The transcriptional program underlying the physiology of clostridial sporulation

A detailed microarray analysis of transcription during sporulation of the strict anaerobe and endospore former Clostridium acetobutylicum is presented

Anaerobic Detoxification of Acetic Acid in a Thermophilic Ethanologen

The liberation of acetate from hemicellulose negatively impacts fermentations of cellulosic biomass, limiting the concentrations of substrate that can be effectively processed. Solvent-producing bacteria have the capacity to convert acetate to the less toxic product acetone, but to the best of our knowledge, this trait has not been transferred to an organism that produces ethanol at high yield. We have engineered a five-step metabolic pathway to convert acetic acid to acetone in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum.

Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018

<p>Abstract</p> <p>Background</p> <p><it>Clostridium acetobutylicum</it>, a gram-positive and spore-forming anaerobe, is a major strain for the fermentative production of acetone, butanol and ethanol. But a previously isolated hyper-butanol producing strain <it>C. acetobutylicum </it>EA 2018 does not produce spores and has greater capability of solvent production, especially for butanol, than the type strain <it>C. acetobutylicum </it>ATCC 824.</p> <p>Results</p> <p>Complete genome of <it>C. acetobutylicum </it>EA 2018 was sequenced using Roche 454 pyrosequencing. Genomic comparison with ATCC 824 identified many variations which may contribute to the hyper-butanol producing characteristics in the EA 2018 strain, including a total of 46 deletion sites and 26 insertion sites. In addition, transcriptomic profiling of gene expression in EA 2018 relative to that of ATCC824 revealed expression-level changes of several key genes related to solvent formation. For example, <it>spo0A </it>and <it>adhEII </it>have higher expression level, and most of the acid formation related genes have lower expression level in EA 2018. Interestingly, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, might accelerate utilization of substrate xylose.</p> <p>Conclusions</p> <p>Comparative analysis of <it>C. acetobutylicum </it>hyper-butanol producing strain EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels, for the first time, provides molecular-level understanding of non-sporulation, higher solvent production and enhanced xylose utilization in the mutant EA 2018. The information could be valuable for further genetic modification of <it>C. acetobutylicum </it>for more effective butanol production.</p

A General Framework for Designing and Validating Oligomer-Based DNA Microarrays and Its Application to Clostridium acetobutylicum▿ †

While DNA microarray analysis is widely accepted as an essential tool for modern biology, its use still eludes many researchers for several reasons, especially when microarrays are not commercially available. In that case, the design, construction, and use of microarrays for a sequenced organism constitute substantial, time-consuming, and expensive tasks. Recently, it has become possible to construct custom microarrays using industrial manufacturing processes, which offer several advantages, including speed of manufacturing, quality control, no up-front setup costs, and need-based microarray ordering. Here, we describe a strategy for designing and validating DNA microarrays manufactured using a commercial process. The 22K microarrays for the solvent producer Clostridium acetobutylicum ATCC 824 are based on in situ-synthesized 60-mers employing the Agilent technology. The strategy involves designing a large library of possible oligomer probes for each target (i.e., gene or DNA sequence) and experimentally testing and selecting the best probes for each target. The degenerate C. acetobutylicum strain M5 lacking the pSOL1 megaplasmid (with 178 annotated open reading frames [genes]) was used to estimate the level of probe cross-hybridization in the new microarrays and to establish the minimum intensity for a gene to be considered expressed. Results obtained using this microarray design were consistent with previously reported results from spotted cDNA-based microarrays. The proposed strategy is applicable to any sequenced organism

Morphological and gene expression changes undergoes during exponential, transitional, and stationary phases

