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Draft Genome Sequence of a Novel Acidophilic Iron-Oxidizing Firmicutes Species, “Acidibacillus ferrooxidans” (SLC66T) Ivan Ñancucheo,a,b Renato Oliveira,a Hivana Dall’Agnol,a,c D. Barrie Johnson,d Barry Grail,d Roseanne Holanda,d Gisele Lopes Nunes,a Sara Cuadros-Orellana,e Guilherme Oliveiraa Vale Institute of Technology, Belém, Pará, Brazila; Facultad de Ingeniería y Tecnología, Universidad San Sebastián, Concepción, Chileb; Universidade Federal do Maranhão, São Luís, Maranhão, Brazilc; College of Natural Sciences, Bangor University, Bangor, United Kingdomd; René Rachou Research Center, Fiocruz, Belo Horizonte, Minas Gerais, Brazile
Here, we present the draft genome sequence of the type strain of “Acidibacillus ferrooxidans,” a mesophilic, heterotrophic, and acidophilic bacterium that was isolated from mine spoilage subjected to accelerated weathering in humidity cell tests carried out by the former U.S. Bureau of Mines in Salt Lake City, UT. Received 23 March 2016 Accepted 4 April 2016 Published 19 May 2016 Citation Ñancucheo I, Oliveira R, Dall’Agnol H, Johnson DB, Grail B, Holanda R, Nunes GL, Cuadros-Orellana S, Oliveira G. 2016. Draft genome sequence of a novel acidophilic iron-oxidizing Firmicutes species, “Acidibacillus ferrooxidans” (SLC66T). Genome Announc 4(3):00383-16. doi:10.1128/genomeA.00383-16. Copyright © 2016 Ñancucheo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Guilherme Oliveira, [email protected].
icroorganisms that have an optimum pH for growth of ⬍3 are classed as extreme acidophiles; many of these contribute to the biogeochemical cycling of iron and sulfur in these environments. Acidophilic bacteria, as a group, vary significantly in how they assimilate carbon. Many species are obligate autotrophic, others are obligate heterotrophic, and some (facultative autotrophs) fix inorganic carbon (CO2) even though they preferentially assimilate organic carbon (1). Some species of acidophilic prokaryotes, mostly notably iron-oxidizing Acidithiobacillus and Leptospirillum spp. (Proteobacteria), are responsible for the bioleaching of sulfide minerals. Known species of Gram-positive acidophilic bacteria are affiliated with two phyla, Firmicutes and Actinobacteria, and include species that are facultative autotrophs and obligate heterotrophs. Here, we report the draft genome sequence of strain SLC66T, the type strain of the novel species “Acidibacillus ferrooxidans” (2, 3). This acidophile had been isolated from mine waste regolithic material subjected to accelerated weathering in humidity cells at pH 2.9 and 25°C (4, 5). A. ferrooxidans is one of two proposed species of the novel genus “Acidibacillus”, the other being “A. sulfuroxidans.” Acidibacillus is the third genus of acidophilic Firmicutes to be described, the others being Alicyclobacillus and Sulfobacillus. A. ferrooxidans is classified as facultative chemolithoheterotroph and can obtain energy from both inorganic and organic electron donors, but it requires an organic carbon source, with complex organic materials (e.g., yeast extract) being the most effective carbon. A. ferrooxidans is mesophilic and acidophilic (growth optimal at 30°C and pH 2.9), in contrast to the moderate thermophile A. sulfuroxidans (growth optimal at 43°C and pH 1.8). A. ferrooxidans catalyzes the dissimilatory oxidation of ferrous iron (and therefore the oxidative dissolution of sulfidic minerals, such as pyrite), while A. sulfuroxidans oxidizes both reduced iron and sulfur. Both species can couple the oxidation of organic carbon with the reduction of ferric iron in the absence of oxygen. Sequencing of the genome of SLC66T resulted in 2,122,638 paired-end reads obtained using the Illumina MiSeq platform,
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May/June 2016 Volume 4 Issue 3 e00383-16
with 34.7⫻ coverage of the expected genome size. The reads were trimmed and filtered using the Fastx Toolkit (http://hannonlab .cshl.edu/fastx_toolkit/), with a Phred score of 20, resulting in a total of 1,996,872 reads. The assembly of the genome sequence was performed with the Roche Newbler assembler version 2.9, ABySS 1.9 (6), and IDBAUD 1.1.1 (7). The contigs were integrated using the MIX (8) pipeline, resulting in a total of 175 contigs. The draft genome of strain SCL66T comprises a single chromosome of 3.2 Mb, with an overall G⫹C content of 51.97%. PATRIC (9) was used for automated annotation and identification of rRNA and tRNA, along with RNAmmer (10) and tRNAscan-SE (11), respectively. The draft genome harbored 4 rRNA genes, 53 tRNA genes, and a total of 3,497 protein-coding genes, from which 1,830 are genes with known functions and 1,667 are genes coding for hypothetical proteins. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at GenBank under the accession no. LVKL00000000. The version described in this paper is version LVKL00000000.1. ACKNOWLEDGMENTS This work was funded by Vale. CNPq supported R.O. and G.L.N. (grant 459913/2014-0). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.
