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

Charlotte M. Wilson; Miguel Rodriguez; Courtney M. Johnson; Stanton L. Martin; Tzu Ming Chu; Russ D. Wolfinger; Loren J. Hauser; Miriam L. Land; Dawn M. Klingeman; Mustafa H. Syed; Arthur J. Ragauskas; Timothy J. Tschaplinski; Jonathan R. Mielenz; Steven D. Brown

doi:10.1186/1754-6834-6-179

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

Charlotte M. Wilson, Miguel Rodriguez, Courtney M. Johnson, Stanton L. Martin, Tzu Ming Chu, Russ D. Wolfinger, Loren J. Hauser, Miriam L. Land, Dawn M. Klingeman, Mustafa H. Syed, Arthur J. Ragauskas, Timothy J. Tschaplinski, Jonathan R. Mielenz, Steven D. Brown

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Abstract

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.

Original language	English
Article number	179
Journal	Biotechnology for Biofuels
Volume	6
Issue number	1
DOIs	https://doi.org/10.1186/1754-6834-6-179
State	Published - Dec 2 2013

Funding

The authors gratefully acknowledge Brian Davison (ORNL) for critical review of the manuscript. The authors thank Kelsey Yee (ORNL), Janet Westpheling (University of Georgia, Athens, GA, USA), Lee Lynd (Dartmouth College, Hanover, NH, USA), and Edward Bayer (Weizmann Institute of Science, Rehovot, Israel) for helpful discussions. Sagar Utturkar (University of Tennessee, Knoxville, TN, USA) provided technical assistance with sequence data. This work was supported by the Office of Biological and Environmental Research in the DOE Office of Science through the BESC, a DOE Bioenergy Research Center. ORNL is managed by UT-Battelle, LLC, Oak Ridge, TN, USA, for the DOE under contract DE-AC05-00OR22725.

Keywords

Biomass
Elemental composition
Genome
Microarray
Normalization
Phosphate
RNA-seq
Reannotation
Transcriptomics

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10.1186/1754-6834-6-179

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Wilson, C. M., Rodriguez, M., Johnson, C. M., Martin, S. L., Chu, T. M., Wolfinger, R. D., Hauser, L. J., Land, M. L., Klingeman, D. M., Syed, M. H., Ragauskas, A. J., Tschaplinski, T. J., Mielenz, J. R., & Brown, S. D. (2013). Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass. Biotechnology for Biofuels, 6(1), Article 179. https://doi.org/10.1186/1754-6834-6-179

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

abstract = "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.",

keywords = "Biomass, Elemental composition, Genome, Microarray, Normalization, Phosphate, RNA-seq, Reannotation, Transcriptomics",

author = "Wilson, {Charlotte M.} and Miguel Rodriguez and Johnson, {Courtney M.} and Martin, {Stanton L.} and Chu, {Tzu Ming} and Wolfinger, {Russ D.} and Hauser, {Loren J.} and Land, {Miriam L.} and Klingeman, {Dawn M.} and Syed, {Mustafa H.} and Ragauskas, {Arthur J.} and Tschaplinski, {Timothy J.} and Mielenz, {Jonathan R.} and Brown, {Steven D.}",

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doi = "10.1186/1754-6834-6-179",

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Wilson, CM, Rodriguez, M, Johnson, CM, Martin, SL, Chu, TM, Wolfinger, RD, Hauser, LJ, Land, ML, Klingeman, DM, Syed, MH, Ragauskas, AJ, Tschaplinski, TJ, Mielenz, JR & Brown, SD 2013, 'Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass', Biotechnology for Biofuels, vol. 6, no. 1, 179. https://doi.org/10.1186/1754-6834-6-179

TY - JOUR

T1 - Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass

AU - Wilson, Charlotte M.

AU - Rodriguez, Miguel

AU - Johnson, Courtney M.

AU - Martin, Stanton L.

AU - Chu, Tzu Ming

AU - Wolfinger, Russ D.

AU - Hauser, Loren J.

AU - Land, Miriam L.

AU - Klingeman, Dawn M.

AU - Syed, Mustafa H.

AU - Ragauskas, Arthur J.

AU - Tschaplinski, Timothy J.

AU - Mielenz, Jonathan R.

AU - Brown, Steven D.

PY - 2013/12/2

Y1 - 2013/12/2

N2 - 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.

AB - 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.

KW - Biomass

KW - Elemental composition

KW - Genome

KW - Microarray

KW - Normalization

KW - Phosphate

KW - RNA-seq

KW - Reannotation

KW - Transcriptomics

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DO - 10.1186/1754-6834-6-179

M3 - Article

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SN - 1754-6834

VL - 6

JO - Biotechnology for Biofuels

JF - Biotechnology for Biofuels

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ER -

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

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