Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum

Jones, Alexander J.; Fast, Alan G.; Clupper, Michael; Papoutsakis, Eleftherios T.; Kelly, Robert M.

doi:10.1128/aem.00597-18

Title: Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum

Journal Article · Fri May 04 00:00:00 EDT 2018 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.00597-18· OSTI ID:1541704

Jones, Alexander J. ^[1];

^[2]; Clupper, Michael ^[3];

^[4]; Kelly, Robert M. ^[5]

Univ. of Delaware, Newark, DE (United States). Dept. of Biological Sciences, and Molecular Biotechnology Lab.; BD Biosciences, Sparks, MD (United States)
Univ. of Delaware, Newark, DE (United States). Dept. of Chemical & Biomolecular Engineering, and Molecular Biotechnology Lab.
Univ. of Delaware, Newark, DE (United States). Dept. of Biological Sciences, and Molecular Biotechnology Lab.
Univ. of Delaware, Newark, DE (United States). Dept. of Biological Sciences, Dept. of Chemical & Biomolecular Engineering, and Molecular Biotechnology Lab.
North Carolina State Univ., Raleigh, NC (United States)

The recently revived Clostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology.C. acetobutylicum has been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad, ctfA, ctfB, and adc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, “degenerate” cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion of solB resulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact on adc mRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reduced adhE1 and ctfA-ctfB mRNA levels, and abolished the ctfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. Finally, key predicted SolB target is the ribosome binding site (RBS) of the ctfA transcript, and this was verified by expressing variants of the ctfA-ctfB genes to demonstrate the importance of SolB for acetone formation.

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Research Organization:: Univ. of Delaware, Newark, DE (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: SC0007092

OSTI ID:: 1541704

Journal Information:: Applied and Environmental Microbiology, Vol. 84, Issue 14; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 11 works

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References (55)

Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications Tracy, Bryan P.; Jones, Shawn W.; Fast, Alan G. Current Opinion in Biotechnology, Vol. 23, Issue 3 https://doi.org/10.1016/j.copbio.2011.10.008	journal	June 2012
Sequence and arrangement of genes encoding enzymes of the acetone-production pathway of Clostridium acetobutylicum ATCC 824 Petersen, Daniel J.; Cary, Jeffrey W.; Vanderleyden, Jos Gene, Vol. 123, Issue 1 https://doi.org/10.1016/0378-1119(93)90545-E	journal	January 1993
Genome-Wide Identification of Regulatory RNAs in the Human Pathogen Clostridium difficile Soutourina, Olga A.; Monot, Marc; Boudry, Pierre PLoS Genetics, Vol. 9, Issue 5 https://doi.org/10.1371/journal.pgen.1003493	journal	May 2013
Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. Nair, R. V.; Bennett, G. N.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 176, Issue 3 https://doi.org/10.1128/jb.176.3.871-885.1994	journal	February 1994
Northern, Morphological, and Fermentation Analysis of spo0A Inactivation and Overexpression in Clostridium acetobutylicum ATCC 824 Harris, L. M.; Welker, N. E.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 184, Issue 13 https://doi.org/10.1128/JB.184.13.3586-3597.2002	journal	July 2002
Target activation by regulatory RNAs in bacteria Papenfort, Kai; Vanderpool, Carin K. FEMS Microbiology Reviews, Vol. 39, Issue 3 https://doi.org/10.1093/femsre/fuv016	journal	May 2015
Sequences affecting the regulation of solvent production in Clostridium acetobutylicum Scotcher, Miles C.; Huang, Ke-xue; Harrison, Mary L. Journal of Industrial Microbiology and Biotechnology, Vol. 30, Issue 7 https://doi.org/10.1007/s10295-003-0057-x	journal	July 2003
Analyzing real-time PCR data by the comparative CT method Schmittgen, Thomas D.; Livak, Kenneth J. Nature Protocols, Vol. 3, Issue 6 https://doi.org/10.1038/nprot.2008.73	journal	June 2008
Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia Ravagnani, Adriana; Jennert, Katrin C. B.; Steiner, Elisabeth Molecular Microbiology, Vol. 37, Issue 5 https://doi.org/10.1046/j.1365-2958.2000.02071.x	journal	September 2000
IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions Busch, Anke; Richter, Andreas S.; Backofen, Rolf Bioinformatics, Vol. 24, Issue 24 https://doi.org/10.1093/bioinformatics/btn544	journal	October 2008
Regulation by Small RNAs in Bacteria: Expanding Frontiers Storz, Gisela; Vogel, Jörg; Wassarman, Karen M. Molecular Cell, Vol. 43, Issue 6 https://doi.org/10.1016/j.molcel.2011.08.022	journal	September 2011
Inactivation of E and G in Clostridium acetobutylicum Illuminates Their Roles in Clostridial-Cell-Form Biogenesis, Granulose Synthesis, Solventogenesis, and Spore Morphogenesis Tracy, B. P.; Jones, S. W.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 193, Issue 6 https://doi.org/10.1128/JB.01380-10	journal	January 2011
Application of new metabolic engineering tools for Clostridium acetobutylicum Lütke-Eversloh, Tina Applied Microbiology and Biotechnology, Vol. 98, Issue 13 https://doi.org/10.1007/s00253-014-5785-5	journal	May 2014
Overexpression of two stress-responsive, small, non-coding RNAs, 6S and tmRNA, imparts butanol tolerance in Clostridium acetobutylicum Jones, Alexander J.; Venkataramanan, Keerthi P.; Papoutsakis, Terry FEMS Microbiology Letters, Vol. 363, Issue 8 https://doi.org/10.1093/femsle/fnw063	journal	March 2016
Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production Lütke-Eversloh, Tina; Bahl, Hubert Current Opinion in Biotechnology, Vol. 22, Issue 5 https://doi.org/10.1016/j.copbio.2011.01.011	journal	October 2011
DNA Array-Based Transcriptional Analysis of Asporogenous, Nonsolventogenic Clostridium acetobutylicum Strains SKO1 and M5 Tomas, C. A.; Alsaker, K. V.; Bonarius, H. P. J. Journal of Bacteriology, Vol. 185, Issue 15 https://doi.org/10.1128/JB.185.15.4539-4547.2003	journal	July 2003
Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis. Fischer, R. J.; Helms, J.; Dürre, P. Journal of Bacteriology, Vol. 175, Issue 21 https://doi.org/10.1128/jb.175.21.6959-6969.1993	journal	November 1993
The Vienna RNA Websuite Gruber, A. R.; Lorenz, R.; Bernhart, S. H. Nucleic Acids Research, Vol. 36, Issue Web Server https://doi.org/10.1093/nar/gkn188	journal	May 2008
The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain. Cornillot, E.; Nair, R. V.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 179, Issue 17 https://doi.org/10.1128/jb.179.17.5442-5447.1997	journal	September 1997