FUNDING INFORMATION This work, including the efforts of Ivan Ñancucheo, Renato Oliveira, Hivana Dall’Agnol, D. Barrie Johnson, Barry M. Grail, Roseanne Holanda, Gisele Lopes Nunes, Sara Cuadros-Orellana, and Guilherme Oliveira, was funded by Vale. This work, including the efforts of Renato Oliveira, Gisele Lopes Nunes, and Roseanne Holanda, was funded by MCTI | Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-459913/2014-0). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Genome Announcements
genomea.asm.org 1
Ñancucheo et al.
REFERENCES 1. Barrie Johnson D, Hallberg KB. 2009. Carbon, iron and sulfur metabolism in acidophilic micro-organisms. Adv Microb Physiol 54:201–255. http://dx.doi.org/10.1016/S0065-2911(08)00003-9. ˇ ancucheo I, DallⴕAgnol H, Grail 2. Holanda R, Hedrich S, Falagán C, N BM, Johnson DB. 2015. Characteristics of Acidibacillus spp.: a novel genus of acidophilic iron-oxidising Firmicutes. AMR 1130:36 –39. http:// dx.doi.org/10.4028/www.scientific.net/AMR.1130.36. 3. Dall’Agnol H, Ñancucheo I, Johnson B, Oliveira R, Leite L, Pylro V, Holanda R, Grail B, Carvalho N, Nunes GL, Tzotzos G, Fernandes GR, Dutra J, Orellana SC, Oliveira G. 2016. Draft genome sequence of “Acidibacillus ferrooxidans” ITV01, a novel acidophilic firmicute isolated from a chalcopyrite mine drainage site in Brazil. Genome Announc 4(2):e0174815. http://dx.doi.org/10.1128/genomeA.01748-15. 4. Johnson DB, Bacelar-Nicolau P, Okibe N, Yahya A, Hallberg KB. 2001. Role of pure and mixed cultures of Gram-positive eubacteria in mineral leaching, p. 461– 470. In Ciminelli VST, Garcia O, Jr (ed), Biohydrometallurgy: fundamentals, technology and sustainable development, part A (process metallurgy), 1st ed. Elsevier, Amsterdam, The Netherlands. ˇ ancucheo I, Oliveira G, Grail BM, Johnson 5. Holanda R, Hedrich S, N DB. 2016. Isolation and characterisation of mineral-oxidizing “Acidibacillus” spp. from mine sites and geothermal environments in different global locations. Res Microbiol, in press. 6. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I. 2009.
2 genomea.asm.org
7.
8.
9.
10.
11.
ABySS: a parallel assembler for short read sequence data. Genome Res 19:1117–1123. http://dx.doi.org/10.1101/gr.089532.108. Peng Y, Leung HC, Yiu SM, Chin FY. 2012. IDBA-UD: a de novo assembler for single cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28:1420 –1428. http://dx.doi.org/10.1093/ bioinformatics/bts174. Soueidan H, Maurier F, Groppi A, Sirand-Pugnet P, Tardy F, Citti C, Dupuy V, Nikolski M. 2013. Finishing bacterial genome assemblies with mix. BMC Bioinformatics 14(Suppl 15):S16. http://dx.doi.org/10.1186/ 1471-2105-14-S15-S16. Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, Gillespie JJ, Gough R, Hix D, Kenyon R, Machi D, Mao C, Nordberg EK, Olson R, Overbeek R, Pusch GD, Shukla M, Schulman J, Stevens RL, Sullivan DE, Vonstein V, Warren A, Will R, Wilson MJC, Yoo HS, Zhang C, Zhang Y, Sobral BW. 2014. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res 42(Database issue):D581–D591. http://dx.doi.org/10.1093/nar/ gkt1099. Lagesen K, Hallin P, Rødland EA, Staerfeldt H-H, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35:3100 –3108. http://dx.doi.org/10.1093/ nar/gkm160. Lowe TM, Eddy SR. 1997. TRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964. http://dx.doi.org/10.1093/nar/25.5.0955.
