Caldanaerobius Polysaccharolyticus Research Articles

Biochemical and Structural Insights into Xylan Utilization by the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

Molecular Basis for the Selectivity and Specificity of Ligand Recognition by the Family 16 Carbohydrate-binding Modules from Thermoanaerobacterium polysaccharolyticum ManA

Enzymes that hydrolyze complex polysaccharides into simple sugars are modular in architecture and consist of single or multiple catalytic domains fused to targeting modules called carbohydrate-binding modules (CBMs). CBMs bind to their ligands with high affinity and increase the efficiency of the catalytic components by targeting the enzymes to its substrate. Here we utilized a multidisciplinary approach to characterize each of the two family 16 carbohydrate-binding domain components of the highly active mannanase from the thermophile Thermoanaerobacterium polysaccharolyticum. These represent the first crystal structures of family 16 CBMs. Calorimetric analysis showed that although these CBMs demonstrate high specificity toward beta-1,4-linked sugars, they can engage both cello- and mannopolysaccharides. To elucidate the molecular basis for this specificity and selectivity, we have determined high resolution crystal structures of each of the two CBMs, as well as of binary complexes of CBM16-1 bound to either mannopentaose or cellopentaose. These results provide detailed molecular insights into ligand recognition and yield a framework for rational engineering experiments designed to expand the natural repertoire of these targeting modules.

Description of Caldanaerobius fijiensis gen. nov., sp. nov., an inulin-degrading, ethanol-producing, thermophilic bacterium from a Fijian hot spring sediment, and reclassification of Thermoanaerobacterium polysaccharolyticum and Thermoanaerobacterium zeae as Caldanaerobius polysaccharolyticus comb. nov. and Caldanaerobius zeae comb. nov.

An obligately anaerobic, spore-forming, Gram-type-positive but Gram-staining-negative thermophilic bacterium, strain JW/YJL-F3(T), was isolated from a Fijian hot spring sediment sample. Cells of strain JW/YJL-F3(T) were straight to slightly curved rods, 0.5-1.2 microm in diameter and 1.5-19 microm long. The temperature range for growth was between 40 and 67 degrees C, with an optimum at 60-63 degrees C. The pH(25 degrees C) range for growth was 4.5-8.4 with an optimum of 6.8. The salinity range for growth was 0-0.5 %. Strain JW/YJL-F3(T) utilized a range of substrates including arabinose, cellobiose, galactose, glucose, inulin, lactose, maltose, mannose, raffinose, ribose, trehalose, xylose and yeast extract as carbon and energy sources. The major fermentation end products from glucose were ethanol, acetate and formate. Strain JW/YJL-F3(T) converted thiosulfate to elemental sulfur, producing sulfur globules. The DNA G+C content was 37.6 mol% as determined by HPLC. Phylogenetic analysis using the 16S rRNA gene sequence indicated that the isolate is distantly related to the clade of the genus Thermoanaerobacterium. However, Thermoanaerobacterium polysaccharolyticum (96.7 % similarity to the type strain) and Thermoanaerobacterium zeae were the closest relatives, forming a separate, well-supported clade together with the novel isolate. Because Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae and strain JW/YJL-F3(T) have different features from other Thermoanaerobacterium species, including a higher G+C content and formate production, and are placed distantly from the remaining species of Thermoanaerobacterium (greater than 10 % distance) in the 16S rRNA gene sequence analysis, we propose to place the new isolate JW/YJL-F3(T) and Thermoanaerobacterium polysaccharolyticum and Thermoanaerobacterium zeae into the novel genus Caldanaerobius gen. nov. as Caldanaerobius fijiensis gen. nov., sp. nov. (the type species), Caldanaerobius polysaccharolyticus comb. nov. and Caldanaerobius zeae comb. nov., respectively. The type strain of Caldanaerobius fijiensis is JW/YJL-F3(T) (=ATCC BAA-1278(T) =DSM 17918(T)).

Molecular characterization and utilization of the CAK1 filamentous viruslike particle derived from Clostridium beijerinckii

An examination of the replication origin and stability determinant associated with the CAK1 filamentous viruslike particle recovered from Clostridium beijerinckii NCIMB 6444 was carried out. Seven deletion derivatives, pCKE, pCEP1, pDT5, pCKP, pDTH102, pYL102E and pYL102, were constructed and transformed into C. beijerinckii NCIMB 8052. The successful transformation of pCKE, pDT5, pCKP, pDTH102, pYL102E and pYL102 into C. beijerinckii 8052, together with the corresponding recovery of single-stranded DNA from Escherichia coli indicated that the double- and single-stranded replication origins are located on a 0.4-kb CAK1 DNA fragment. Sequence analysis of the putative 0.4-kb replication origin region of CAK1 reveals a nick site containing 22 base pairs that has homology with plasmids pC194 and pUB110 and suggests the presence of a 2.0-kb DNA region involved in stability. The putative Rep protein of CAK1 contains three conserved motifs and three essential residues of the catalytic site in agreement with Rep proteins associated with the pC194 family. The utility of the developed CAK1-derived phagemid designated pYL102E was evaluated by using it to examine heterologous expression of: (1) the manA gene derived from Thermoanaerobacterium polysaccharolyticum in E. coli and C. beijerinckii NCIMB 8052 and (2) the sol operon derived from Clostridium acetobutylicum DSM 792 in C. beijerinckii SA-2.

