- Email: [email protected]
Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598
Journal of Biotechnology 244 (2017) 1–3
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598 Karel Sedlar a,∗ , Jan Kolek b , Ivo Provaznik a , Petra Patakova b a b
Department of Biomedical Engineering, Brno University of Technology, Technicka 12, 616 00 Brno, Czechia Department of Biotechnology, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague, Czechia
a r t i c l e
i n f o
Article history: Received 7 October 2016 Received in revised form 9 January 2017 Accepted 15 January 2017 Available online 19 January 2017 Keywords: Clostridium pasteurianum Clostridium beijerinckii Reclassification Phylogenomic analysis
a b s t r a c t The complete genome sequence of non-type strain Clostridium pasteurianum NRRL B-598 was introduced last year; it is an oxygen tolerant, spore-forming, mesophilic heterofermentative bacterium with high hydrogen production and acetone-butanol fermentation ability. The basic genome statistics have shown its similarity to C. beijerinckii rather than the C. pasteurianum species. Here, we present a comparative analysis of the strain with several other complete clostridial genome sequences. Besides a 16S rRNA gene sequence comparison, digital DNA–DNA hybridization (dDDH) and phylogenomic analysis confirmed an inaccuracy of the taxonomic status of strain Clostridium pasteurianum NRRL B-598. Therefore, we suggest its reclassification to be Clostridium beijerinckii NRRL B-598. This is a specific strain and is not identical to other C. beijerinckii strains. This misclassification explains its unexpected behavior, different from other C. pasteurianum strains; it also permits better understanding of the bacterium for a future genetic manipulation that might increase its biofuel production potential. © 2017 Elsevier B.V. All rights reserved.
Clostridia forms a large and diverse group of typically rod-shape, spore-forming anaerobes. The present study, as well as previous molecular studies, suggests that the genus Clostridium represents a polyphyletic group with uncertain phylogenetic affinities. Therefore, misclassification of its representatives, including type strains, is not unusual (Rainey and Lawson, 2016; Moon et al., 2008). Although taxonomic dissection for Clostridia based on the 16S rRNA gene sequence was proposed, it has also shown that the genus is not phylogenetically coherent and taxonomic placing of newly sequenced genomes can be questionable (Collins et al., 1994; Stackebrandt et al., 1999). For distantly related prokaryotes, this technique can predict genome-wide levels of similarity very well; but for closely related ones, other marker genes are needed (Lan et al., 2016). In particular, for strains with ambiguous properties, genome-wide studies like DNA–DNA hybridization are needed for the definitive assignment (Janda and Abbott, 2007). The strain C. pasteurianum NRRL B-598 is a spore-forming, mesophilic heterofermentative bacterium with acetone-butanol fermentation ability (Lipovsky et al., 2016). It is available from the Agricultural Research Service Culture Collection (NRRL). The bacterium is unable to utilize glycerol, but it ferments a wide range
∗ Corresponding author. E-mail addresses: [email protected] (K. Sedlar), [email protected] (J. Kolek), [email protected] (I. Provaznik), [email protected] (P. Patakova). http://dx.doi.org/10.1016/j.jbiotec.2017.01.003 0168-1656/© 2017 Elsevier B.V. All rights reserved.
