Transcription profiling by array of Clostridium acetobutylicum on glucose and xylose substrates under exponential, continuous and diauxie growth.
ABSTRACT: Clostridium acetobutylicum was grown in a batch-culture with minimal medium containing glucose and xylose as substrate. Diauxie growth was observed after glucose was consumed. Following the organism grows on xylose. Transcriptional analysis was done to pursue the cellular processes during the switch from growth on glucose to growth on xylose. We compared DNA-Microarray data from cells grown during the exponential phase on glucose (A), with cells growing during the start of diauxie growth lag (B), during the end of diauxie growth lag (C) and during exponential growth on xylose (D). We used cells grown in a continuous culture with glucose as substrate as common reference for the samples A-D.
Project description:In order to identify genes which contribute on the uptake of glucose into cells of the mutant R. eutropha G+1, a genome wide transcription analyses was done. Transcripts of strain H16 and the glucose-utilizing mutant R. eutropha G+1, cultivated in mineral salts medium supplemented with either fructose or glucose were compared.
Project description:Oxidative stress is harmful for organism and occurs when the cells exposed to superoxid, hydrogen peroxide and alkylhydroperoxides. In microorganism, the glutathione- and thioredoxin-dependent reduction systems are universal and play an important role in response to defending oxidative stress. The _-glutamylcysteine synthetase (_-GCS) is an essential enzyme to biosynthesize the tripeptide glutathione (GSH) in organism. Similarly, thioredoxin reductase is an important enzyme in thioredoxin-dependent reduction system. In Clostridium acetobutylicum, the _-glutamylcysteine synthetase (encoded by CAC1539, gcs) and thioredoxin reductase (encoded by CAC1548, trxB) were inactivated using ClosTron technology. The gcs mutant grew insufficiently and consumed less glucose in the phosphate-limited continuous culture and exhibited more sensitive to oxidative stress. The trxB mutant just exhibited lower growth rate and less glucose uptake in the solventogenic phase, compared to wild type. The DNA microarrays were performed to investigate the transcripome difference between wild type and the mutants. In gcs mutant, the genes related to chemotaxis and flagella biosynthesis proteins were induced significantly and in the trxB mutant, the sporulation genes were induced largely. Based on the phenotypes and transcriptome comparison results, the relationship between GSH- and Trx-dependent induction systems was discussed in Clostridium acetobutylicum.
Project description:Alkaline hemicellulytic bacteria Bacillus sp. N16-5 has abroad substrate spectrum and exhibits great growth ability on complex carbohydrates. In order to get insight into its carbohydrate utilization mechanism, global transcriptional profiles were separately determined for growth on glucose, fructose, mannose, galactose, arabinose, xylose, galactomannan, xylan, pectin and carboxymethyl cellulose by using one-color microarrays. Substrate induced gene expression was measured when culture was grown on glucose, fructose, mannose, galactose, arabinose, xylose, galactomannan, xylan and CMC to mid-logarithmic phase.
Project description:Clostridium acetobutylicum is a Gram-positive, endospore-forming bacterium that is considered as a strict anaerobe. It ferments sugars to the organic acids acetate and butyrate or shifts to formation of the solvents - ethanol, butanol and acetone. In most bacteria the major regulator of iron homeostasis is Fur (ferric uptake regulator). Analysis of the genome of Clostridium acetobutylicum has revealed three genes encoding Fur-like proteins. The amino acid sequece of one of them showed 70% similarity to the Fur protein of the closely related Bacillus subtilis.<br>Thus, to gain insight into the role of Fur and the mechanisms for maintenance of iron homeostasis in this strict anaerobic organism, we determined its transcriptional profile in response to iron limitation and inactivation of fur.
Project description:The batch fermentation of Clostridium acetobutylicum is characterized by an acetogenic growth phase during exponential growth when mainly acetate and butyrate are fermentation products. Then, at the end of exponential growth and during stationary phase, the organism switches to solventogenic growth and large amounts of acetone, ethanol and butanol are produced. These growth phases can be studied independent from each other in a phosphate-limited continuous culture. In transcription analysis of continuous cultures using DNA microarrays it became evident that, among others, operons involved in sulfur assimilation are strongly up-regulated during solventogenesis. Using the ClosTron technique we constructed two knock-out mutants in the genes CAC0105 and CAC0930 annotated as involved in sulfate reduction and cysteine biosynthesis. Complementation experiments were carried out with sulfite and cysteine to prove the predicted function. The fermentation experiments of wild type and mutants using phosphate-limited and sulfur-limited continuous culture demonstrated that less sulfur source was consumed in solventogenic phase and the efficiency of cysteine uptake became lower. DNA microarrays were performed to study the difference of transcriptional expression when the wild type was challenged with insufficient sulfur source and the mccB (CAC0930) mutant was inactivated in the continuous culture. The result provided insights into understanding the sulfur metabolism regulatory.
Project description:Background: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of fuels. To release the major fermentable sugars such as glucose and xylose, pretreatment and enzymatic hydrolysis of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and eventually the production rate and yield. Acetate is one of the major inhibitors liberated from hemicelluloses during dilute acid pretreatment. An understanding of the toxic effects of acetate on the fermentation microorganism is critical to improving biofuel yields in the process. In addition, the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results: We have observed previously that some pretreatment inhibitors affect growth and performance in Zymomonas mobilis 8b differently when different sugars (e.g. glucose or xylose) are used as substrate. To investigate this phenomenon at the cellular level, microarray technology was used to investigate the acetate stress responses of Z. mobilis 8b when using single carbon sources of glucose or xylose, and mixed sugars of glucose and xylose. We designed a microarray based on the most up-to-date genome annotation for both coding sequences and intergenic regions. In the presence of acetate, 8b still can utilize all the glucose (though xylose utilization was inhibited) with similar ethanol yield although the growth, final biomass, and ethanol production rate were reduced. The presence of acetate caused genes related to biosynthesis, flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Our result indicates that Z. mobilis utilized different mechanism for xylose utilization compared to that of glucose, with even more dramatic results than those caused by treatment of the culture with the inhibitor acetate. Our study also suggests that redox imbalance caused by stressful conditions may trigger a metabolic reaction that leads to the accumulation of toxic intermediates such as xylitol, but Z. mobilis appears to be capable of managing its carbon and energy metabolism through the control of individual reactions to overcome the inhibition caused by stressful conditions. Several target gene candidates based on transcriptomic result have been selected for genetic manipulation and a TonB-dependent receptor knockout mutant was confirmed to have advantage on acetate tolerance. Conclusions: We have gained insights into the molecular responses of the model ethanologenic bacterium Z. mobilis to the inhibitor acetate when grown in different sugar sources. These insights will facilitate future metabolic modeling studies and help further strain metabolic engineering efforts for better xylose utilization and acetate tolerance. Two series of microarray studies using total RNA extracted from Zymomonas mobilis subsp mobilis 8b (an xylose-utilizing recombinant) were carried out to investigate the effect of carbon source and acetate on Z. mobilis. One study compared the acetate effect in either glucose or xylose at exponential phase and another study investigated the acetate effect in mixed sugar of glucose and xylose at three growth phases of exponential, transition, and stationary. Tthree biological replicates were used for each condition.
Project description:Xylose induced effects on metabolism and gene expression during anaerobic growth of an engineered Saccharomyces cerevisiae on mixed glucose-xylose medium were quantified. Gene expression of S. cerevisiae harbouring an XR-XDH pathway for xylose utilisation was analysed from early cultivation when mainly glucose was metabolised, to times when xylose was co-consumed in the presence of low glucose concentrations, and finally, to glucose depletion and solely xylose being consumed. Cultivations on glucose as a sole carbon source were used as a control. Genome-scale dynamic flux balance analysis models were developed and simulated to analyse the metabolic dynamics of S. cerevisiae in the cultivations. Model simulations quantitatively estimated xylose dependent dynamics of fluxes and challenges to the metabolic network utilisation. Increased relative xylose utilisation was predicted to induce two-directionality of glycolytic flux and a redox challenge already at low glucose concentrations. Xylose effects on gene expression were observed also when glucose was still abundant. Remarkably, xylose was observed to specifically delay the glucose-dependent repression of particular genes in mixed glucose-xylose cultures compared to glucose cultures. The delay occurred during similar metabolic flux activities in the both cultures. Xylose is abundantly present together with glucose in lignocellulosic streams that would be available for the valorisation to biochemicals or biofuels. Yeast S. cerevisiae has superior characteristics for a host of the bioconversion except that it strongly prefers glucose and the co-consumption of xylose is yet a challenge. Further, since xylose is not a natural substrate of S. cerevisiae, the regulatory response it induces in an engineered yeast strain cannot be expected to have evolved for its utilisation. Dynamic cultivation experiments on mixed glucose-xylose medium having glucose cultures as control integrated with mathematical modelling allowed to resolve specific effects of xylose on the gene expression and metabolism of engineered S. cerevisiae in the presence of varying amounts of glucose. Overall design: S. cerevisiae harbouring an XR-XDH pathway for xylose is grown in two conditions, growth on D-glucose vs growth on both D-glucose and D-xylose. The strains were grown in bioreactors and samples for microarrays were taken from 5 time points during the cultivation (3.5h, 6.5h, 9.5h, 12.5h, and 24.5h). Three replicate cultures were performed for each strain. The total number of microarrays is 30. Each arrays measures 5,777 genes from Saccharomyces cerevisiae with eight 60-mer probes per gene, with three-fold technical redundancy.