Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass

Biotechnology for Biofuels

Charlotte M Wilson²  Miguel Rodriguez²  Courtney M Johnson²  Stanton L Martin¹  Tzu Ming Chu¹  Russ D Wolfinger¹  Loren J Hauser²  Miriam L Land²  Dawn M Klingeman²  Mustafa H Syed²  Arthur J Ragauskas³  Timothy J Tschaplinski²  Jonathan R Mielenz²  Steven D Brown² 

[1] SAS Institute, Cary, NC 27513, USA

[2] BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

[3] School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA

关键词: Transcriptomics;    Normalization;    Phosphate;    Microarray;    RNA-seq;    Elemental composition;    Biomass;    Reannotation;    Genome;   

Others  :  794351
DOI  :  10.1186/1754-6834-6-179

 received in 2013-08-02, accepted in 2013-11-19,  发布年份 2013

【 摘 要 】

Background

The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. The aim of this study was to investigate C. thermocellum genes required to ferment biomass substrates and to conduct a robust comparison of DNA microarray and RNA sequencing (RNA-seq) analytical platforms.

Results

C. thermocellum ATCC 27405 fermentations were conducted with a 5 g/L solid substrate loading of either pretreated switchgrass or Populus. Quantitative saccharification and inductively coupled plasma emission spectroscopy (ICP-ES) for elemental analysis revealed composition differences between biomass substrates, which may have influenced growth and transcriptomic profiles. High quality RNA was prepared for C. thermocellum grown on solid substrates and transcriptome profiles were obtained for two time points during active growth (12 hours and 37 hours postinoculation). A comparison of two transcriptomic analytical techniques, microarray and RNA-seq, was performed and the data analyzed for statistical significance. Large expression differences for cellulosomal genes were not observed. We updated gene predictions for the strain and a small novel gene, Cthe_3383, with a putative AgrD peptide quorum sensing function was among the most highly expressed genes. RNA-seq data also supported different small regulatory RNA predictions over others. The DNA microarray gave a greater number (2,351) of significant genes relative to RNA-seq (280 genes when normalized by the kernel density mean of M component (KDMM) method) in an analysis of variance (ANOVA) testing method with a 5% false discovery rate (FDR). When a 2-fold difference in expression threshold was applied, 73 genes were significantly differentially expressed in common between the two techniques. Sulfate and phosphate uptake/utilization genes, along with genes for a putative efflux pump system were some of the most differentially regulated transcripts when profiles for C. thermocellum grown on either pretreated switchgrass or Populus were compared.

Conclusions

Our results suggest that a high degree of agreement in differential gene expression measurements between transcriptomic platforms is possible, but choosing an appropriate normalization regime is essential.