of substrates containing glucose, xylose, arabinose, mannose, saccharose, lactose, cellobiose or starch. Furthermore, it can liquefy gelatin, decompose casein, produce polysaccharide capsules when grown on starch, and form stock polysaccharide granulose prior to the sporulation. The inability to grow on glycerol supports the misclassification of the strain. This is because this ability is generally considered to be one of the main distinguishing phenotypic traits for C. pasteurianum strains (Jensen et al., 2012). The strain is also naturally chloramphenicol/thiamphenicol resistant which has never been observed for the C. pasteurianum species, but it sometimes occurs at C. beijerinckii (Kolek et al., 2016a). First analyses of the genome showed a similarity of the genes involved in solvetogenesis to C. beijerinckii (Sedlar et al., 2014). However, a previous draft genome assembly did not contain any 16S rRNA gene sequence (Kolek et al., 2014); its correct taxonomic identification was therefore impossible. Last year, we presented its complete genome (Sedlar et al., 2015). This is available at DDBJ/EMBL/GenBank under accession No. CP011966. The complete genome sequence allowed us to perform a 16S rRNA gene sequence analysis, in addition to genome-wide analyses that proved misclassification of the strain. Therefore, we suggested its reclassification to be C. beijerinckii NRRL B-598. Analysis of the 16S rRNA gene sequences showed that the strain C. pasteurianum (beijerinckii) NRRL B-598 shares only 92% similarity with the type strain C. pasteurianum ATCC 6013 (Rotta et al.,
2
K. Sedlar et al. / Journal of Biotechnology 244 (2017) 1–3
Fig. 1. Phylogenetic position of the strain C. pasteurianum (beijerinckii) NRRL B-598 based on genome-wide sequence tree. Branches determining the position of the strain are supported with high bootstrap values. The tree was drawn in scale and the scale bar represents the estimated number of amino acid changes per site for a unit of branch length utilizing CAT model.
Table 1 dDDH values among C. pasteurianum and C. beijerinckii strains. Values DDH >70% indicates that the strains belongs to the same species, values DDH >79% indicates the same subspecies.
C. pasteurianum NRRL B-598 C. pasteurianum ATCC 6013 (=DSM525) C. pasteurianum BC1 C. beijerinckii NCIMB 8052 C. beijerinckii ATCC 35702 C. beijerinckii NCIMB 14988
C. pasteurianum NRRL B-598
C. pasteurianum ATCC 6013 (=DSM525)
C. pasteurianum BC1
C. beijerinckii NCIMB 8052
C. beijerinckii ATCC 35702
C. beijerinckii NCIMB 14988
–
24.90
26.70
78.40
78.40
75.40
–
28.00
25.30
25.30
23.30
–
26.20
26.20
24.40
–
100
74.50
–
74.50
2015), while similarity to the well described strain C. beijerinckii NCIMB 8052 is as high as 99% (sequence of the type strain is not available). Beside the fact that it has a 99% similarity to other C. beijerinckii strains (e.g. ATCC 35702 or NCIMB 14988), and therefore nothing about strain identity can be revealed, this high similarity (98–99%) is also shared with other clostridial species. Examples include: C. saccharoperbutylacetonicum N1-4(HMT), C. butyricum subsp. convexa JCM 7840, C. saccharobutylicum DSM 13864, and others. Therefore, for reaching maximum accuracy, we decided to infer taxonomy of most related strains on a genome-wide scale using complete genomes rather than using only 16S rRNA gene sequences. The analysis was built by a PhyloPhlAn 0.99 (Segata et al., 2013) comparing >400 selected protein sequences conserved across a bacterial domain. The genes were identified using an internal PhyloPhlAn database by translated mapping with USEARCH 8.1 (Edgar, 2010). The final tree was reconstructed using FastTree 2.1 (Price et al., 2010) from protein subsequences of the genes concatenating their most informative amino-acid positions, each aligned using Muscle 3.8 (Edgar, 2004). The topology was computed using neighbor-joining algorithm with utilization of Jukes-Cantor evolution model. Moreover, CAT model and gamma correction were used to optimize and rescale the tree. The resulting tree, visualized using MEGA 6.06 (Tamura et al., 2013), is shown in Fig 1.
