BACKGROUND: One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. RESULTS: In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term "RNA binding" among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term "protein binding" which corresponded to activation of heat-shock proteins that were identified within this cluster. CONCLUSIONS: We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the corresponding phenotypic response. We identified genes whose regulation was affected by the addition of butanol to the cultivation medium and inferred related molecular functions that were significantly influenced. Additionally, using high-quality genome assembly and custom-made gene ontology annotation, we demonstrated that this settled terminology, widely used for the analysis of model organisms, could also be applied to non-model organisms and for research in the field of biofuels.

Department of Biomedical Engineering Brno University of Technology Technicka 12 616 00 Brno Czech Republic

Department of Biotechnology University of Chemistry and Technology Prague Technicka 5 166 28 Prague Czech Republic

Institut für Informatik Ludwig Maximilians Universität München Amalienstraße 17 80333 Munich Germany

Zobrazit více v PubMed

Kujawska A, Kujawski J, Bryjak M, Kujawski W. ABE fermentation products recovery methods—a review. Renew Sustain Energy Rev. 2015;48:648–661. doi: 10.1016/j.rser.2015.04.028. DOI

Patakova P, Kolek J, Sedlar K, Koscova P, Branska B, Kupkova K, et al. Comparative analysis of high butanol tolerance and production in clostridia. Biotechnol Adv. 2018;36:721–738. doi: 10.1016/j.biotechadv.2017.12.004. PubMed DOI

Green EM. Fermentative production of butanol-the industrial perspective. Curr Opin Biotechnol. 2011;22:337–343. doi: 10.1016/j.copbio.2011.02.004. PubMed DOI

Charubin K, Bennett RK, Fast AG, Papoutsakis ET. Engineering Clostridium organisms as microbial cell-factories: challenges & opportunities. Metab Eng. 2018;50:173–191. doi: 10.1016/j.ymben.2018.07.012. PubMed DOI

Joseph RC, Kim NM, Sandoval NR. Recent developments of the synthetic biology toolkit for Clostridium. Front Microbiol. 2018;9:154. doi: 10.3389/fmicb.2018.00154/full. PubMed DOI PMC

Sedlar K, Kolek J, Skutkova H, Branska B, Provaznik I, Patakova P. Complete genome sequence of Clostridium pasteurianum NRRL B-598, a non-type strain producing butanol. J Biotechnol. 2015;214:113–114. doi: 10.1016/j.jbiotec.2015.09.022. PubMed DOI

Sedlar K, Kolek J, Provaznik I, Patakova P. Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598. J Biotechnol. 2017;244:1–3. doi: 10.1016/j.jbiotec.2017.01.003. PubMed DOI

Kolek J, Sedlar K, Provaznik I, Patakova P. Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation. Biotechnol Biofuels. 2016;9:14. doi: 10.1186/s13068-016-0436-y. PubMed DOI PMC

Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, et al. Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol. 2001;183:4823–4838. doi: 10.1128/JB.183.16.4823-4838.2001. PubMed DOI PMC

Wang Y, Li X, Mao Y, Blaschek HP. Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq. BMC Genomics. 2011;12:479. doi: 10.1186/1471-2164-12-479. PubMed DOI PMC

Poehlein A, Grosse-Honebrink A, Zhang Y, Minton NP, Daniel R. Complete genome sequence of the nitrogen-fixing and solvent-producing Clostridium pasteurianum DSM 525. Genome Announc. 2015;3:e01591-14. doi: 10.1128/genomeA.01591-14. PubMed DOI PMC

Sandoval-Espinola WJ, Makwana ST, Chinn MS, Thon MR, Andrea Azcárate-Peril M, Bruno-Bárcena JM. Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052. Microbiology. 2013;159:2558–2570. doi: 10.1099/mic.0.069534-0. PubMed DOI PMC

Poehlein A, Solano JDM, Flitsch SK, Krabben P, Winzer K, Reid SJ, et al. Microbial solvent formation revisited by comparative genome analysis. Biotechnol Biofuels. 2017;10:58. doi: 10.1186/s13068-017-0742-z. PubMed DOI PMC

Venkataramanan KP, Jones SW, McCormick KP, Kunjeti SG, Ralston MT, Meyers BC, et al. The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum. BMC Genomics. 2013;14:849. doi: 10.1186/1471-2164-14-849. PubMed DOI PMC

Wang Y, Li X, Blaschek HP. Effects of supplementary butyrate on butanol production and the metabolic switch in Clostridium beijerinckii NCIMB 8052: genome-wide transcriptional analysis with RNA-Seq. Biotechnol Biofuels. 2013;6:138. doi: 10.1186/1754-6834-6-138. PubMed DOI PMC

