformatics (GCB) 99, pp. 45–56. Garcia-Balboa, C., Bedoya, I.C., Gonzalez, F., Blazquez, M.L., Munoz, J.A., Ballester, A., 2010. Bio-reduction of Fe (III) ores using three pure strains of Aeromonas hydrophila, Serratia fonticola and Clostridium celerecrescens and a natural consortium. Bioresour. Technol. 101, 7864–7871. Kanehisa, M., Goto, S., Kawashima, S., Okuno, Y., Hattori, M., 2004. The KEGG resource for deciphering the genome. Nucleic Acids Res. 32, 277–280. Palop, M.L., Valles, S., Pinaga, F., Flors, A., 1989. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int. J. Syst. Evol. Microbiol. 39, 68–71. Ren, Z., Ward, T.E., Logan, B.E., Regan, J.M., 2007. Characterization of the cellulolytic and hydrogen‐producing activities of six mesophilic Clostridium species. J. Appl. Microbiol. 103, 2258–2266. Zaybak, Z., Pisciotta, J.M., Tokash, J.C., Logan, B.E., 2013. Enhanced start-up of anaerobic facultatively autotrophic biocathodes in bioelectrochemical systems. J. Biotechnol. 168, 478–485.
Please cite this article as: Honkalas, V.S., et al., Draft genome sequence of Clostridium celerecrescens 152B isolated from sub-seafloor methane hydrate deposits, Mar. Genomics (2015), http://dx.doi.org/10.1016/j.margen.2015.01.008