–
The results of a PhyloPhlAn analysis show that C. pasteurianum NRRL B-598 is closely related to C. beijerinckii strains. To determine whether the strain is identical to another C. beijerinckii strain, we performed another genome-wide analysis utilizing dDDH (digital DNA–DNA hybridization) (Auch et al., 2010). This technique replaces the wet-lab DDH by in silico comparison using complete genome sequences. The dDDH was computed using GGDC (Genome-to-Genome Distance Calculator) (Meier-Kolthoff et al., 2013). The results, comparing the studied strain with other C. pasteurianum and C. beijerinckii strains, are summarized in Table 1. The values indicate that C. pasteurianum NRRL B-598 belongs to the C. beijerinckii species. This is because similarities to other C. beijerinckii species are above the 70% cutoff value for species delineation, while similarities to C. pasteurianum are far below this value. The analysis also suggests that strains C. beijerinckii NCIMB 8052 and C. beijerinckii ATCC 35702 are identical, while C. pasteurianum NRRL B-598 represents a standalone C. beijerinckii strain, because none of its similarities to other C. beijerinckii strains reached the cutoff value of 79%. Although C. beijerinckii and C. pasteurianum genomes have similar GC content, C. beijerinckii genomes are larger with a higher number of genes, as shown in Table 2. Another substantial difference between C. beijerinckii and C. pasteurianum genomes can be found in sol operon, which forms
K. Sedlar et al. / Journal of Biotechnology 244 (2017) 1–3
3
Table 2 Basic genome statistics for the three selected strains.
Length (bp) GC content (%) Total number of genes Protein conding genes (CDS)
C. pasteurianum NRRL B-598
C. beijerinckii NCIMB 8052
C. pasteurianum ATCC 6013
6,186,879 29.8 5365 5002
6,000,632 29.9 5231 5023
4,352,101 29.9 3980 3791
an important part of the pathway involved in solventogenesis. In C. pasteurianum, sol operon consists of the adhE gene (alcohol/acetaldehyde dehydrogenase), ctfA (CoA transferase subunit A), ctfB (CoA transferase subunit B), and adc (acetoacetate decarboxylase). Moreover, the adc gene is carried by the opposite strand from the other genes in operon. Sol operon in C. beijerinckii strains and C. pasteurianum NRRL B-598 genomes lack a adhE gene and contain a ald (aldehyde dehydrogenase) gene instead. Moreover, all genes in operon are carried by the same strand. Besides mentioned differences, the strain NRRL B-598 also exhibits some unique deviations from C. beijerinckii NCIMB 8052 (and other clostridia). For example, a specific DNA methylation pattern and restriction system requires Dam and Dcm methylation free DNA molecules for its transformation (Kolek et al., 2016a). Furthermore, both strains exhibit slightly different behavior in solvent production, sporulation and other cultivation characteristics (Kolek et al., 2016b). The analyses demonstrate undisputable evidence that C. pasteurianum NRRL B-598 is misclassified. Therefore, we suggest its reclassification to be C. beijerinckii NRRL B-598. This correction helps to explain why development of the novel transformation method – totally different from the method for C. pasteurianum strains – was needed. It also helps us to better understand the behavior of the strain and allows us to properly plan for its future genetic manipulation, in order to increase its biofuel production. Acknowledgements Computational resources were partially provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme “Projects of Large Research, Development, and Innovations Infrastructures”. The study was also partly financed by specific university research (MSMT No 21/2012 and MSMT No 20-SVV/2016). References Auch, A.F., von Jan, M., Klenk, H.-P., Göker, M., 2010. Digital DNA–DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2, 117–134, http://dx.doi.org/10.4056/sigs.531120. Collins, M., Lawson, P., Willems, A., Cordoba, J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H., Farrow, J., 1994. The phylogeny of the genus clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Evol. Microbiol. 44, 812–826, http://dx.doi.org/10.1099/00207713-44-4-812. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797, http://dx.doi.org/10.1093/ nar/gkh340. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461, http://dx.doi.org/10.1093/bioinformatics/ btq461. Janda, J., Abbott, S., 2007. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J. Clin. Microbiol. 45, 2761–2764, http://dx.doi.org/10.1128/JCM.01228-07.
