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Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq.

ABSTRACT: BACKGROUND: Clostridium beijerinckii is an important solvent producing microorganism. The genome of C. beijerinckii NCIMB 8052 has recently been sequenced. Although transcriptome structure is important in order to reveal the functional and regulatory architecture of the genome, the physical structure of transcriptome for this strain, such as the operon linkages and transcript boundaries are not well understood. RESULTS: In this study, we conducted a single-nucleotide resolution analysis of the C. beijerinckii NCIMB 8052 transcriptome using high-throughput RNA-Seq technology. We identified the transcription start sites and operon structure throughout the genome. We confirmed the structure of important gene operons involved in metabolic pathways for acid and solvent production in C. beijerinckii 8052, including pta-ack, ptb-buk, hbd-etfA-etfB-crt (bcs) and ald-ctfA-ctfB-adc (sol) operons; we also defined important operons related to chemotaxis/motility, transcriptional regulation, stress response and fatty acids biosynthesis along with others. We discovered 20 previously non-annotated regions with significant transcriptional activities and 15 genes whose translation start codons were likely mis-annotated. As a consequence, the accuracy of existing genome annotation was significantly enhanced. Furthermore, we identified 78 putative silent genes and 177 putative housekeeping genes based on normalized transcription measurement with the sequence data. We also observed that more than 30% of pseudogenes had significant transcriptional activities during the fermentation process. Strong correlations exist between the expression values derived from RNA-Seq analysis and microarray data or qRT-PCR results. CONCLUSIONS: Transcriptome structural profiling in this research provided important supplemental information on the accuracy of genome annotation, and revealed additional gene functions and regulation in C. beijerinckii.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3271303 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq.

Wang Yi Y Li Xiangzhen X Mao Yuejian Y Blaschek Hans P HP

BMC genomics 20110930

<h4>Background</h4>Clostridium beijerinckii is an important solvent producing microorganism. The genome of C. beijerinckii NCIMB 8052 has recently been sequenced. Although transcriptome structure is important in order to reveal the functional and regulatory architecture of the genome, the physical structure of transcriptome for this strain, such as the operon linkages and transcript boundaries are not well understood.<h4>Results</h4>In this study, we conducted a single-nucleotide resolution anal ...[more]

PMID: 21962126

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Project description:BACKGROUND: Clostridium beijerinckii is a prominent solvent-producing microbe that has great potential for biofuel and chemical industries. Although transcriptional analysis is essential to understand gene functions and regulation and thus elucidate proper strategies for further strain improvement, limited information is available on the genome-wide transcriptional analysis for C. beijerinckii. RESULTS: The genome-wide transcriptional dynamics of C. beijerinckii NCIMB 8052 over a batch fermentation process was investigated using high-throughput RNA-Seq technology. The gene expression profiles indicated that the glycolysis genes were highly expressed throughout the fermentation, with comparatively more active expression during acidogenesis phase. The expression of acid formation genes was down-regulated at the onset of solvent formation, in accordance with the metabolic pathway shift from acidogenesis to solventogenesis. The acetone formation gene (adc), as a part of the sol operon, exhibited highly-coordinated expression with the other sol genes. Out of the > 20 genes encoding alcohol dehydrogenase in C. beijerinckii, Cbei_1722 and Cbei_2181 were highly up-regulated at the onset of solventogenesis, corresponding to their key roles in primary alcohol production. Most sporulation genes in C. beijerinckii 8052 demonstrated similar temporal expression patterns to those observed in B. subtilis and C. acetobutylicum, while sporulation sigma factor genes sigE and sigG exhibited accelerated and stronger expression in C. beijerinckii 8052, which is consistent with the more rapid forespore and endspore development in this strain. Global expression patterns for specific gene functional classes were examined using self-organizing map analysis. The genes associated with specific functional classes demonstrated global expression profiles corresponding to the cell physiological variation and metabolic pathway switch. CONCLUSIONS: The results from this work provided insights for further C. beijerinckii strain improvement employing system biology-based strategies and metabolic engineering approaches.

Project description:BACKGROUND:Butanol (n-butanol) has high values as a promising fuel source and chemical feedstock. Biobutanol is usually produced by the solventogenic clostridia through a typical biphasic (acidogenesis and solventogenesis phases) acetone-butanol-ethanol (ABE) fermentation process. It is well known that the acids produced in the acidogenic phase are significant and play important roles in the switch to solventogenesis. However, the mechanism that triggers the metabolic switch is still not clear. RESULTS:Sodium butyrate (40 mM) was supplemented into the medium for the ABE fermentation with Clostridium beijerinckii NCIMB 8052. With butyrate addition (reactor R1), solvent production was triggered early in the mid-exponential phase and completed quickly in?<?50 h, while in the control (reactor R2), solventogenesis was initiated during the late exponential phase and took?>?90 h to complete. Butyrate supplementation led to 31% improvement in final butanol titer, 58% improvement in sugar-based yield, and 133% improvement in butanol productivity, respectively. The butanol/acetone ratio was 2.4 versus 1.8 in the control, indicating a metabolic shift towards butanol production due to butyrate addition. Genome-wide transcriptional dynamics was investigated with RNA-Seq analysis. In reactor R1, gene expression related to solventogenesis was induced about 10 hours earlier when compared to that in reactor R2. Although the early sporulation genes were induced after the onset of solventogenesis in reactor R1 (mid-exponential phase), the sporulation events were delayed and uncoupled from the solventogenesis. In contrast, in reactor R2, sporulation genes were induced at the onset of solventogenesis, and highly expressed through the solventogenesis phase. The motility genes were generally down-regulated to lower levels prior to stationary phase in both reactors. However, in reactor R2 this took much longer and gene expression was maintained at comparatively higher levels after entering stationary phase. CONCLUSIONS:Supplemented butyrate provided feedback inhibition to butyrate formation and may be re-assimilated through the reversed butyrate formation pathway, thus resulting in an elevated level of intracellular butyryl phosphate, which may act as a phosphate donor to Spo0A and then trigger solventogenesis and sporulation events. High-resolution genome-wide transcriptional analysis with RNA-Seq revealed detailed insights into the biochemical effects of butyrate on solventogenesis related-events at the gene regulation level.

Project description:Furfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulosic biomass to monomeric sugars, but the molecular response of Clostridium beijerinckii NCIMB 8052 to this compound is unknown. To discern the effect of furfural on C. beijerinckii and to gain insights into the molecular mechanisms of action and detoxification, we studied the physiological changes of furfural-stressed cultures during acetone-butanol-ethanol (ABE) fermentation, and profiled differentially expressed genes by genome-wide transcriptional analysis. C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control. The growth and ABE by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase. By comparing gene expression of furfural-stressed cultures to that of the unstressed control, at both the acidogenic and solventogenic phases, we ascertained that furfural induces expression of several genes including those that code for heat shock proteins, redox enzymes and cofactor associated proteins, and ATP-binding cassette transporters, and represses genes belonging to the phosphotransferase system, two-component system, chemotaxis and cell motility. Based on these results, we discuss the underpinning for furfural-mediated change in ABE fermentation by the solventogenic Clostridium species. C. beijerinckii 8052 pre-culture was incubated anaerobically to attain acidogenic or solventogenic growth phase. The culture was then subdivided into two bottles. One bottle was challenged with furfural and the other bottle was left unchallenged. After 3 h growth, C. beijerinckii 8052 samples were collected, during which the original concentration of furfural in the growth medium was reduced to more than half. Total RNA was isolated, purified, converted to enriched mRNA, and dye-coupled (Alexa Fluor 555) complementary cRNA. This was followed by hybridization and microarray data analysis.

Project description:BackgroundSolventogenic clostridia offer a sustainable alternative to petroleum-based production of butanol--an important chemical feedstock and potential fuel additive or replacement. C. beijerinckii is an attractive microorganism for strain design to improve butanol production because it (i) naturally produces the highest recorded butanol concentrations as a byproduct of fermentation; and (ii) can co-ferment pentose and hexose sugars (the primary products from lignocellulosic hydrolysis). Interrogating C. beijerinckii metabolism from a systems viewpoint using constraint-based modeling allows for simulation of the global effect of genetic modifications.ResultsWe present the first genome-scale metabolic model (iCM925) for C. beijerinckii, containing 925 genes, 938 reactions, and 881 metabolites. To build the model we employed a semi-automated procedure that integrated genome annotation information from KEGG, BioCyc, and The SEED, and utilized computational algorithms with manual curation to improve model completeness. Interestingly, we found only a 34% overlap in reactions collected from the three databases--highlighting the importance of evaluating the predictive accuracy of the resulting genome-scale model. To validate iCM925, we conducted fermentation experiments using the NCIMB 8052 strain, and evaluated the ability of the model to simulate measured substrate uptake and product production rates. Experimentally observed fermentation profiles were found to lie within the solution space of the model; however, under an optimal growth objective, additional constraints were needed to reproduce the observed profiles--suggesting the existence of selective pressures other than optimal growth. Notably, a significantly enriched fraction of actively utilized reactions in simulations--constrained to reflect experimental rates--originated from the set of reactions that overlapped between all three databases (P = 3.52 × 10-9, Fisher's exact test). Inhibition of the hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed.ConclusionsMicrobial production of butanol by C. beijerinckii offers a promising, sustainable, method for generation of this important chemical and potential biofuel. iCM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production.

Dataset Information

Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq.

Publications

Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq.

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