Genome Announcements
May/June 2016 Volume 4 Issue 3 e00383-16
Draft Genome Sequence of a Novel Acidophilic Iron-Oxidizing Firmicutes Species, “Acidibacillus ferrooxidans” (SLC66T) Ivan Ñancucheo,a,b Renato Oliveira,a Hivana Dall’Agnol,a,c D. Barrie Johnson,d Barry Grail,d Roseanne Holanda,d Gisele Lopes Nunes,a Sara Cuadros-Orellana,e Guilherme Oliveiraa Vale Institute of Technology, Belém, Pará, Brazila; Facultad de Ingeniería y Tecnología, Universidad San Sebastián, Concepción, Chileb; Universidade Federal do Maranhão, São Luís, Maranhão, Brazilc; College of Natural Sciences, Bangor University, Bangor, United Kingdomd; René Rachou Research Center, Fiocruz, Belo Horizonte, Minas Gerais, Brazile
Here, we present the draft genome sequence of the type strain of “Acidibacillus ferrooxidans,” a mesophilic, heterotrophic, and acidophilic bacterium that was isolated from mine spoilage subjected to accelerated weathering in humidity cell tests carried out by the former U.S. Bureau of Mines in Salt Lake City, UT. Received 23 March 2016 Accepted 4 April 2016 Published 19 May 2016 Citation Ñancucheo I, Oliveira R, Dall’Agnol H, Johnson DB, Grail B, Holanda R, Nunes GL, Cuadros-Orellana S, Oliveira G. 2016. Draft genome sequence of a novel acidophilic iron-oxidizing Firmicutes species, “Acidibacillus ferrooxidans” (SLC66T). Genome Announc 4(3):00383-16. doi:10.1128/genomeA.00383-16. Copyright © 2016 Ñancucheo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Guilherme Oliveira, [email protected].
icroorganisms that have an optimum pH for growth of ⬍3 are classed as extreme acidophiles; many of these contribute to the biogeochemical cycling of iron and sulfur in these environments. Acidophilic bacteria, as a group, vary significantly in how they assimilate carbon. Many species are obligate autotrophic, others are obligate heterotrophic, and some (facultative autotrophs) fix inorganic carbon (CO2) even though they preferentially assimilate organic carbon (1). Some species of acidophilic prokaryotes, mostly notably iron-oxidizing Acidithiobacillus and Leptospirillum spp. (Proteobacteria), are responsible for the bioleaching of sulfide minerals. Known species of Gram-positive acidophilic bacteria are affiliated with two phyla, Firmicutes and Actinobacteria, and include species that are facultative autotrophs and obligate heterotrophs. Here, we report the draft genome sequence of strain SLC66T, the type strain of the novel species “Acidibacillus ferrooxidans” (2, 3). This acidophile had been isolated from mine waste regolithic material subjected to accelerated weathering in humidity cells at pH 2.9 and 25°C (4, 5). A. ferrooxidans is one of two proposed species of the novel genus “Acidibacillus”, the other being “A. sulfuroxidans.” Acidibacillus is the third genus of acidophilic Firmicutes to be described, the others being Alicyclobacillus and Sulfobacillus. A. ferrooxidans is classified as facultative chemolithoheterotroph and can obtain energy from both inorganic and organic electron donors, but it requires an organic carbon source, with complex organic materials (e.g., yeast extract) being the most effective carbon. A. ferrooxidans is mesophilic and acidophilic (growth optimal at 30°C and pH 2.9), in contrast to the moderate thermophile A. sulfuroxidans (growth optimal at 43°C and pH 1.8). A. ferrooxidans catalyzes the dissimilatory oxidation of ferrous iron (and therefore the oxidative dissolution of sulfidic minerals, such as pyrite), while A. sulfuroxidans oxidizes both reduced iron and sulfur. Both species can couple the oxidation of organic carbon with the reduction of ferric iron in the absence of oxygen. Sequencing of the genome of SLC66T resulted in 2,122,638 paired-end reads obtained using the Illumina MiSeq platform,
M
May/June 2016 Volume 4 Issue 3 e00383-16
with 34.7⫻ coverage of the expected genome size. The reads were trimmed and filtered using the Fastx Toolkit (http://hannonlab .cshl.edu/fastx_toolkit/), with a Phred score of 20, resulting in a total of 1,996,872 reads. The assembly of the genome sequence was performed with the Roche Newbler assembler version 2.9, ABySS 1.9 (6), and IDBAUD 1.1.1 (7). The contigs were integrated using the MIX (8) pipeline, resulting in a total of 175 contigs. The draft genome of strain SCL66T comprises a single chromosome of 3.2 Mb, with an overall G⫹C content of 51.97%. PATRIC (9) was used for automated annotation and identification of rRNA and tRNA, along with RNAmmer (10) and tRNAscan-SE (11), respectively. The draft genome harbored 4 rRNA genes, 53 tRNA genes, and a total of 3,497 protein-coding genes, from which 1,830 are genes with known functions and 1,667 are genes coding for hypothetical proteins. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at GenBank under the accession no. LVKL00000000. The version described in this paper is version LVKL00000000.1. ACKNOWLEDGMENTS This work was funded by Vale. CNPq supported R.O. and G.L.N. (grant 459913/2014-0). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.
