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Clostridium thermocellum transcriptomic profiles after exposure to furfural or heat stress.

ABSTRACT: BACKGROUND:The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It is capable of both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. A previous C. thermocellum ethanol stress study showed that the largest transcriptomic response was in genes and proteins related to nitrogen uptake and metabolism. RESULTS:In this study, C. thermocellum was grown to mid-exponential phase and treated with furfural or heat to a final concentration of 3 g.L-1 or 68°C respectively to investigate general and specific physiological and regulatory stress responses. Samples were taken at 10, 30, 60 and 120 min post-shock, and from untreated control fermentations, for transcriptomic analyses and fermentation product determinations and compared to a published dataset from an ethanol stress study. Urea uptake genes were induced following furfural stress, but not to the same extent as ethanol stress and transcription from these genes was largely unaffected by heat stress. The largest transcriptomic response to furfural stress was genes for sulfate transporter subunits and enzymes in the sulfate assimilatory pathway, although these genes were also affected late in the heat and ethanol stress responses. Lactate production was higher in furfural treated culture, although the lactate dehydrogenase gene was not differentially expressed under this condition. Other redox related genes such as a copy of the rex gene, a bifunctional acetaldehyde-CoA/alcohol dehydrogenase and adjacent genes did show lower expression after furfural stress compared to the control, heat and ethanol fermentation profiles. Heat stress induced expression from chaperone related genes and overlap was observed with the responses to the other stresses. This study suggests the involvement of C. thermocellum genes with functions in oxidative stress protection, electron transfer, detoxification, sulfur and nitrogen acquisition, and DNA repair mechanisms in its stress responses and the use of different regulatory networks to coordinate and control adaptation. CONCLUSIONS:This study has identified C. thermocellum gene regulatory motifs and aspects of physiology and gene regulation for further study. The nexus between future systems biology studies and recently developed genetic tools for C. thermocellum offers the potential for more rapid strain development and for broader insights into this organism's physiology and regulation.

SUBMITTER: Wilson CM

PROVIDER: S-EPMC3848806 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Clostridium thermocellum transcriptomic profiles after exposure to furfural or heat stress.

Wilson Charlotte M CM Yang Shihui S Rodriguez Miguel M Ma Qin Q Johnson Courtney M CM Dice Lezlee L Xu Ying Y Brown Steven D SD

Biotechnology for biofuels 20130912 1

<h4>Background</h4>The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It is capable of both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. Intolerance to stresses routinely encountered during industrial fermentations may hinder the commercial development of this organism. A previous C. thermocellum ethanol stress study showed that the largest transcriptomic response w ...[more]

PMID: 24028713

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Project description:The thermophilic anaerobe Clostridium thermocellum is a candidate consolidated bioprocessing (CBP) biocatalyst for cellulosic ethanol production. It expresses enzymes for both cellulose solubilization and its fermentation to produce lignocellulosic ethanol. To gain insights into the C. thermocellum genes, using an updated version of the C. thermocellum ATCC 27405 genome annotation, that are required for specific growth on the cellulosic feedstocks of either pretreated switchgrass or Populus, duplicate fermentations were conducted with a 5 g/L solid substrate loading. High quality RNA was extracted using a method we report for C. thermocellum grown on solid substrates. Transcriptome profiles were obtained at two time points during actively growing fermentations (12 h and 37 h post inoculation). A comparison of two transcriptomic analytical techniques, microarray and RNAseq, was performed and the data analyzed for statistical significance. When thresholds for genes passing significance of FDR>0.05 were applied, microarray (2351 genes) had a greater number of significant genes relative to RNA-seq (280 genes when normalized by KDMM). When a 2-fold difference in expression threshold was applied, seventy-three genes were significantly differentially expressed in common between the two techniques. We identified genes differentially expressed when C. thermocellum ATC 27405 was grown on the two biomass substrates, with two putative efflux/transport systems highly differentially regulated (>5-fold). This study has revealed consistency between these two transcriptomics analytical platforms that gives confidence in our switch from the DNA microarray platform to an RNAseq based platform for routine transcriptomics analyses. To gain insights into the C. thermocellum genes, using an updated version of the C. thermocellum ATCC 27405 genome annotation, that are required for specific growth on the cellulosic feedstocks of either pretreated switchgrass or Populus, duplicate fermentations were conducted with a 5 g/L solid substrate loading. High quality RNA was extracted using a method we report for C. thermocellum grown on solid substrates. Transcriptome profiles were obtained at two time points during actively growing fermentations (12 h and 37 h post inoculation). A comparison of two transcriptomic analytical techniques, microarray and RNAseq, was performed and the data analyzed for statistical significance.

Project description:Background: The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results: A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions: Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligo-saccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cells’ movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered.

Project description:Background: The ability of Clostridium thermocellum ATCC 27405 wild-type strain to hydrolyze cellulose and ferment the degradation products directly to ethanol and other metabolic byproducts makes it an attractive candidate for consolidated bioprocessing of cellulosic biomass to biofuels. In this study, whole-genome microarrays were used to investigate the expression of C. thermocellum mRNA during growth on crystalline cellulose in controlled replicate batch fermentations. Results: A time-series analysis of gene expression revealed changes in transcript levels of ~40% of genes (~1300 out of 3198 ORFs encoded in the genome) during transition from early-exponential to late-stationary phase. K-means clustering of genes with statistically significant changes in transcript levels identified six distinct clusters of temporal expression. Broadly, genes involved in energy production, translation, glycolysis and amino acid, nucleotide and coenzyme metabolism displayed a decreasing trend in gene expression as cells entered stationary phase. In comparison, genes involved in cell structure and motility, chemotaxis, signal transduction and transcription showed an increasing trend in gene expression. Hierarchical clustering of cellulosome-related genes highlighted temporal changes in composition of this multi-enzyme complex during batch growth on crystalline cellulose, with increased expression of several genes encoding hydrolytic enzymes involved in degradation of non-cellulosic substrates in stationary phase. Conclusions: Overall, the results suggest that under low substrate availability, growth slows due to decreased metabolic potential and C. thermocellum alters its gene expression to (i) modulate the composition of cellulosomes that are released into the environment with an increased proportion of enzymes than can efficiently degrade plant polysaccharides other than cellulose, (ii) enhance signal transduction and chemotaxis mechanisms perhaps to sense the oligo-saccharide hydrolysis products, and nutrient gradients generated through the action of cell-free cellulosomes and, (iii) increase cellular motility for potentially orienting the cellsM-bM-^@M-^Y movement towards positive environmental signals leading to nutrient sources. Such a coordinated cellular strategy would increase its chances of survival in natural ecosystems where feast and famine conditions are frequently encountered. Total RNA was extracted from the cell pellets and the reverse transcribed cDNA was hybridized to oligo-arrays containing duplicated probes representing ~90% of the annotated ORFs in C. thermocellum ATCC27405 genome.Dual-channel dye swap experimental design was used to analyze the time-course of gene expression during cellulose fermentation using two biological replicate fermentation. The 6hr sample as the reference, to which all other time-point samples (8, 10, 12, 14, 16hr) were compared.

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Clostridium thermocellum transcriptomic profiles after exposure to furfural or heat stress.

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Clostridium thermocellum transcriptomic profiles after exposure to furfural or heat stress.

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