Wang Y, Li X, Mao Y, Blaschek HP. Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. BMC Genomics. 2012;13:102. doi: 10.1186/1471-2164-13-102. PubMed DOI PMC

Patakova P, Branska B, Sedlar K, Vasylkivska M, Jureckova K, Kolek J, et al. Acidogenesis, solventogenesis, metabolic stress response and life cycle changes in Clostridium beijerinckii NRRL B-598 at the transcriptomic level. Sci Rep. 2019;9:1371. doi: 10.1038/s41598-018-37679-0. PubMed DOI PMC

Sedlar K, Koscova P, Vasylkivska M, Branska B, Kolek J, Kupkova K, et al. Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq. BMC Genomics. 2018;19:415. doi: 10.1186/s12864-018-4805-8. PubMed DOI PMC

Kolek J, Diallo M, Vasylkivska M, Branska B, Sedlar K, López-Contreras AM, et al. Comparison of expression of key sporulation, solventogenic and acetogenic genes in C. beijerinckii NRRL B-598 and its mutant strain overexpressing spo0A. Appl Microbiol Biotechnol. 2017;101:8279–8291. doi: 10.1007/s00253-017-8555-3. PubMed DOI

Tomas CA, Beamish J, Papoutsakis ET. Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J Bacteriol. 2004;186:2006–2018. doi: 10.1128/JB.186.7.2006-2018.2004. PubMed DOI PMC

Alsaker KV, Paredes C, Papoutsakis ET. Metabolite stress and tolerance in the production of biofuels and chemicals: gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotechnol Bioeng. 2010;105:1131–1147. PubMed

Kolek J, Patakova P, Melzoch K, Sigler K, Rezanka T. Changes in membrane plasmalogens of Clostridium pasteurianum during butanol fermentation as determined by lipidomic analysis. PLoS ONE. 2015;10:e0122058. doi: 10.1371/journal.pone.0122058. PubMed DOI PMC

Lipovsky J, Patakova P, Paulova L, Pokorny T, Rychtera M, Melzoch K. Butanol production by Clostridium pasteurianum NRRL B-598 in continuous culture compared to batch and fed-batch systems. Fuel Process Technol. 2016;144:139–144. doi: 10.1016/j.fuproc.2015.12.020. DOI

Bateman A, Martin M-J, Orchard S, Magrane M, Alpi E, Bely B, et al. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2018;47:D506–D515. PubMed PMC

Mitchell AL, Attwood TK, Babbitt PC, Blum M, Bork P, Bridge A, et al. InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 2018;47:D351–D360. doi: 10.1093/nar/gky1100. PubMed DOI PMC

Carbon S, Douglass E, Dunn N, Good B, Harris NL, Lewis SE, et al. The gene ontology resource: 20 years and still GOing strong. Nucleic Acids Res. 2018;47:D330–D338. PubMed PMC

Sweeney BA, Petrov AI, Burkov B, Finn RD, Bateman A, Szymanski M, et al. RNAcentral: a hub of information for non-coding RNA sequences. Nucleic Acids Res. 2018;47:D221–D229. PubMed PMC

Altschul S, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Alexa A, Rahnenfuhrer J. topGO: enrichment analysis for gene ontology. R package version 2.30.1. 2016.

Van Der Maaten L, Hinton G. Visualizing Data using t-SNE. J Mach Learn Res. 2008;620:267–284.

Au KF, Underwood JG, Lee L, Wong WH. Improving PacBio long read accuracy by short read alignment. PLoS ONE. 2012;7:e46679. doi: 10.1371/journal.pone.0046679. PubMed DOI PMC

Xu M, Zhao J, Yu L, Tang I-C, Xue C, Yang S-T. Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production. Appl Microbiol Biotechnol. 2015;99:1011–1022. doi: 10.1007/s00253-014-6249-7. PubMed DOI

Huerta-Cepas J, Forslund K, Coelho LP, Szklarczyk D, Jensen LJ, von Mering C, et al. Fast genome-wide functional annotation through orthology assignment by eggNOG-Mapper. Mol Biol Evol. 2017;34:2115–2122. doi: 10.1093/molbev/msx148. PubMed DOI PMC

Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, et al. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 2008;36:3420–3435. doi: 10.1093/nar/gkn176. PubMed DOI PMC

Skunca N, Altenhoff A, Dessimoz C. Quality of computationally inferred gene ontology annotations. PLoS Comput Biol. 2012;8:e1002533. doi: 10.1371/journal.pcbi.1002533. PubMed DOI PMC