Jensen, T.Ø., Kvist, T., Mikkelsen, M.J., Christensen, P.V., Westermann, P., 2012. Fermentation of crude glycerol from biodiesel production by Clostridium pasteurianum. J. Ind. Microbiol. Biotechnol. 39, 709–717, http://dx.doi.org/10. 1007/s10295-011-1077-6. Kolek, J., Sedlar, K., Provaznik, I., Patakova, P., 2014. Draft genome sequence of clostridium pasteurianum NRRL B-598, a potential butanol or hydrogen producer. Genome Announc. 2, e00192–e00214, http://dx.doi.org/10.1128/ genomeA.00192-14. Kolek, J., Branska, B., Drahokoupil, M., Patakova, P., Melzoch, K., 2016a. Evaluation of viability, metabolic activity and spore quantity in clostridial cultures during ABE fermentation. FEMS Microbiol. Lett. 6, fnw031, http://dx.doi.org/10.1093/ femsle/fnw031. Kolek, J., Sedlar, K., Provaznik, I., Patakova, P., 2016b. Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation. Biotechnol. Biofuels 9, 1–14, http://dx.doi.org/10.1186/s13068016-0436-y. Lan, Y., Rosen, G., Hershberg, R., 2016. Marker genes that are less conserved in their sequences are useful for predicting genome-wide similarity levels between closely related prokaryotic strains. Microbiome 4, 1–35, http://dx.doi.org/10. 1186/s40168-016-0162-5. Lipovsky, J., Patakova, P., Paulova, L., Pokorny, T., Rychtera, M., Melzoch, K., 2016. Butanol production by Clostridium pasteurianum NRRL B-598 in continuous culture compared to batch and fed-batch systems. Fuel Process. Technol. 144, 139–144, http://dx.doi.org/10.1016/j.fuproc.2015.12.020. Meier-Kolthoff, J.P., Auch, A.F., Klenk, H.-P., Göker, M., 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinf. 14, 1–14, http://dx.doi.org/10.1186/14712105-14-60. Moon, C., Pacheco, D., Kelly, W., Leahy, S., Li, D., Kopecny, J., Attwood, G., 2008. Reclassification of Clostridium proteoclasticum as Butyrivibrio proteoclasticus comb. nov., a butyrate-producing ruminal bacterium. Int. J. Syst. Evol. Microbiol. 58, 2041–2045, http://dx.doi.org/10.1099/ijs.0.65845-0. Price, M.N., Dehal, P.S., Arkin, A.P., 2010. FastTree 2 – Approximately maximum-likelihood trees for large alignments. PLoS One 5, e9490, http://dx. doi.org/10.1371/journal.pone.0009490. Rainey, F., Lawson, P., 2016. Proposal to restrict the genus Clostridium Prazmowski to Clostridium butyricum and related species. Int. J. Syst. Evol. Microbiol. 60, 1009–1016, http://dx.doi.org/10.1099/ijsem.0.000824. Rotta, C., Poehlein, A., Schwarz, K., McClure, P., Daniel, R., Minton, N., 2015. Closed genome sequence of Clostridium pasteurianum ATCC 6013. Genome Announc. 3, e01596–e01614, http://dx.doi.org/10.1128/genomeA.01596-14. Sedlar, K., Skutkova, H., Kolek, J., Patakova, P., Provaznik, I., 2014. Identification and characterization of sol operon in clostridium pasteurianum NRRL B-598 genome. In: Proceedings of the 2nd International Conference on Chemical Technology. Czech Society of Industrial Chemistry, Mikulov, pp. 435–439, http://dx.doi.org/10.13140/2.1.3688.6402. Sedlar, K., Kolek, J., Skutkova, H., Branska, B., Provaznik, I., Patakova, P., 2015. Complete genome sequence of Clostridium pasteurianum NRRL B-598, a non-type strain producing butanol. J. Biotechnol. 214, 113–114, http://dx.doi. org/10.1016/j.jbiotec.2015.09.022. Segata, N., Börnigen, D., Morgan, X.C., Huttenhower, C., 2013. PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nat. Commun. 4, 2304, http://dx.doi.org/10.1038/ncomms3304. Stackebrandt, E., Kramer, I., Swiderski, J., Hippe, H., 1999. Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol. Med. Microbiol. 24, 253–258, http://dx.doi.org/10.1111/j.1574-695X.1999.tb01291. x. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6. 0. Mol. Biol. Evol. 30, 2725–2729, http://dx.doi.org/10.1093/molbev/mst197.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598 Karel Sedlar a,∗ , Jan Kolek b , Ivo Provaznik a , Petra Patakova b a b
