Unknown,Transcriptomics,Genomics,Proteomics

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Comparative analysis of extremely thermophilic Caldicellulosiruptor species reveals common and differentiating cellular strategies for plant biomass utilization

ABSTRACT: 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.

ORGANISM(S): Caldicellulosiruptor kronotskyensis

SUBMITTER: Jeffrey Zurawski

PROVIDER: E-GEOD-68810 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Phylogenetic, microbiological and comparative genomic analysis was used to examine the diversity among members of the genus Caldicellulosiruptor with an eye towards the capacity of these extremely thermophilic bacteria for degrading the complex carbohydrate content of plant biomass. Seven species from this genus (C. saccharolyticus, C. bescii (formerly Anaerocellum thermophilum), C. hydrothermalis, C. owensensis, C. kronotskyensis, C. lactoaceticus, and C. kristjanssonii) were compared on the basis of 16S rRNA phylogeny and cross-species DNA-DNA hybridization to a whole genome C. saccharolyticus oligonucleotide microarray. Growth physiology of the seven Caldicellulosiruptor species on a range of carbohydrates showed that, while all could be cultivated on acid pre-treated switchgrass, only C. saccharolyticus, C. besci, C. kronotskyensis, and C. lactoaceticus were capable of hydrolyzing Whatman No. 1 filter paper. Two-dimensional gel electrophoresis of the secretomes from cells grown on microcrystalline cellulose revealed that species capable of crystalline cellulose hydrolysis also had diverse secretome fingerprints. The two-dimensional secretome of C. saccharolyticus revealed a prominent S-layer protein that appears to be also indicative of highly cellulolytic Caldicellulosiruptor species, suggesting a possible role in cell-substrate interaction. These growth physiology results were also linked to glycoside hydrolase and carbohydrate-binding module inventories for the seven bacteria, deduced from draft genome sequence information. These preliminary inventories indicated that the absence of a single glycoside hydrolase family and carbohydrate binding motif family appear to be responsible for some Caldicellulosiruptor species’ diminished cellulolytic capabilities. Overall, the genus Caldicellulosiruptor appears to contain more genomic and physiological diversity than previously reported, and is well suited for biomass deconstruction applications. Six dye-flip experiments were conducted using C. saccharolyticus genomic DNA as the reference in each dye-flip, and one of six different Caldicellulosiruptor spp. as a tester in each dye-flip

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:Thermomacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, a M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supra-normal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to up-regulation of 55 genes. Genome re-sequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism or transport. These mutations included 7 non-synonymous substitutions, 4 insertions and 1 deletion. One of the insertion mutations mapped to pseudogene, Msed_1517, and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that include the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula was naturally lacking this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low affinity high velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated spontaneous arsenate resistant mutants derived from CuR1 all underwent mutation in pitA and non-selectively became copper resistant. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. The study comprises 5 samples, described in detail below. WT_CuR1: Differential transcriptional response of Metallosphaera sedula DSM 5348, WT, to the supra-normal copper resistant spontaneous Metallosphaera sedula mutant, CuR1 under normal growth conditions. This experiment was done to analyze the differential transcription of WT cells compared with CuR1 cells at mid log phase. WT-15_CuR1-15: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 15 minutes post copper challenge. The copper cultures were harvested 15 minutes after the shock. WT-60_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 60 minutes post copper challenge. The copper cultures were harvested 60 minutes after the shock. WT-15_WT-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively. CuR1-15_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula CuR1 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively.

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Comparative analysis of extremely thermophilic Caldicellulosiruptor species reveals common and differentiating cellular strategies for plant biomass utilization

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