Branska B, Pechacova Z, Kolek J, Vasylkivska M, Patakova P. Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions. Biotechnol Biofuels. 2018;11:99. doi: 10.1186/s13068-018-1096-x. PubMed DOI PMC

Whitley D, Goldberg SP, Jordan WD. Heat shock proteins: a review of the molecular chaperones. J Vasc Surg. 1999;29:748–751. doi: 10.1016/S0741-5214(99)70329-0. PubMed DOI

Mann MS, Dragovic Z, Schirrmacher G, Lütke-Eversloh T. Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress. Biotechnol Lett. 2012;34:1643–1649. doi: 10.1007/s10529-012-0951-2. PubMed DOI

Liao Z, Zhang Y, Luo S, Suo Y, Zhang S, Wang J. Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium. J Biotechnol. 2017;252:1–10. doi: 10.1016/j.jbiotec.2017.04.031. PubMed DOI

Tomas CA, Welker NE, Papoutsakis ET. Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program. Appl Environ Microbiol. 2003;69:4951–4965. doi: 10.1128/AEM.69.8.4951-4965.2003. PubMed DOI PMC

Reyes LH, Almario MP, Kao KC. Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli. PLoS ONE. 2011;6:e17678. doi: 10.1371/journal.pone.0017678. PubMed DOI PMC

Xue C, Zhao J, Chen L, Yang ST, Bai F. Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum. Biotechnol Adv. 2017;35:310–322. doi: 10.1016/j.biotechadv.2017.01.007. PubMed DOI

Steiner E, Scott J, Minton NP, Winzer K. An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum. Appl Environ Microbiol. 2012;78:1113–1122. doi: 10.1128/AEM.06376-11. PubMed DOI PMC

Setlow B, Setlow P. Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide. Appl Environ Microbiol. 1993;59:3418–3423. PubMed PMC

Setlow P. I will survive: DNA protection in bacterial spores. Trends Microbiol. 2007;15:172–180. doi: 10.1016/j.tim.2007.02.004. PubMed DOI

Raju D, Waters M, Setlow P, Sarker MR. Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat. BMC Microbiol. 2006;6:50. doi: 10.1186/1471-2180-6-50. PubMed DOI PMC

Wetzel D, Fischer R-J. Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC. Microbiology. 2015;161:2098–2109. doi: 10.1099/mic.0.000162. PubMed DOI

Ewels P, Magnusson M, Lundin S, Kaller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32:3047–3048. doi: 10.1093/bioinformatics/btw354. PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Li H, Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25:1754–1760. doi: 10.1093/bioinformatics/btp324. PubMed DOI PMC

Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9:e112963. doi: 10.1371/journal.pone.0112963. PubMed DOI PMC

Kopylova E, Noé L, Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics. 2012;28:3211–3217. doi: 10.1093/bioinformatics/bts611. PubMed DOI

Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41:D590–D596. doi: 10.1093/nar/gks1219. PubMed DOI PMC

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. doi: 10.1093/bioinformatics/bts635. PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Liao Y, Smyth GK, Shi W. FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–930. doi: 10.1093/bioinformatics/btt656. PubMed DOI

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550. doi: 10.1186/s13059-014-0550-8. PubMed DOI PMC

Van Der Maaten L. Accelerating t-SNE using tree-based algorithms. J Mach Learn Res. 2014;15:1–21.

Wickham H. ggplot2 elegant graphics for data analysis. Cham: Springer; 2009.

Chen H, Boutros PC. VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinform. 2011;12:35. doi: 10.1186/1471-2105-12-35. PubMed DOI PMC

Binns D, Dimmer E, Huntley R, Barrell D, O’Donovan C, Apweiler R. QuickGO: a web-based tool for gene ontology searching. Bioinformatics. 2009;25:3045–3046. doi: 10.1093/bioinformatics/btp536. PubMed DOI PMC

Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21:3674–3676. doi: 10.1093/bioinformatics/bti610. PubMed DOI

Fang H, Gough J. The ‘dnet’ approach promotes emerging research on cancer patient survival. Genome Med. 2014;6:64. doi: 10.1186/s13073-014-0064-8. PubMed DOI PMC

Csárdi G, Nepusz T. The igraph software package for complex network research. InterJournal Complex Syst. 2006;1695:1–9.

Augusta: From RNA-Seq to gene regulatory networks and Boolean models

Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data

Deeper below the surface-transcriptional changes in selected genes of Clostridium beijerinckii in response to butanol shock

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance