Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyrate posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butanol.
Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyric acid posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butyric acid.
Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyrate posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butanol. C. acetobutylicum cultures were grown, three biological replicates, to mid-exponential phase and then stressed with four levels of butanol (0 mM - No stress; 30 mM - Low stress; 60 mM - Medium stress; & 90 mM - High stress). Samples were collected following the stress at 0, 15, 30, 45, 60 and 75 min, post stress. These sampling times, which are of the order of the doubling time of these cells, were meant to capture largely the direct and immediate impact of these stresses on gene expression. The RNA extracted from two biological replicates were used for microarray hybridization foloowing cDNA generationa nd labelling using Agilent 44K arrays, while the third was used for q-RT-PCR validation. For each stress level, 6 time points with 2 biological replicates and dye swaps (Cy3/Cy5) were prepared for comparison. The hybridization was perfomed against an equal amount of oppositely labeled cDNA from common reference pool prepared using equal amounts of labeled cDNA from all four stress levels.
Project description:Clostridium acetobutylicum is a typical bacterium of major importance to industrial butanol production. In order to dissect the regulatory network pertaining to the industrial application of this bacterium, catabolite control protein A (CcpA) was investigated for its global function by DNA microarray.It showed that CcpA of C. acetobutylicum controls hundreds of genes, not only carbon metabolism, but also solvent production and sporulation in the life cycle.The results here demonstrated that CcpA is an important pleiotropic regulator related to some specific physiological and biochemical process in butanol-producing C. acetobutylicum.
Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyric acid posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butyric acid. C. acetobutylicum cultures were grown, three biological replicates, to mid-exponential phase and then stressed with four levels of butyric acid (0 mM - No stress; 30 mM - Low stress; 40 mM - Medium stress; & 50 mM - High stress). Butyric acid was pH adjusted with 10M KOH to match the pH of the cultures, prior to addition. Samples were collected following the stress at 0, 15, 30, 45, 60 and 75 min, post stress. These sampling times, which are of the order of the doubling time of these cells, were meant to capture largely the direct and immediate impact of these stresses on gene expression. The RNA extracted from two biological replicates were used for microarray hybridization foloowing cDNA generation and labelling using Agilent 44K arrays, while the third was used for q-RT-PCR validation. For each stress level, 6 time points with 2 biological replicates and dye swaps (Cy3/Cy5) were prepared for comparison. The hybridization was perfomed against an equal amount of oppositely labeled cDNA from common reference pool prepared using equal amounts of labeled cDNA from all four stress levels.
Project description:Clostridium acetobutylicum, the endospore-forming anaerobe best known for its ABE (acetone-butanol-ethanol) fermentation, has received renewed attention recently for the biological production of butanol, both for bulk chemical production and as a potential biofuel. With butanol production in mind, most of the recent research on C. acetobutylicum has focused on increasing butanol production, tolerance to butanol, and optimizing it for various substrates. However, an equally important trait, though less understood, is its sporulation program, which it coupled to solvent formation. The model organism for endospore formation is Bacillus subtilis, but significant physiological, metabolic, and genomic differences exist between the two organisms. Despite these differences, the major sporulation-related transcription/sigma factors are conserved between the two species. These transcription/sigma factors are activated in a cascade manner such that Spo0A becomes active first, followed by σF, then σE, then σG, and finally σK. The goal of this study is to determine the regulons of 4 of these transcription/sigma factors (Spo0A, σF, σE, and σG) and compare them to those in B. subtilis. To accomplish this goal, individual mutant strains were created for Spo0A, σF, σE, and σG, in which the transcription/sigma factor is silenced. These mutants were then compared transcriptionally using microarrays to determine the regulon of each transcription/sigma factor. To help avoid false positives, comparisons were made between strains in which the downstream transcription/sigma factor is silenced (e.g., for the Spo0A regulon, the Spo0A mutant was compared against the σF mutant and the σE mutant, since they are both upregulated by Spo0A) rather than just the WT. For each regulon, 4 timepoints were taken, since it is very difficult to synchronize sporulation in C. acetobutylicum cultures, and dye-swaps were prepared for each timepoint.
Project description:Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, but such effects are not well understood at the genomic level. Using DNA microarrays, the Clostridium acetobutylicum transcriptional stress response to butanol was analyzed. Keywords: stress response
Project description:Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, with a variety of genetic resistance mechanisms previously identified. Using random genomic libraries and DNA microarrays, library inserts were preferentially enriched by culturing in media containing butanol, with successive transfers into fresh media containing incrementally increasing butanol concentrations. Keywords: genomic library preferential enrichment
Project description:Clostridium acetobutylicum is a typical bacterium of major importance to industrial butanol production. In order to dissect the regulatory network pertaining to the industrial application of this bacterium, catabolite control protein A (CcpA) was investigated for its global function by DNA microarray.It showed that CcpA of C. acetobutylicum controls hundreds of genes, not only carbon metabolism, but also solvent production and sporulation in the life cycle.The results here demonstrated that CcpA is an important pleiotropic regulator related to some specific physiological and biochemical process in butanol-producing C. acetobutylicum. In order to enable a global understanding of the regulatory roles of CcpA when fermenting mixed sugars, which is of great significance in utilization of lignocellulosic hydrolysates, D-glucose plus D-xylose were used as the carbon sources in fermentation for microarray analysis. Microarray analysis was performed at four time points:the time point M and L were chosen both in acidogenic phase, while the time point T and S were chosen in shift phase (from acidogenesis to solventogenesis) and solventogenic phase, respectively.One-color microarray assays were performed.Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US). The ratio of transcript level between wildtype and mutant can been achieved using the formula: 2^(value of wildtype)/2^(value of ccpA mutant).