Department of Biomedical Engineering, Brno University of Technology, Technicka 12, 616 00 Brno, Czechia Department of Biotechnology, University of Chemistry and Technology Prague, Technicka 5, 166 28 Prague, Czechia
a r t i c l e
i n f o
Article history: Received 7 October 2016 Received in revised form 9 January 2017 Accepted 15 January 2017 Available online 19 January 2017 Keywords: Clostridium pasteurianum Clostridium beijerinckii Reclassification Phylogenomic analysis
a b s t r a c t The complete genome sequence of non-type strain Clostridium pasteurianum NRRL B-598 was introduced last year; it is an oxygen tolerant, spore-forming, mesophilic heterofermentative bacterium with high hydrogen production and acetone-butanol fermentation ability. The basic genome statistics have shown its similarity to C. beijerinckii rather than the C. pasteurianum species. Here, we present a comparative analysis of the strain with several other complete clostridial genome sequences. Besides a 16S rRNA gene sequence comparison, digital DNA–DNA hybridization (dDDH) and phylogenomic analysis confirmed an inaccuracy of the taxonomic status of strain Clostridium pasteurianum NRRL B-598. Therefore, we suggest its reclassification to be Clostridium beijerinckii NRRL B-598. This is a specific strain and is not identical to other C. beijerinckii strains. This misclassification explains its unexpected behavior, different from other C. pasteurianum strains; it also permits better understanding of the bacterium for a future genetic manipulation that might increase its biofuel production potential. © 2017 Elsevier B.V. All rights reserved.
Clostridia forms a large and diverse group of typically rod-shape, spore-forming anaerobes. The present study, as well as previous molecular studies, suggests that the genus Clostridium represents a polyphyletic group with uncertain phylogenetic affinities. Therefore, misclassification of its representatives, including type strains, is not unusual (Rainey and Lawson, 2016; Moon et al., 2008). Although taxonomic dissection for Clostridia based on the 16S rRNA gene sequence was proposed, it has also shown that the genus is not phylogenetically coherent and taxonomic placing of newly sequenced genomes can be questionable (Collins et al., 1994; Stackebrandt et al., 1999). For distantly related prokaryotes, this technique can predict genome-wide levels of similarity very well; but for closely related ones, other marker genes are needed (Lan et al., 2016). In particular, for strains with ambiguous properties, genome-wide studies like DNA–DNA hybridization are needed for the definitive assignment (Janda and Abbott, 2007). The strain C. pasteurianum NRRL B-598 is a spore-forming, mesophilic heterofermentative bacterium with acetone-butanol fermentation ability (Lipovsky et al., 2016). It is available from the Agricultural Research Service Culture Collection (NRRL). The bacterium is unable to utilize glycerol, but it ferments a wide range
∗ Corresponding author. E-mail addresses: [email protected] (K. Sedlar), [email protected] (J. Kolek), [email protected] (I. Provaznik), [email protected] (P. Patakova). http://dx.doi.org/10.1016/j.jbiotec.2017.01.003 0168-1656/© 2017 Elsevier B.V. All rights reserved.
