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Transcriptional analysis of the response of Caldicellulosiruptor saccharolyticus to different H2 partial pressures

ABSTRACT: Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments a broad range of substrates to mainly acetate, CO2, and hydrogen gas (H2). Its high hydrogen-producing capacity make this bacterium an attractive candidate for microbial biohydrogen production. However, increased H2 levels tend to inhibit hydrogen formation and leads to the formation of other reduced end products like lactate and ethanol. To investigate the organism’s strategy for dealing with elevated H2 levels and to identify alternative pathways involved in the disposal of the reducing equivalents, the effect of the hydrogen partial pressure (PH2) on fermentation performance was studied. For this purpose cultures were grown under high and low PH2 in a glucose limited chemostat setup. Transcriptome analysis revealed the up-regulation of genes involved in the disposal of reducing equivalents under high PH2, like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings were in line with the observed shift in fermentation profiles from acetate production under low PH2 to a mixed production of acetate, lactate and ethanol under high PH2. In addition, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. The presented transcription data provides experimental evidence for the involvement of the redox sensing Rex protein in gene regulation under high PH2 cultivation conditions. Overall, these findings indicate that the PH2 dependent changes in the fermentation pattern of C. saccharolyticus are, in addition to the known regulation at the enzyme/metabolite level, also regulated at the transcription level.

ORGANISM(S): Caldicellulosiruptor saccharolyticus

PROVIDER: GSE40430 | GEO | 2013/08/28

SECONDARY ACCESSION(S): PRJNA173931

REPOSITORIES: GEO

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Project description:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments a broad range of substrates to mainly acetate, CO2, and hydrogen gas (H2). Its high hydrogen-producing capacity make this bacterium an attractive candidate for microbial biohydrogen production. However, increased H2 levels tend to inhibit hydrogen formation and leads to the formation of other reduced end products like lactate and ethanol. To investigate the organismM-bM-^@M-^Ys strategy for dealing with elevated H2 levels and to identify alternative pathways involved in the disposal of the reducing equivalents, the effect of the hydrogen partial pressure (PH2) on fermentation performance was studied. For this purpose cultures were grown under high and low PH2 in a glucose limited chemostat setup. Transcriptome analysis revealed the up-regulation of genes involved in the disposal of reducing equivalents under high PH2, like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings were in line with the observed shift in fermentation profiles from acetate production under low PH2 to a mixed production of acetate, lactate and ethanol under high PH2. In addition, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. The presented transcription data provides experimental evidence for the involvement of the redox sensing Rex protein in gene regulation under high PH2 cultivation conditions. Overall, these findings indicate that the PH2 dependent changes in the fermentation pattern of C. saccharolyticus are, in addition to the known regulation at the enzyme/metabolite level, also regulated at the transcription level. Two conditions: low H2 partial pressure and high H2 partial pressure, both at steady state growth were harvested for a dye-flip microarray experimental design. Biological replicates were harvested for both conditions and combined prior to cDNA synthesis. Both conditions were labeled with cy3 and cy5 dyes allowing for a technical replicate of hybridization in addition to the biological replicates.

Project description:Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO2 and H2. C. bescii, C. kronotskyensis and C. saccharolyticus solubilized 38%, 36% and 29% (by weight) of unpretreated switchgrass (5 g/l), repectively, which was about half of the concentration of crystalline cellulose (Avicel, 5 g/l) that was solubilized under the same conditions. The lower yields with C. saccharolyticus were unexpected, given that its genome encodes the same GH9-GH48 multi-domain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing Cellulose to switchgrass showed that many carbohydrate ABC transporters and multi-domain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagella biosynthesis were up-regulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation. A dye swap was completed with each of three Caldicellulosiruptor species: C. bescii, C. kronotskyensis and C. saccharolyticus growing on cellulose and switchgrass. Half of the RNA sample for one condition was labeled with Cy3 and the other half Cy5. The two differentially labeled samples were run on two different slides and analyzed to investigate differences in transcription during growth on cellulose and switchgrass.

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Transcriptional analysis of the response of Caldicellulosiruptor saccharolyticus to different H2 partial pressures

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