【 授权许可】

   
2013 Wilson et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Lynd LR, Van Zyl WH, McBride JE, Laser M: Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 2005, 16:577-583.
• [2]Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS: Microbial cellulose utilization: Fundamentals and biotechnology. Microbiol Mol Biol Rev 2002, 66:506-577.
• [3]Demain AL, Newcomb M, Wu JH: Cellulase, clostridia, and ethanol. Microbiol Mol Biol Rev 2005, 69:124-154.
• [4]Alper H, Stephanopoulos G: Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat Rev Microbiol 2009, 7:715-723.
• [5]Farrell AE, Plevin RJ, Turner BT, Jones AD, O’Hare M, Kammen DM: Ethanol can contribute to energy and environmental goals. Science 2006, 311:506-508.
• [6]Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G: Bio-ethanol - the fuel of tomorrow from the residues of today. Trends Biotechnol 2006, 24:549-556.
• [7]Himmel ME, Ding S-Y, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD: Biomass recalcitrance: Engineering plants and enzymes for biofuels production. Science 2007, 315:804-807.
• [8]Stephanopoulos G: Challenges in engineering microbes for biofuels production. Science 2007, 315:801-804.
• [9]Delcher A, Bratke K, Powers E, Salzberg S: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 2007, 23:673-679.
• [10]Badger J, Olsen G: CRITICA: coding region identification tool invoking comparative analysis. Mol Bio Evol 1999, 16:512-524.
• [11]Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ: Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinforma 2010, 11:119. BioMed Central Full Text
• [12]Hauser LJ, Land ML, Brown SD, Larimer F, Keller KL, Rapp-Giles BJ, Price MN, Lin M, Bruce DC, Detter JC, Tapia R, Han CS, Goodwin LA, Cheng JF, Pitluck S, Copeland A, Lucas S, Nolan M, Lapidus AL, Palumbo AV, Wall JD: The complete genome sequence and updated annotation of Desulfovibrio alaskensis G20. J Bacteriol 2011, 193:4268-4269.
• [13]Yang S, Pappas KM, Hauser LJ, Land ML, Chen GL, Hurst GB, Pan C, Kouvelis VN, Typas MA, Pelletier DA, Klingeman DM, Chang YJ, Samatova NF, Brown SD: Improved genome annotation for Zymomonas mobilis. Nat Biotechnol 2009, 27:893-894.
• [14]Raman B, Pan C, Hurst GB, Rodriguez M Jr, McKeown CK, Lankford PK, Samatova NF, Mielenz JR: Impact of pretreated switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: A quantitative proteomic analysis. PLoS One 2009, 4:e5271.
• [15]Gold ND, Martin VJJ: Global view of the Clostridium thermocellum cellulosome revealed by quantitative proteomic analysis. J Bacteriol 2007, 189:6787-6795.
• [16]Riederer A, Takasuka TE, Makino S-I, Stevenson DM, Bukhman YV, Elsen NL, Fox BG: Global gene expression patterns in Clostridium thermocellum as determined by microarray analysis of chemostat cultures on cellulose or cellobiose. Appl Environ Microbiol 2011, 77:1243-1253.
• [17]Fontes CM, Gilbert HJ: Cellulosomes: Highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates. Ann Rev Biochem 2010, 79:655-681.
• [18]Department of Energy (DOE), Office of Energy Efficiency and Renewable Energy (EERE), Office of the Biomass Program: U.S. Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. Oak Ridge, TN: Oak Ridge National Laboratory; 2011:227.
• [19]Leimena MM, Wels M, Bongers RS, Smid EJ, Zoetendal EG, Kleerebezem M: Comparative analysis of Lactobacillus plantarum WCFS1 transcriptomes by using DNA microarray and next-generation sequencing technologies. Appl Environ Microbiol 2012, 78:4141-4148.
• [20]Passalacqua KD, Varadarajan A, Ondov BD, Okou DT, Zwick ME, Bergman NH: Structure and complexity of a bacterial transcriptome. J Bacteriol 2009, 191:3203-3211.
• [21]Yoder-Himes DR, Chain PS, Zhu Y, Wurtzel O, Rubin EM, Tiedje JM, Sorek R: Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing. Proc Natl Acad Sci U S A 2009, 106:3976-3981.
• [22]Jourdren L, Bernard M, Dillies MA, Le Crom S: Eoulsan: a cloud computing-based framework facilitating high throughput sequencing analyses. Bioinformatics 2012, 28:1542-1543.
• [23]Shendure J, Ji H: Next-generation DNA sequencing. Nat Biotechnol 2008, 26:1135-1145.
• [24]Oshlack A, Robinson MD, Young MD: From RNA-seq reads to differential expression results. Genome Biol 2010, 11:220. BioMed Central Full Text