of substrates containing glucose, xylose, arabinose, mannose, saccharose, lactose, cellobiose or starch. Furthermore, it can liquefy gelatin, decompose casein, produce polysaccharide capsules when grown on starch, and form stock polysaccharide granulose prior to the sporulation. The inability to grow on glycerol supports the misclassification of the strain. This is because this ability is generally considered to be one of the main distinguishing phenotypic traits for C. pasteurianum strains (Jensen et al., 2012). The strain is also naturally chloramphenicol/thiamphenicol resistant which has never been observed for the C. pasteurianum species, but it sometimes occurs at C. beijerinckii (Kolek et al., 2016a). First analyses of the genome showed a similarity of the genes involved in solvetogenesis to C. beijerinckii (Sedlar et al., 2014). However, a previous draft genome assembly did not contain any 16S rRNA gene sequence (Kolek et al., 2014); its correct taxonomic identification was therefore impossible. Last year, we presented its complete genome (Sedlar et al., 2015). This is available at DDBJ/EMBL/GenBank under accession No. CP011966. The complete genome sequence allowed us to perform a 16S rRNA gene sequence analysis, in addition to genome-wide analyses that proved misclassification of the strain. Therefore, we suggested its reclassification to be C. beijerinckii NRRL B-598. Analysis of the 16S rRNA gene sequences showed that the strain C. pasteurianum (beijerinckii) NRRL B-598 shares only 92% similarity with the type strain C. pasteurianum ATCC 6013 (Rotta et al.,
2
K. Sedlar et al. / Journal of Biotechnology 244 (2017) 1–3
Fig. 1. Phylogenetic position of the strain C. pasteurianum (beijerinckii) NRRL B-598 based on genome-wide sequence tree. Branches determining the position of the strain are supported with high bootstrap values. The tree was drawn in scale and the scale bar represents the estimated number of amino acid changes per site for a unit of branch length utilizing CAT model.
Table 1 dDDH values among C. pasteurianum and C. beijerinckii strains. Values DDH >70% indicates that the strains belongs to the same species, values DDH >79% indicates the same subspecies.
C. pasteurianum NRRL B-598 C. pasteurianum ATCC 6013 (=DSM525) C. pasteurianum BC1 C. beijerinckii NCIMB 8052 C. beijerinckii ATCC 35702 C. beijerinckii NCIMB 14988
C. pasteurianum NRRL B-598
C. pasteurianum ATCC 6013 (=DSM525)
C. pasteurianum BC1
C. beijerinckii NCIMB 8052
C. beijerinckii ATCC 35702
C. beijerinckii NCIMB 14988
–
24.90
26.70
78.40
78.40
75.40
–
28.00
25.30
25.30
23.30
–
26.20
26.20
24.40
–
100
74.50
–
74.50
2015), while similarity to the well described strain C. beijerinckii NCIMB 8052 is as high as 99% (sequence of the type strain is not available). Beside the fact that it has a 99% similarity to other C. beijerinckii strains (e.g. ATCC 35702 or NCIMB 14988), and therefore nothing about strain identity can be revealed, this high similarity (98–99%) is also shared with other clostridial species. Examples include: C. saccharoperbutylacetonicum N1-4(HMT), C. butyricum subsp. convexa JCM 7840, C. saccharobutylicum DSM 13864, and others. Therefore, for reaching maximum accuracy, we decided to infer taxonomy of most related strains on a genome-wide scale using complete genomes rather than using only 16S rRNA gene sequences. The analysis was built by a PhyloPhlAn 0.99 (Segata et al., 2013) comparing >400 selected protein sequences conserved across a bacterial domain. The genes were identified using an internal PhyloPhlAn database by translated mapping with USEARCH 8.1 (Edgar, 2010). The final tree was reconstructed using FastTree 2.1 (Price et al., 2010) from protein subsequences of the genes concatenating their most informative amino-acid positions, each aligned using Muscle 3.8 (Edgar, 2004). The topology was computed using neighbor-joining algorithm with utilization of Jukes-Cantor evolution model. Moreover, CAT model and gamma correction were used to optimize and rescale the tree. The resulting tree, visualized using MEGA 6.06 (Tamura et al., 2013), is shown in Fig 1.