Growth and acid and solvent production curves as they relate to morphological and transcriptional changes during sporulation. The gray bar indicates the beginning of the transitional phase as determined by solvent production. Awith microarray sample (filled squares); A(open squares); butyrate (filled circles); butanol (filled triangles). Roman numerals correspond with those in (b), and bars and numbers along the top correspond to the clusters in (c). Morphological changes during sporulation. When stained with Syto-9 (green) and PI (red), vegetative cells take on a predominantly red color (I and II). At peak butanol production, swollen, cigar-shaped clostridial-form cells appear (arrow in III), which stain almost equally with both dyes, and persist until late stationary phase. Towards the end of solvent production (IV), endospore (arrow 1) forms are visible, and clostridial (arrow 2) forms are still present. As the culture enters late stationary phase (V and VI), cells stain almost exclusively green, regardless of morphology. All cell types are still present, including free spores (arrows in V and VI), and vegetative cells identified by their motility. Average expression profiles for each K-means cluster generated using a moving average trendline with period 3. Expression of the 814 genes (rows) at 25 timepoints (columns, hours 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66). Genes with higher expression than the reference RNA are shown in red and those with lower expression as green. Saturated expression levels: ten-fold difference.<p><b>Copyright information:</b></p><p>Taken from "The transcriptional program underlying the physiology of clostridial sporulation"</p><p>http://genomebiology.com/2008/9/7/R114</p><p>Genome Biology 2008;9(7):R114-R114.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2530871.</p><p></p

Expression profiles of sigma factors with unknown function and the effects of down-regulation

Expression profiles of CAC3267 (open triangles), CAP0167 (filled squares), and CAP0157 (open circles) as ratios compared to the first expressed timepoint. Gray bar indicates the onset of transitional phase. Expression profiles of CAC0550 (filled circles), CAC2052 (open squares), and CAC1766 (filled triangles) as ratios compared to the first expressed timepoint. Gray bar indicates the onset of transitional phase. Ranked expression intensities of the sigma factors. White denotes a rank of 1, while dark blue denotes a rank of 100 (see scale). Gray squares indicate timepoints at which the intensity did not exceed the threshold value. Microscopy time-course of asRNA strains compared to WT and plasmid control strains. Microscopy samples from WT (I) and pSOS95del (II) cultures (as controls) and three asRNA strains taken for two timepoints over a course of 72 hours. At 72 hours, WT (I) and pSOS95del (II) exhibit the typical clostridial forms (white arrows), while asCAP0166 (III) shows advanced differentiation with forespores and endospores (orange arrows) already visible. Strains asCAP0166 (III), asCAP0167 (IV), and asCAC1766 (V) show a novel, extra-swollen clostridial form (yellow arrows).<p><b>Copyright information:</b></p><p>Taken from "The transcriptional program underlying the physiology of clostridial sporulation"</p><p>http://genomebiology.com/2008/9/7/R114</p><p>Genome Biology 2008;9(7):R114-R114.</p><p>Published online 16 Jul 2008</p><p>PMCID:PMC2530871.</p><p></p

Final Report on Development of Thermoanaerobacterium saccharolyticum for the conversion of lignocellulose to ethanol

This project addressed the need for economical technology for the conversion of lignocellulosic biomass to fuels, specifically the conversion of pretreated hardwood to ethanol. The technology developed is a set of strains of the bacterium Thermoanaerobacterium saccharolyticum and an associated fermentation process for pretreated hardwood. Tools for genetic engineering and analysis of the organism were developed, including a markerless mutation method, a complete genome sequence and a set of gene expression profiles that show the activity of its genes under a variety of conditions relevant to lignocellulose conversion. Improved strains were generated by selection and genetic engineering to be able to produce higher amounts of ethanol (up to 70 g/L) and to be able to better tolerate inhibitory compounds from pretreated hardwood. Analysis of these strains has generated useful insight into the genetic basis for desired properties of biofuel producing organisms. Fermentation conditions were tested and optimized to achieve ethanol production targets established in the original project proposal. The approach proposed was to add cellulase enzymes to the fermentation, a method called Simultaneous Saccharification and Fermentation (SSF). We had reason to think SSF would be an efficient approach because the optimal temperature and pH for the enzymes and bacterium are very close. Unfortunately, we discovered that commercially available cellulases are inactivated in thermophilic SSF by a combination of low redox potential and ethanol. Despite this, progress was made against the fermentation targets using bacterial cellulases. Thermoanaerobacterium saccharolyticum may still prove to be a commercially viable technology should cellulase enzyme issues be addressed. Moreover, the organism was demonstrated to produce ethanol at approximately theoretical yield from oligomeric hemicellulose extracts, an ability that may prove to be uniquely valuable in pretreatment configurations in which cellulose and hemicellulose are separated