• [25]Soneson C, Delorenzi M: A comparison of methods for differential expression analysis of RNA-seq data. BMC Bioinforma 2013, 14:91. BioMed Central Full Text
• [26]Mardis ER: Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 2008, 9:387-402.
• [27]Luo C, Hu G-Q, Zhu H: Genome reannotation of Escherichia coli CFT073 with new insights into virulence. BMC Genomics 2009, 10:552. BioMed Central Full Text
• [28]Siezen RJ, Francke C, Renckens B, Boekhorst J, Wels M, Kleerebezem M, Van Hijum SAFT: Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome. J Bacteriol 2012, 194:195-196.
• [29]Wood V, Rutherford KM, Ivens A, Rajandream MA, Barrell B: A re-annotation of the Saccharomyces cerevisiae genome. Comp Funct Genomics 2001, 2:143-154.
• [30]Yang S, Giannone RJ, Dice L, Yang ZK, Engle NL, Tschaplinski TJ, Hettich RL, Brown SD: Elucidation of the Clostridium thermocellum ATCC27405 ethanol shock responses using an integrated transcriptomic, proteomic and metabolomic profiling approach. BMC Genomics 2012, 13:336. BioMed Central Full Text
• [31]Mazumder K, York WS: Structural analysis of arabinoxylans isolated from ball-milled switchgrass biomass. Carbohydr Res 2010, 345:2183-2193.
• [32]Nataf Y, Bahari L, Kahel-Raifer H, Borovok I, Lamed R, Bayer EA, Sonenshein AL, Shoham Y: Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors. Proc Natl Acad Sci U S A 2010, 107:18646-18651.
• [33]Nataf Y, Yaron S, Stahl F, Lamed R, Bayer EA, Scheper TH, Sonenshein AL, Shoham Y: Cellodextrin and laminaribiose ABC transporters in Clostridium thermocellum. J Bacteriol 2009, 191:203-209.
• [34]Yang S, Giannone RJ, Dice L, Yang ZK, Engle NL, Tschaplinski TJ, Hettich RL, Brown SD: Clostridium thermocellum ATCC27405 transcriptomic, metabolomic and proteomic profiles after ethanol stress. BMC Genomics 2012, 13:336. BioMed Central Full Text
• [35]Esvelt KM, Wang HH: Genome-scale engineering for systems and synthetic biology. Mol Syst Biol 2013, 9:641.
• [36]Chen Y, Indurthi DC, Jones SW, Papoutsakis ET: Small RNAs in the Genus Clostridium. mBio 2011, 2:e00340-10. doi:10.1128/mBio.00340-10
• [37]van den Berg BH, McCarthy FM, Lamont SJ, Burgess SC: Re-annotation is an essential step in systems biology modeling of functional genomics data. Plos One 2010, 5:e10642.
• [38]Salzberg SL: Genome re-annotation: a wiki solution? Genome Biol 2007, 8:102. BioMed Central Full Text
• [39]Dillies MA, Rau A, Aubert J, Hennequet-Antier C, Jeanmougin M, Servant N, Keime C, Marot G, Castel D, Estelle J, Guernec G, Jagla B, Jouneau L, Laloë D, Le Gall C, Schaëffer B, Le Crom S, Guedj M, Jaffrézic F, French StatOmique Consortium: A comprehensive evaluation of normalization methods for Illumina high-throughput RNA sequencing data analysis. Brief Bioinform 2013, 14:671-683.
• [40]Oshlack A, Wakefield MJ: Transcript length bias in RNA-seq data confounds systems biology. Biol Direct 2009, 4:14. BioMed Central Full Text
• [41]Pickrell JK, Marioni JC, Pai AA, Degner JF, Engelhardt BE, Nkadori E, Veyrieras JB, Stephens M, Gilad Y, Pritchard JK: Understanding mechanisms underlying human gene expression variation with RNA sequencing. Nature 2010, 464:768-772.
• [42]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5:621-628.
• [43]Bullard JH, Purdom E, Hansen KD, Dudoit S: Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinforma 2010, 11:94. BioMed Central Full Text
• [44]Ebringerova A: Structural diversity and application potential of hemicelluloses. Macromol Symp 2005, 232:1-12.
• [45]Izydorczyk MS, Biliaderis CG: Cereal arabinoxylans: Advances in structure and physicochemical properties. Carbohyd Polym 1995, 28:33-48.
• [46]Klinke HB, Thomsen AB, Ahring BK: Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol 2004, 66:10-26.
• [47]Yee KL, Rodriguez MJ, Tschaplinski TJ, Engle NL, Martin MZ, Fu C, Wang ZY, Hamilton-Brehm SD, Mielenz JR: Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach. Biotechnol Biofuels 2012, 5:81. BioMed Central Full Text
• [48]Pu Y, Hu F, Huang F, Davison BH, Ragauskas AJ: Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnol Biofuels 2013, 6:15. BioMed Central Full Text
• [49]DeMartini JD, Pattathil S, Miller JS, Li H, Hahn MG, Wyman CE: Investigating plant cell wall components that affect biomass recalcitrance in poplar and switchgrass.Energy. Environ Sci 2013, 6:898-909.