–
The results of a PhyloPhlAn analysis show that C. pasteurianum NRRL B-598 is closely related to C. beijerinckii strains. To determine whether the strain is identical to another C. beijerinckii strain, we performed another genome-wide analysis utilizing dDDH (digital DNA–DNA hybridization) (Auch et al., 2010). This technique replaces the wet-lab DDH by in silico comparison using complete genome sequences. The dDDH was computed using GGDC (Genome-to-Genome Distance Calculator) (Meier-Kolthoff et al., 2013). The results, comparing the studied strain with other C. pasteurianum and C. beijerinckii strains, are summarized in Table 1. The values indicate that C. pasteurianum NRRL B-598 belongs to the C. beijerinckii species. This is because similarities to other C. beijerinckii species are above the 70% cutoff value for species delineation, while similarities to C. pasteurianum are far below this value. The analysis also suggests that strains C. beijerinckii NCIMB 8052 and C. beijerinckii ATCC 35702 are identical, while C. pasteurianum NRRL B-598 represents a standalone C. beijerinckii strain, because none of its similarities to other C. beijerinckii strains reached the cutoff value of 79%. Although C. beijerinckii and C. pasteurianum genomes have similar GC content, C. beijerinckii genomes are larger with a higher number of genes, as shown in Table 2. Another substantial difference between C. beijerinckii and C. pasteurianum genomes can be found in sol operon, which forms
K. Sedlar et al. / Journal of Biotechnology 244 (2017) 1–3
3
Table 2 Basic genome statistics for the three selected strains.
Length (bp) GC content (%) Total number of genes Protein conding genes (CDS)
C. pasteurianum NRRL B-598
C. beijerinckii NCIMB 8052
C. pasteurianum ATCC 6013
6,186,879 29.8 5365 5002
6,000,632 29.9 5231 5023
4,352,101 29.9 3980 3791
an important part of the pathway involved in solventogenesis. In C. pasteurianum, sol operon consists of the adhE gene (alcohol/acetaldehyde dehydrogenase), ctfA (CoA transferase subunit A), ctfB (CoA transferase subunit B), and adc (acetoacetate decarboxylase). Moreover, the adc gene is carried by the opposite strand from the other genes in operon. Sol operon in C. beijerinckii strains and C. pasteurianum NRRL B-598 genomes lack a adhE gene and contain a ald (aldehyde dehydrogenase) gene instead. Moreover, all genes in operon are carried by the same strand. Besides mentioned differences, the strain NRRL B-598 also exhibits some unique deviations from C. beijerinckii NCIMB 8052 (and other clostridia). For example, a specific DNA methylation pattern and restriction system requires Dam and Dcm methylation free DNA molecules for its transformation (Kolek et al., 2016a). Furthermore, both strains exhibit slightly different behavior in solvent production, sporulation and other cultivation characteristics (Kolek et al., 2016b). The analyses demonstrate undisputable evidence that C. pasteurianum NRRL B-598 is misclassified. Therefore, we suggest its reclassification to be C. beijerinckii NRRL B-598. This correction helps to explain why development of the novel transformation method – totally different from the method for C. pasteurianum strains – was needed. It also helps us to better understand the behavior of the strain and allows us to properly plan for its future genetic manipulation, in order to increase its biofuel production. Acknowledgements Computational resources were partially provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme “Projects of Large Research, Development, and Innovations Infrastructures”. The study was also partly financed by specific university research (MSMT No 21/2012 and MSMT No 20-SVV/2016). References Auch, A.F., von Jan, M., Klenk, H.-P., Göker, M., 2010. Digital DNA–DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand. Genomic Sci. 2, 117–134, http://dx.doi.org/10.4056/sigs.531120. Collins, M., Lawson, P., Willems, A., Cordoba, J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H., Farrow, J., 1994. The phylogeny of the genus clostridium: proposal of five new genera and eleven new species combinations. Int. J. Syst. Evol. Microbiol. 44, 812–826, http://dx.doi.org/10.1099/00207713-44-4-812. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797, http://dx.doi.org/10.1093/ nar/gkh340. Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461, http://dx.doi.org/10.1093/bioinformatics/ btq461. Janda, J., Abbott, S., 2007. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J. Clin. Microbiol. 45, 2761–2764, http://dx.doi.org/10.1128/JCM.01228-07.
