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Phylogenetic, microbiological and glycoside hydrolase diversities within the extremely thermophilic, plant biomass-degrading genus Caldicellulosiruptor

ABSTRACT: 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.

ORGANISM(S): Caldicellulosiruptor bescii Caldicellulosiruptor owensensis Caldicellulosiruptor saccharolyticus Caldicellulosiruptor hydrothermalis Caldicellulosiruptor acetigenus Caldicellulosiruptor kronotskyensis

PROVIDER: GSE23606 | GEO | 2010/11/29

SECONDARY ACCESSION(S): PRJNA129969

REPOSITORIES: GEO

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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: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.

Project description:Background (Pseudo)Bacteroides cellulosolvens is a cellulolytic bacterium producing the most extensive and intricate cellulosomal system known in nature. Recently, an elaborate architecture of B. cellulosolvens cellulosomal system was revealed from its genome sequence analysis, and first evidence on the interactions between its structural and enzymatic components were detected in vitro. Yet, the cellulolytic potential of the bacterium in carbohydrate deconstruction may reside only within complete high-molecular weight protein complexes, which are secreted from the bacterium. Results The current proteome-wide work reveals patterns of protein expression of various cellulosomal components, and explores the differential expression signature upon bacterial growth two carbon sources, either cellobiose or microcrystalline cellulose. Mass spectrometry analysis of the bacterial secretome fraction revealed the expression of 24 scaffoldin structural units and 166 dockerin-bearing enzymes, in addition to free enzymatic subunits. All these components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Various glycoside hydrolase (GH) family memebers were represented among 102 carbohydrate-degrading enzymes, including the most abundant GH48 exoglucanase. Specific cellulosomal components were associatred with different carbon sources, in cellulosomal fractions of different molecular weights. Overall, microcrystalline cellulose-derived cellulosomes showed higher expression levels of the structural and enzymatic components, and exhibited the highest degradation activity on five different carbohydrates. The cellulosomal activity of B. cellulosolvens showed high degradation rates that are very promising in biotechnological terms and were compatible with the activity levels exhibited by C. thermocellum purified cellulosomes. Conclusions The current research demonstrates the involvement of key cellulosomal factors participating in the mechanism of carbohydrate degradation by B. cellulosolvens. The powerful ability of the bacterium to exhibit different degradation strategies of various carbon sourcesis revealed. Thus, a novel components reservoir of degradation machineries that may serve for subsequent cellulosomal research, as a pool for designing new cellulolytic cocktails for biotechnological purposes.

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Phylogenetic, microbiological and glycoside hydrolase diversities within the extremely thermophilic, plant biomass-degrading genus Caldicellulosiruptor

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