Small RNAs in the Genus Clostridium Chen, Y.; Indurthi, D. C.; Jones, S. W. mBio, Vol. 2, Issue 1 https://doi.org/10.1128/mBio.00340-10	journal	December 2010
TargetRNA: a tool for predicting targets of small RNA action in bacteria Tjaden, B. Nucleic Acids Research, Vol. 36, Issue Web Server https://doi.org/10.1093/nar/gkn264	journal	May 2008
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 Alsaker, Keith V.; Paredes, Carlos; Papoutsakis, Eleftherios T. Biotechnology and Bioengineering https://doi.org/10.1002/bit.22628	journal	January 2010
Nutritional Factors Affecting the Ratio of Solvents Produced by Clostridium acetobutylicum Bahl, H.; Gottwald, M.; Kuhn, A. Applied and Environmental Microbiology, Vol. 52, Issue 1 https://doi.org/10.1128/AEM.52.1.169-172.1986	journal	January 1986
K of Clostridium acetobutylicum Is the First Known Sporulation-Specific Sigma Factor with Two Developmentally Separated Roles, One Early and One Late in Sporulation Al-Hinai, M. A.; Jones, S. W.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 196, Issue 2 https://doi.org/10.1128/JB.01103-13	journal	November 2013
The transcriptional program underlying the physiology of clostridial sporulation Jones, Shawn W.; Paredes, Carlos J.; Tracy, Bryan Genome Biology, Vol. 9, Issue 7 https://doi.org/10.1186/gb-2008-9-7-r114	journal	January 2008
New insights into small RNA-dependent translational regulation in prokaryotes Desnoyers, Guillaume; Bouchard, Marie-Pier; Massé, Eric Trends in Genetics, Vol. 29, Issue 2 https://doi.org/10.1016/j.tig.2012.10.004	journal	February 2013
Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production. Nair, R. V.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 176, Issue 18 https://doi.org/10.1128/jb.176.18.5843-5846.1994	journal	September 1994
A genomic-library based discovery of a novel, possibly synthetic, acid-tolerance mechanism in Clostridium acetobutylicum involving non-coding RNAs and ribosomal RNA processing Borden, Jacob R.; Jones, Shawn W.; Indurthi, Dinesh Metabolic Engineering, Vol. 12, Issue 3, p. 268-281 https://doi.org/10.1016/j.ymben.2009.12.004	journal	May 2010
mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. Gerischer, U.; Dürre, P. Journal of Bacteriology, Vol. 174, Issue 2 https://doi.org/10.1128/jb.174.2.426-433.1992	journal	January 1992
A Secondary Structure in the 5' Untranslated Region of adhE mRNA Required for RNase G-Dependent Regulation Ito, Kazutaka; Hamasaki, Kohshin; Kayamori, Aya Bioscience, Biotechnology, and Biochemistry, Vol. 77, Issue 12 https://doi.org/10.1271/bbb.130618	journal	December 2013
Overexpression of groESL in Clostridium acetobutylicum Results in Increased Solvent Production and Tolerance, Prolonged Metabolism, and Changes in the Cell's Transcriptional Program Tomas, C. A.; Welker, N. E.; Papoutsakis, E. T. Applied and Environmental Microbiology, Vol. 69, Issue 8 https://doi.org/10.1128/AEM.69.8.4951-4965.2003	journal	August 2003
A comparative genomic view of clostridial sporulation and physiology Paredes, Carlos J.; Alsaker, Keith V.; Papoutsakis, Eleftherios T. Nature Reviews Microbiology, Vol. 3, Issue 12 https://doi.org/10.1038/nrmicro1288	journal	October 2005
Novel System for Efficient Isolation of Clostridium Double-Crossover Allelic Exchange Mutants Enabling Markerless Chromosomal Gene Deletions and DNA Integration Al-Hinai, Mohab A.; Fast, Alan G.; Papoutsakis, Eleftherios T. Applied and Environmental Microbiology, Vol. 78, Issue 22 https://doi.org/10.1128/AEM.02214-12	journal	September 2012
Control of Butanol Formation in Clostridium acetobutylicum by Transcriptional Activation Thormann, K.; Feustel, L.; Lorenz, K. Journal of Bacteriology, Vol. 184, Issue 7 https://doi.org/10.1128/JB.184.7.1966-1973.2002	journal	April 2002
Solventogenesis in Clostridium acetobutylicum fermentations related to carboxylic acid and proton concentrations Hüsemann, Michael H. W.; Papoutsakis, Eleftherios T. Biotechnology and Bioengineering, Vol. 32, Issue 7 https://doi.org/10.1002/bit.260320702	journal	September 1988
Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids. Wiesenborn, D. P.; Rudolph, F. B.; Papoutsakis, E. T. Applied and Environmental Microbiology, Vol. 55, Issue 2 https://doi.org/10.1128/AEM.55.2.323-329.1989	journal	January 1989
The effect of pH on nitrogen supply, cell lysis, and solvent production in fermentations ofClostridium acetobutylicum Roos, Joseph W.; McLaughlin, Joseph K.; Papoutsakis, Eleftherios T. Biotechnology and Bioengineering, Vol. 27, Issue 5 https://doi.org/10.1002/bit.260270518	journal	May 1985