Jensen, T.Ø., Kvist, T., Mikkelsen, M.J., Christensen, P.V., Westermann, P., 2012. Fermentation of crude glycerol from biodiesel production by Clostridium pasteurianum. J. Ind. Microbiol. Biotechnol. 39, 709–717, http://dx.doi.org/10. 1007/s10295-011-1077-6. Kolek, J., Sedlar, K., Provaznik, I., Patakova, P., 2014. Draft genome sequence of clostridium pasteurianum NRRL B-598, a potential butanol or hydrogen producer. Genome Announc. 2, e00192–e00214, http://dx.doi.org/10.1128/ genomeA.00192-14. Kolek, J., Branska, B., Drahokoupil, M., Patakova, P., Melzoch, K., 2016a. Evaluation of viability, metabolic activity and spore quantity in clostridial cultures during ABE fermentation. FEMS Microbiol. Lett. 6, fnw031, http://dx.doi.org/10.1093/ femsle/fnw031. Kolek, J., Sedlar, K., Provaznik, I., Patakova, P., 2016b. Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation. Biotechnol. Biofuels 9, 1–14, http://dx.doi.org/10.1186/s13068016-0436-y. Lan, Y., Rosen, G., Hershberg, R., 2016. Marker genes that are less conserved in their sequences are useful for predicting genome-wide similarity levels between closely related prokaryotic strains. Microbiome 4, 1–35, http://dx.doi.org/10. 1186/s40168-016-0162-5. Lipovsky, J., Patakova, P., Paulova, L., Pokorny, T., Rychtera, M., Melzoch, K., 2016. Butanol production by Clostridium pasteurianum NRRL B-598 in continuous culture compared to batch and fed-batch systems. Fuel Process. Technol. 144, 139–144, http://dx.doi.org/10.1016/j.fuproc.2015.12.020. Meier-Kolthoff, J.P., Auch, A.F., Klenk, H.-P., Göker, M., 2013. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinf. 14, 1–14, http://dx.doi.org/10.1186/14712105-14-60. Moon, C., Pacheco, D., Kelly, W., Leahy, S., Li, D., Kopecny, J., Attwood, G., 2008. Reclassification of Clostridium proteoclasticum as Butyrivibrio proteoclasticus comb. nov., a butyrate-producing ruminal bacterium. Int. J. Syst. Evol. Microbiol. 58, 2041–2045, http://dx.doi.org/10.1099/ijs.0.65845-0. Price, M.N., Dehal, P.S., Arkin, A.P., 2010. FastTree 2 – Approximately maximum-likelihood trees for large alignments. PLoS One 5, e9490, http://dx. doi.org/10.1371/journal.pone.0009490. Rainey, F., Lawson, P., 2016. Proposal to restrict the genus Clostridium Prazmowski to Clostridium butyricum and related species. Int. J. Syst. Evol. Microbiol. 60, 1009–1016, http://dx.doi.org/10.1099/ijsem.0.000824. Rotta, C., Poehlein, A., Schwarz, K., McClure, P., Daniel, R., Minton, N., 2015. Closed genome sequence of Clostridium pasteurianum ATCC 6013. Genome Announc. 3, e01596–e01614, http://dx.doi.org/10.1128/genomeA.01596-14. Sedlar, K., Skutkova, H., Kolek, J., Patakova, P., Provaznik, I., 2014. Identification and characterization of sol operon in clostridium pasteurianum NRRL B-598 genome. In: Proceedings of the 2nd International Conference on Chemical Technology. Czech Society of Industrial Chemistry, Mikulov, pp. 435–439, http://dx.doi.org/10.13140/2.1.3688.6402. Sedlar, K., Kolek, J., Skutkova, H., Branska, B., Provaznik, I., Patakova, P., 2015. Complete genome sequence of Clostridium pasteurianum NRRL B-598, a non-type strain producing butanol. J. Biotechnol. 214, 113–114, http://dx.doi. org/10.1016/j.jbiotec.2015.09.022. Segata, N., Börnigen, D., Morgan, X.C., Huttenhower, C., 2013. PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nat. Commun. 4, 2304, http://dx.doi.org/10.1038/ncomms3304. Stackebrandt, E., Kramer, I., Swiderski, J., Hippe, H., 1999. Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol. Med. Microbiol. 24, 253–258, http://dx.doi.org/10.1111/j.1574-695X.1999.tb01291. x. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular evolutionary genetics analysis version 6. 0. Mol. Biol. Evol. 30, 2725–2729, http://dx.doi.org/10.1093/molbev/mst197.