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Draft Genome Sequence of the Strict Anaerobe Clostridium neopropionicum X4 (DSM 3847T) Matthias H. Beck,a Anja Poehlein,b Frank R. Bengelsdorf,a Bettina Schiel-Bengelsdorf,a Rolf Daniel,b Peter Dürrea Institut für Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germanya; Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Georg-August University Göttingen, Göttingen, Germanyb M.H.B. and A.P. contributed equally to this work.
Here, we report the draft genome sequence of Clostridium neopropionicum X4 (DSM 3847T), a strictly anaerobic bacterium capable of fermenting ethanol and CO2 to propionate, acetate, and propanol. The genome consists of a single chromosome (3.19 Mb). Received 29 February 2016 Accepted 2 March 2016 Published 14 April 2016 Citation Beck MH, Poehlein A, Bengelsdorf FR, Schiel-Bengelsdorf B, Daniel R, Dürre P. 2016. Draft genome sequence of the strict anaerobe Clostridium neopropionicum X4 (DSM 3847T). Genome Announc 4(2):e00209-16. doi:10.1128/genomeA.00209-16. Copyright © 2016 Beck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Peter Dürre, [email protected].
C
lostridium neopropionicum X4 was first isolated in 1982 from a mesophilic industrial anaerobic digester treating vegetable cannery wastewaters (1). Later, it was further characterized as an obligately anaerobic endospore-forming bacterium, being the first representative of propionate fermentation from ethanol with simultaneous acetate production (1, 2). Tholozan et al. (2) reported the formation of [2-13C]propionate, [1-13C]acetate, [2-13C]propanol, and traces of [3-13C]butyrate as products of [1-13C]ethanol fermentation, suggesting the absence of a randomizing pathway (2). The respective reactions of ethanol conversion to these metabolic end products are all exergonic (2). Meanwhile, C. neopropionicum was suggested to be assigned to the genus Tyzzerella (3). The MasterPure complete DNA purification kit (Epicentre, Madison, WI, USA) was used to isolate chromosomal DNA. Illumina Nextera XT shotgun sequencing libraries were generated from the extracted DNA, and sequencing was performed with an Illumina MiSeq machine, as recommended by the manufacturer (Illumina, San Diego, CA, USA). Sequencing resulted in 2,669,816 300-bp paired-end reads. Reads were trimmed using Trimmomatic 0.35 (4) to remove sequences with quality scores ⬍20 (Illumina 1.9 encoding) and adaptor sequences. The remaining 2,530,048 reads were used for the de novo assembly performed with the SPAdes genome assembler software version 3.6.2 (5). The assembly resulted in 29 contigs (⬎500 bp) and an average coverage of 182-fold. The genome of C. neopropionicum probably consists of a single circular chromosome (3.19 Mb), with an overall G⫹C content of 40.17%. Automatic gene prediction was performed by using the software tool Prodigal (6). Genes coding for rRNA and tRNA were identified using RNAmmer (7) and tRNAscan (8), respectively. The Integrated Microbial Genomes-Expert Review (IMG-ER) system (9) was used for automatic annotation, which was subsequently manually curated by using the Swiss-Prot, TrEMBL, and InterPro databases (10). The genome harbored 6 rRNA genes, 58 tRNA genes, 2,090
March/April 2016 Volume 4 Issue 2 e00209-16
protein-coding genes with predicted functions, and 839 genes coding for hypothetical proteins. Genome analysis revealed the presence of several genes encoding alcohol dehydrogenases, including also an adhE gene (CLNEO_13930), which codes for a bifunctional aldehyde/alcohol dehydrogenase and a gene (CLNEO_02960) that putatively represents the NADP⫹dependent ethanol dehydrogenase gene postulated by Tholozan et al. (2). Genes encoding key enzymes of the acrylate pathway (propionate-coenzyme A [CoA] transferase [Pct], lactoyl-CoA dehydratase [Lcd], and acrylyl-CoA reductase [Acr]) were also identified and show high sequence similarity to the corresponding genes described for Clostridium propionicum. These genes are organized in two clusters (lcdCAB-pct and acrCBA), which showed an arrangement identical to that in C. propionicum (11). Whole-genome comparison based on computed average nucleotide identity (ANI) revealed a similarity of ⬎80% between C. neopropionicum and C. propionicum, which is clearly in contrast to earlier findings of only 18% DNA/DNA homology (2). However, our analysis and former 16S rRNA gene-based studies (12) suggest that C. neopropionicum and C. propionicum are highly related. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. LRVM00000000. The version described in this paper is version LRVM01000000. ACKNOWLEDGMENTS This work was supported by the ERA-IB5 program (project CO2CHEM, grant 031A566A). We thank Kathleen Gollnow and Frauke-Dorothee Meyer for technical support.
REFERENCES 1. Samain E, Albagnac G, Dubourguier HC, Touzel JP. 1982. Characterization of a new propionic acid bacterium that ferments ethanol and displays a growth factor-dependent association with a Gram-negative ho-
Genome Announcements
genomea.asm.org 1
Beck et al.
2.
3. 4. 5.
6.
7.
moacetogen. FEMS Microbiol Lett 15:69 –74. http://dx.doi.org/10.1111/ j.1574-6968.1982.tb00040.x. Tholozan JL, Touzel JP, Samain E, Grivet JP, Prensier G, Albagnac G. 1992. Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway. Arch Microbiol 157:249 –257. http://dx.doi.org/10.1007/BF00245158. Yutin N, Galperin MY. 2013. A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ Microbiol 15:2631–2641. http://dx.doi.org/10.1111/1462-2920.12173. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114 –2120. http:// dx.doi.org/10.1093/bioinformatics/btu170. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: A new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 19:455– 477. http://dx.doi.org/10.1089/ cmb.2012.0021. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. http://dx.doi.org/ 10.1186/1471-2105-11-119. Lagesen K, Hallin P, Rødland EA, Stærfeldt H-H, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal
2 genomea.asm.org
8.
9.
10.
11.
12.
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. Markowitz VM, Chen IA, Palaniappan K, Chu K, Szeto E, Pillay M, Ratner A, Huang J, Woyke T, Huntemann M, Anderson I, Billis K, Varghese N, Mavromatis K, Pati A, Ivanova NN, Kyrpides NC. 2014. IMG4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res 42:D560 –D567. http://dx.doi.org/10.1093/nar/ gkt963. Zdobnov EM, Apweiler R. 2001. InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17: 847– 848. http://dx.doi.org/10.1093/bioinformatics/17.9.847. Poehlein A, Schlien K, Chowdhury N, Gottschalk G, Buckel W, Daniel R. 2016. Complete genome sequence of the amino acid-fermenting Clostridium propionicum X2 (DSM 1682). Genome Announc 4(2):e0029416. http://dx.doi.org/10.1128/genomeA.00294-16. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, Cai J, Hippe H, Farrow JA. 1994. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44:812– 826. http://dx.doi.org/10.1099/00207713 -44-4-812.
Genome Announcements
March/April 2016 Volume 4 Issue 2 e00209-16
Draft Genome Sequence of the Strict Anaerobe Clostridium neopropionicum X4 (DSM 3847T) Matthias H. Beck,a Anja Poehlein,b Frank R. Bengelsdorf,a Bettina Schiel-Bengelsdorf,a Rolf Daniel,b Peter Dürrea Institut für Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germanya; Genomic and Applied Microbiology & Göttingen Genomics Laboratory, Georg-August University Göttingen, Göttingen, Germanyb M.H.B. and A.P. contributed equally to this work.
Here, we report the draft genome sequence of Clostridium neopropionicum X4 (DSM 3847T), a strictly anaerobic bacterium capable of fermenting ethanol and CO2 to propionate, acetate, and propanol. The genome consists of a single chromosome (3.19 Mb). Received 29 February 2016 Accepted 2 March 2016 Published 14 April 2016 Citation Beck MH, Poehlein A, Bengelsdorf FR, Schiel-Bengelsdorf B, Daniel R, Dürre P. 2016. Draft genome sequence of the strict anaerobe Clostridium neopropionicum X4 (DSM 3847T). Genome Announc 4(2):e00209-16. doi:10.1128/genomeA.00209-16. Copyright © 2016 Beck et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Peter Dürre, [email protected].
C
lostridium neopropionicum X4 was first isolated in 1982 from a mesophilic industrial anaerobic digester treating vegetable cannery wastewaters (1). Later, it was further characterized as an obligately anaerobic endospore-forming bacterium, being the first representative of propionate fermentation from ethanol with simultaneous acetate production (1, 2). Tholozan et al. (2) reported the formation of [2-13C]propionate, [1-13C]acetate, [2-13C]propanol, and traces of [3-13C]butyrate as products of [1-13C]ethanol fermentation, suggesting the absence of a randomizing pathway (2). The respective reactions of ethanol conversion to these metabolic end products are all exergonic (2). Meanwhile, C. neopropionicum was suggested to be assigned to the genus Tyzzerella (3). The MasterPure complete DNA purification kit (Epicentre, Madison, WI, USA) was used to isolate chromosomal DNA. Illumina Nextera XT shotgun sequencing libraries were generated from the extracted DNA, and sequencing was performed with an Illumina MiSeq machine, as recommended by the manufacturer (Illumina, San Diego, CA, USA). Sequencing resulted in 2,669,816 300-bp paired-end reads. Reads were trimmed using Trimmomatic 0.35 (4) to remove sequences with quality scores ⬍20 (Illumina 1.9 encoding) and adaptor sequences. The remaining 2,530,048 reads were used for the de novo assembly performed with the SPAdes genome assembler software version 3.6.2 (5). The assembly resulted in 29 contigs (⬎500 bp) and an average coverage of 182-fold. The genome of C. neopropionicum probably consists of a single circular chromosome (3.19 Mb), with an overall G⫹C content of 40.17%. Automatic gene prediction was performed by using the software tool Prodigal (6). Genes coding for rRNA and tRNA were identified using RNAmmer (7) and tRNAscan (8), respectively. The Integrated Microbial Genomes-Expert Review (IMG-ER) system (9) was used for automatic annotation, which was subsequently manually curated by using the Swiss-Prot, TrEMBL, and InterPro databases (10). The genome harbored 6 rRNA genes, 58 tRNA genes, 2,090
March/April 2016 Volume 4 Issue 2 e00209-16
protein-coding genes with predicted functions, and 839 genes coding for hypothetical proteins. Genome analysis revealed the presence of several genes encoding alcohol dehydrogenases, including also an adhE gene (CLNEO_13930), which codes for a bifunctional aldehyde/alcohol dehydrogenase and a gene (CLNEO_02960) that putatively represents the NADP⫹dependent ethanol dehydrogenase gene postulated by Tholozan et al. (2). Genes encoding key enzymes of the acrylate pathway (propionate-coenzyme A [CoA] transferase [Pct], lactoyl-CoA dehydratase [Lcd], and acrylyl-CoA reductase [Acr]) were also identified and show high sequence similarity to the corresponding genes described for Clostridium propionicum. These genes are organized in two clusters (lcdCAB-pct and acrCBA), which showed an arrangement identical to that in C. propionicum (11). Whole-genome comparison based on computed average nucleotide identity (ANI) revealed a similarity of ⬎80% between C. neopropionicum and C. propionicum, which is clearly in contrast to earlier findings of only 18% DNA/DNA homology (2). However, our analysis and former 16S rRNA gene-based studies (12) suggest that C. neopropionicum and C. propionicum are highly related. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. LRVM00000000. The version described in this paper is version LRVM01000000. ACKNOWLEDGMENTS This work was supported by the ERA-IB5 program (project CO2CHEM, grant 031A566A). We thank Kathleen Gollnow and Frauke-Dorothee Meyer for technical support.
REFERENCES 1. Samain E, Albagnac G, Dubourguier HC, Touzel JP. 1982. Characterization of a new propionic acid bacterium that ferments ethanol and displays a growth factor-dependent association with a Gram-negative ho-
Genome Announcements
genomea.asm.org 1
Beck et al.
2.
3. 4. 5.
6.
7.
moacetogen. FEMS Microbiol Lett 15:69 –74. http://dx.doi.org/10.1111/ j.1574-6968.1982.tb00040.x. Tholozan JL, Touzel JP, Samain E, Grivet JP, Prensier G, Albagnac G. 1992. Clostridium neopropionicum sp. nov., a strict anaerobic bacterium fermenting ethanol to propionate through acrylate pathway. Arch Microbiol 157:249 –257. http://dx.doi.org/10.1007/BF00245158. Yutin N, Galperin MY. 2013. A genomic update on clostridial phylogeny: Gram-negative spore formers and other misplaced clostridia. Environ Microbiol 15:2631–2641. http://dx.doi.org/10.1111/1462-2920.12173. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114 –2120. http:// dx.doi.org/10.1093/bioinformatics/btu170. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: A new genome assembly algorithm and its applications to singlecell sequencing. J Comput Biol 19:455– 477. http://dx.doi.org/10.1089/ cmb.2012.0021. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119. http://dx.doi.org/ 10.1186/1471-2105-11-119. Lagesen K, Hallin P, Rødland EA, Stærfeldt H-H, Rognes T, Ussery DW. 2007. RNAmmer: consistent and rapid annotation of ribosomal
2 genomea.asm.org
8.
9.
10.
11.
12.
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. Markowitz VM, Chen IA, Palaniappan K, Chu K, Szeto E, Pillay M, Ratner A, Huang J, Woyke T, Huntemann M, Anderson I, Billis K, Varghese N, Mavromatis K, Pati A, Ivanova NN, Kyrpides NC. 2014. IMG4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res 42:D560 –D567. http://dx.doi.org/10.1093/nar/ gkt963. Zdobnov EM, Apweiler R. 2001. InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17: 847– 848. http://dx.doi.org/10.1093/bioinformatics/17.9.847. Poehlein A, Schlien K, Chowdhury N, Gottschalk G, Buckel W, Daniel R. 2016. Complete genome sequence of the amino acid-fermenting Clostridium propionicum X2 (DSM 1682). Genome Announc 4(2):e0029416. http://dx.doi.org/10.1128/genomeA.00294-16. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, Cai J, Hippe H, Farrow JA. 1994. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44:812– 826. http://dx.doi.org/10.1099/00207713 -44-4-812.
Genome Announcements
March/April 2016 Volume 4 Issue 2 e00209-16
