Project description:Genomic analysis of the model lignocellulosic biomass degrading bacteria C. phytofermentans indicates that it can degrade, transport, and utilize a wide-range of carbohydrates as possible growth substrates. Previous experiments characterized the expression of the degradation and transport machinery using custom whole genome oligonucleotide microarrays. The results indicate that C. phytofermentans utilizes ATP-binding cassette (ABC) transporters for carbohydrate uptake and does not use the sole phosphoenolpyruvate-phosphotransferase system (PTS) for any of the tested substrates. While some ABC transporters are specific for a single carbohydrate, the expression profiles indicate that others may be capable of transporting multiple substrates. Distinct sets of Carbohydrate Active Enzymes (CAZy) genes were also up-regulated on specific substrates indicative of C. phytofermentans ability to selectively degrade plant biomass. We also identified a highly expressed cluster of genes which includes seven extracellular glycoside hydrolases and two ABC transporters with unknown specificity. These results lead to the hypothesis that when grown on plant biomass, C. phytofermentans is capable of degrading and transporting all major carbohydrate components of the plant cell. To test this, C. phytofermentans was grown on cornstover and switchgrass. Results from this expression data and HPLC analysis indicates that C. phytofermentans is utilizing multiple substrates. with multiple sugar ABC transporter clusters and glycoside hydrolases being expressed. Interestingly all of the transporters were initially identified on disaccharides or oligio-saccharides, and none of the transporters identified as monosaccharide specific transporters were expressed. This could be an indication that C. phytofermentans prefers to transport oligiosacchrides over monosaccharides. The results presented here corroborate the genomic data which indicates the breath of the carbohydrate degradation, transport, and utilization machinery of C. phytofermentans.

Project description:Genomic analysis of the model lignocellulosic biomass degrading bacteria C. phytofermentans indicates that it can degrade, transport, and utilize a wide-range of carbohydrates as possible growth substrates. Previous experiments characterized the expression of the degradation and transport machinery using custom whole genome oligonucleotide microarrays. The results indicate that C. phytofermentans utilizes ATP-binding cassette (ABC) transporters for carbohydrate uptake and does not use the sole phosphoenolpyruvate-phosphotransferase system (PTS) for any of the tested substrates. While some ABC transporters are specific for a single carbohydrate, the expression profiles indicate that others may be capable of transporting multiple substrates. Distinct sets of Carbohydrate Active Enzymes (CAZy) genes were also up-regulated on specific substrates indicative of C. phytofermentans ability to selectively degrade plant biomass. We also identified a highly expressed cluster of genes which includes seven extracellular glycoside hydrolases and two ABC transporters with unknown specificity. These results lead to the hypothesis that when grown on plant biomass, C. phytofermentans is capable of degrading and transporting all major carbohydrate components of the plant cell. To test this, C. phytofermentans was grown on cornstover and switchgrass. Results from this expression data and HPLC analysis indicates that C. phytofermentans is utilizing multiple substrates. with multiple sugar ABC transporter clusters and glycoside hydrolases being expressed. Interestingly all of the transporters were initially identified on disaccharides or oligio-saccharides, and none of the transporters identified as monosaccharide specific transporters were expressed. This could be an indication that C. phytofermentans prefers to transport oligiosacchrides over monosaccharides. The results presented here corroborate the genomic data which indicates the breath of the carbohydrate degradation, transport, and utilization machinery of C. phytofermentans. C. phytofermentans was cultured anaerobically on switchgrass and corn stover to determine specific expression patterns. The data in this series consists three independent RNA preparations from replicate cultures.

Project description:The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose.IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

Dataset Information

Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality.

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