• [50]Zhang YH, Lynd LR: Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. Proc Natl Acad Sci U S A 2005, 102:7321-7325.
• [51]Ahsan M, Matsumoto M, Karita S, Kimura T, Sakka K, Ohmiya K: Purification and characterization of the family J catalytic domain derived from the Clostridium thermocellum endoglucanase CelJ. Biosci Biotechnol Biochem 1997, 61:427-431.
• [52]Davidson AL, Dassa E, Orelle C, Chen J: Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 2008, 72:317-364.
• [53]Strobel HJ, Caldwell FC, Dawson KA: Carbohydrate transport by the anaerobic thermophile Clostridium thermocellum LQRI. Appl Environ Microbiol 1995, 61:4012-4015.
• [54]Alexander JK: Purification and specificity of cellobiose phosphorylase from Clostridium thermocellum. J Biol Chem 1968, 243:2899-2904.
• [55]Fischer RJ, Oehmcke S, Meyer U, Mix M, Schwarz K, Fiedler T, Bahl H: Transcription of the pst operon of Clostridium acetobutylicum is dependent on phosphate concentration and pH. J Bacteriol 2006, 188:5469-5478.
• [56]Gebhard S, Tran SL, Cook GM: The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate. Microbiology 2006, 152:3453-3465.
• [57]Antelmann H, Scharf C, Hecker M: Phosphate starvation-inducible proteins of Bacillus subtilis: proteomics and transcriptional analysis. J Bacteriol 2000, 182:4478-4490.
• [58]Aguena M, Yagil M, Spira B: Transcriptional analysis of the pst operon of Escherichia coli. Mol Genet Genomics 2002, 268:518-524.
• [59]El-Nashaar HM, Banowetz GM, Griffith SM, Casler MD, Vogel KP: Genotypic variability in mineral composition of switchgrass. Bioresour Technol 2009, 100:1809-1814.
• [60]Diaz-Ramirez M, Boman C, Sebastian F, Royo J, Xiong SJ, Bostrom D: Ash characterization and transformation behavior of the fixed-bed combustion of novel crops: poplar, brassica, and cassava fuels. Energ Fuel 2012, 26:3218-3229.
• [61]Alvarez-Ortega C, Olivares J, Martinez JL: RND multidrug efflux pumps: what are they good for? Front Microbiol 2013, 4:7.
• [62]Mearls EB, Izquierdo JA, Lynd LR: Formation and characterization of non-growth states in Clostridium thermocellum: spores and L-forms. BMC Microbiol 2012, 12:180. BioMed Central Full Text
• [63]Steiner E, Scott J, Minton NP, Winzer K: An agr quorum sensing system regulates granulose formation and sporulation in Clostridium acetobutylicum. Appl Environ Microbiol 2012, 78:1113-1122.
• [64]Paredes CJ, Alsaker KV, Papoutsakis ET: A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 2005, 3:969-978.
• [65]Shi L, Campbell G, Jones WD, Campagne F, Wen Z, Walker SJ, Su Z, Chu TM, Goodsaid FM, Pusztai L, Shaughnessy JD Jr, Oberthuer A, Thomas RS, Paules RS, Fielden M, Barlogie B, Chen W, Du P, Fischer M, Furlanello C, Gallas BD, Ge X, Megherbi DB, Symmans WF, Wang MD, Zhang J, Bitter H, Brors B, Bushel PR, Bylesjo M, et al.: The MicroArray Quality Control (MAQC)-II study of common practices for the development and validation of microarray-based predictive models. Nat Biotechnol 2010, 28:827-838.
• [66]Shi L, Reid LH, Jones WD, Shippy R, Warrington JA, Baker SC, Collins PJ, de Longueville F, Kawasaki ES, Lee KY, Luo Y, Sun YA, Willey JC, Setterquist RA, Fischer GM, Tong W, Dragan YP, Dix DJ, Frueh FW, Goodsaid FM, Herman D, Jensen RV, Johnson CD, Lobenhofer EK, Puri RK, Schrf U, Thierry-Mieg J, Wang C, Wilson M, MAQC Consortium, et al.: The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol 2006, 24:1151-1161.
• [67]Schell DJ, Farmer J, Newman M, McMillan JD: Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor: investigation of yields, kinetics, and enzymatic digestibilities of solids. Appl Biochem Biotechnol 2003, 105–108:69-85.
• [68]Sannigrahi P, Ragauskas AJ: Characterization of fermentation residues from the production of bio-ethanol from lignocellulosic feedstocks. J Biobased Mater Bio 2011, 5:514-519.
• [69]Kridelbaugh DM, Nelson J, Engle NL, Tschaplinski TJ, Graham DE: Nitrogen and sulfur requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on cellulosic substrates in minimal nutrient media. Bioresour Technol 2013, 130:125-135.
• [70]Syed MH, Karpinets TV, Parang M, Leuze MR, Park BH, Hyatt D, Brown SD, Moulton S, Galloway MD, Uberbacher EC: BESC knowledgebase public portal. Bioinformatics 2012, 28:750-751.