In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Mermelstein, L. D.; Papoutsakis, E. T. Applied and Environmental Microbiology, Vol. 59, Issue 4 https://doi.org/10.1128/AEM.59.4.1077-1081.1993	journal	January 1993
Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations Tummala, S. B.; Junne, S. G.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 185, Issue 12 https://doi.org/10.1128/JB.185.12.3644-3653.2003	journal	June 2003
CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains Wright, Patrick R.; Georg, Jens; Mann, Martin Nucleic Acids Research, Vol. 42, Issue W1 https://doi.org/10.1093/nar/gku359	journal	May 2014
TargetRNA2: identifying targets of small regulatory RNAs in bacteria Kery, Mary Beth; Feldman, Monica; Livny, Jonathan Nucleic Acids Research, Vol. 42, Issue W1 https://doi.org/10.1093/nar/gku317	journal	April 2014
Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress Gaida, S. M.; Al-Hinai, M. A.; Indurthi, D. C. Nucleic Acids Research, Vol. 41, Issue 18 https://doi.org/10.1093/nar/gkt651	journal	July 2013
RNA-based control mechanisms of Clostridium difficile Soutourina, Olga Current Opinion in Microbiology, Vol. 36 https://doi.org/10.1016/j.mib.2017.01.004	journal	April 2017
Stable and enhanced gene expression in Clostridium acetobutylicum using synthetic untranslated regions with a stem-loop Lee, Joungmin; Jang, Yu-Sin; Papoutsakis, Eleftherios T. Journal of Biotechnology, Vol. 230 https://doi.org/10.1016/j.jbiotec.2016.05.020	journal	July 2016
Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum Tummala, S. B.; Welker, N. E.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 185, Issue 6 https://doi.org/10.1128/JB.185.6.1923-1934.2003	journal	March 2003
Engineering solventogenic clostridia Papoutsakis, Eleftherios T. Current Opinion in Biotechnology, Vol. 19, Issue 5, p. 420-429 https://doi.org/10.1016/j.copbio.2008.08.003	journal	October 2008
Transcriptional Analysis of Butanol Stress and Tolerance in Clostridium acetobutylicum Tomas, C. A.; Beamish, J.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 186, Issue 7 https://doi.org/10.1128/JB.186.7.2006-2018.2004	journal	March 2004
Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum Venkataramanan, Keerthi P.; Min, Lie; Hou, Shuyu Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0260-9	journal	June 2015
Transcription factors and genetic circuits orchestrating the complex, multilayered response of Clostridium acetobutylicum to butanol and butyrate stress Wang, Qinghua; Venkataramanan, Keerthi; Huang, Hongzhan BMC Systems Biology, Vol. 7, Issue 1 https://doi.org/10.1186/1752-0509-7-120	journal	January 2013
The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum Venkataramanan, Keerthi P.; Jones, Shawn W.; McCormick, Kevin P. BMC Genomics, Vol. 14, Issue 1 https://doi.org/10.1186/1471-2164-14-849	journal	January 2013
Transcriptional Program of Early Sporulation and Stationary-Phase Events in Clostridium acetobutylicum Alsaker, K. V.; Papoutsakis, E. T. Journal of Bacteriology, Vol. 187, Issue 20 https://doi.org/10.1128/JB.187.20.7103-7118.2005	journal	September 2005
Transport of Substrates and Metabolites and Their Effect on Cell Metabolism (in Butyric-Acid and Methylotrophic Fermentations) Papoutsakis, E. T.; Bussineau, C. M.; Chu, I-M. Annals of the New York Academy of Sciences, Vol. 506, Issue 1 Biochemical E https://doi.org/10.1111/j.1749-6632.1987.tb23808.x	journal	November 1987
Fermentative butanol production by clostridia Lee, Sang Yup; Park, Jin Hwan; Jang, Seh Hee Biotechnology and Bioengineering, Vol. 101, Issue 2, p. 209-228 https://doi.org/10.1002/bit.22003	journal	October 2008
Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum Tummala, Seshu B.; Welker, Neil E.; Papoutsakis, Eleftherios T. Journal of Bacteriology, Vol. 185, Issue 9 https://doi.org/10.1128/jb.185.9.2973.2003	journal	May 2003
mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis Gerischer, Ulrike; Dürre, Peter Universität Ulm https://doi.org/10.18725/oparu-1277	text	January 2009

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59 BASIC BIOLOGICAL SCIENCES
biotechnology & applied microbiology
microbiology
SolB
small RNA
noncoding RNA
Clostridium acetobutylicum
acetone
butanol
ABE
sol locus
metabolic engineering
mRNA stability
solvent formation
RBS
translational regulation

Title: Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum

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