FUNDING INFORMATION This work, including the efforts of Ivan Ñancucheo, Renato Oliveira, Hivana Dall’Agnol, D. Barrie Johnson, Barry M. Grail, Roseanne Holanda, Gisele Lopes Nunes, Sara Cuadros-Orellana, and Guilherme Oliveira, was funded by Vale. This work, including the efforts of Renato Oliveira, Gisele Lopes Nunes, and Roseanne Holanda, was funded by MCTI | Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-459913/2014-0). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Genome Announcements
genomea.asm.org 1
Ñancucheo et al.
REFERENCES 1. Barrie Johnson D, Hallberg KB. 2009. Carbon, iron and sulfur metabolism in acidophilic micro-organisms. Adv Microb Physiol 54:201–255. http://dx.doi.org/10.1016/S0065-2911(08)00003-9. ˇ ancucheo I, DallⴕAgnol H, Grail 2. Holanda R, Hedrich S, Falagán C, N BM, Johnson DB. 2015. Characteristics of Acidibacillus spp.: a novel genus of acidophilic iron-oxidising Firmicutes. AMR 1130:36 –39. http:// dx.doi.org/10.4028/www.scientific.net/AMR.1130.36. 3. Dall’Agnol H, Ñancucheo I, Johnson B, Oliveira R, Leite L, Pylro V, Holanda R, Grail B, Carvalho N, Nunes GL, Tzotzos G, Fernandes GR, Dutra J, Orellana SC, Oliveira G. 2016. Draft genome sequence of “Acidibacillus ferrooxidans” ITV01, a novel acidophilic firmicute isolated from a chalcopyrite mine drainage site in Brazil. Genome Announc 4(2):e0174815. http://dx.doi.org/10.1128/genomeA.01748-15. 4. Johnson DB, Bacelar-Nicolau P, Okibe N, Yahya A, Hallberg KB. 2001. Role of pure and mixed cultures of Gram-positive eubacteria in mineral leaching, p. 461– 470. In Ciminelli VST, Garcia O, Jr (ed), Biohydrometallurgy: fundamentals, technology and sustainable development, part A (process metallurgy), 1st ed. Elsevier, Amsterdam, The Netherlands. ˇ ancucheo I, Oliveira G, Grail BM, Johnson 5. Holanda R, Hedrich S, N DB. 2016. Isolation and characterisation of mineral-oxidizing “Acidibacillus” spp. from mine sites and geothermal environments in different global locations. Res Microbiol, in press. 6. Simpson JT, Wong K, Jackman SD, Schein JE, Jones SJ, Birol I. 2009.
2 genomea.asm.org
7.
8.
9.
10.
11.
ABySS: a parallel assembler for short read sequence data. Genome Res 19:1117–1123. http://dx.doi.org/10.1101/gr.089532.108. Peng Y, Leung HC, Yiu SM, Chin FY. 2012. IDBA-UD: a de novo assembler for single cell and metagenomic sequencing data with highly uneven depth. Bioinformatics 28:1420 –1428. http://dx.doi.org/10.1093/ bioinformatics/bts174. Soueidan H, Maurier F, Groppi A, Sirand-Pugnet P, Tardy F, Citti C, Dupuy V, Nikolski M. 2013. Finishing bacterial genome assemblies with mix. BMC Bioinformatics 14(Suppl 15):S16. http://dx.doi.org/10.1186/ 1471-2105-14-S15-S16. Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, Gillespie JJ, Gough R, Hix D, Kenyon R, Machi D, Mao C, Nordberg EK, Olson R, Overbeek R, Pusch GD, Shukla M, Schulman J, Stevens RL, Sullivan DE, Vonstein V, Warren A, Will R, Wilson MJC, Yoo HS, Zhang C, Zhang Y, Sobral BW. 2014. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res 42(Database issue):D581–D591. http://dx.doi.org/10.1093/nar/ gkt1099. Lagesen K, Hallin P, Rødland EA, Staerfeldt H-H, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35:3100 –3108. http://dx.doi.org/10.1093/ nar/gkm160. Lowe TM, Eddy SR. 1997. TRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964. http://dx.doi.org/10.1093/nar/25.5.0955.
Genome Announcements
May/June 2016 Volume 4 Issue 3 e00383-16