Purification and characterization of a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum.

Food ingredients containing alpha-1,6-galactoside bonds elicit gastrointestinal disturbances in monogastric animals, including humans. Pretreatment of such ingredients with alpha-galactosidase (EC 3.2.1.22) has the potential to alleviate this condition. For this purpose, a thermostable alpha-galactosidase from Thermoanaerobacterium polysaccharolyticum was purified by a combination of anion exchange and size exclusion chromatographies. The enzyme has a monomeric molecular weight of approximately 80 kDa; however, it is active as a dimer. The optimum temperature for enzyme activity is 77.5 degrees C. Approximately 84 and 88% of enzyme activity remained after 36.5 h of incubation at 70 and 65 degrees C, respectively. Optimum activity was observed at pH 8.0, with a broad range of activity from pH 5.0 to 9.0. Different transition metals had weak to strong inhibitory effects on enzyme activity. The K(m) and V(max) of the enzyme are 0.29-0.345 mM and 200-232 micromol/min/mg of protein, respectively. Importantly, enzyme activity was only slightly inhibited by 75-100 mM galactose, an end product of hydrolysis. Enzyme activity was specific for the alpha-1,6-galactosyl bond, and activity was demonstrated on melibiose and soy molasses.

Molecular characterization and utilization of the CAK1 filamentous viruslike particle derived from Clostridium beijerinckii.

An examination of the replication origin and stability determinant associated with the CAK1 filamentous viruslike particle recovered from Clostridium beijerinckii NCIMB 6444 was carried out. Seven deletion derivatives, pCKE, pCEP1, pDT5, pCKP, pDTH102, pYL102E and pYL102, were constructed and transformed into C. beijerinckii NCIMB 8052. The successful transformation of pCKE, pDT5, pCKP, pDTH102, pYL102E and pYL102 into C. beijerinckii 8052, together with the corresponding recovery of single-stranded DNA from Escherichia coli indicated that the double- and single-stranded replication origins are located on a 0.4-kb CAK1 DNA fragment. Sequence analysis of the putative 0.4-kb replication origin region of CAK1 reveals a nick site containing 22 base pairs that has homology with plasmids pC194 and pUB110 and suggests the presence of a 2.0-kb DNA region involved in stability. The putative Rep protein of CAK1 contains three conserved motifs and three essential residues of the catalytic site in agreement with Rep proteins associated with the pC194 family. The utility of the developed CAK1-derived phagemid designated pYL102E was evaluated by using it to examine heterologous expression of: (1) the manA gene derived from Thermoanaerobacterium polysaccharolyticum in E. coli and C. beijerinckii NCIMB 8052 and (2) the sol operon derived from Clostridium acetobutylicum DSM 792 in C. beijerinckii SA-2.

Characterization of two novel saccharolytic, anaerobic thermophiles, Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov., and emendation of the genus Thermoanaerobacterium.

Two anaerobic, thermophilic, Gram-positive, non-spore forming bacteria with an array of polysaccharide-degrading enzymes were isolated from the leachate of a waste pile from a canning factory in Hoopeston, East Central Illinois, USA. The results of 16S rDNA sequence homology indicated that their closest relatives belong to the saccharolytic, thermophilic and anaerobic genera of Thermoanaerobacterium and Thermoanaerobacter. Although, the evolutionary distances between these bacteria and their closest relatives are greater than 11%, there is no defining phenotypic characteristic for the creation of a new genus. It is proposed that these bacteria should be placed in the genus Thermoanaerobacterium, which requires emendment of the genus description with regard to the reduction of thiosulfate to sulfur, because neither isolate is capable of this reduction. Thermoanaerobacterium polysaccharolyticum reduces thiosulfate to sulfide, whereas Thermoanaerobacterium zeae is unable to reduce thiosulfate. The cells of both isolates are rod-shaped and exist as single cells or sometimes in pairs. Cells are motile by means of flagella. Growth occurs between 45 and 72 degrees C, with optimum temperature of 65-68 degrees C at pH 6.8. The pH range for growth is from 4 to 8 at a temperature of 65 degrees C. Both organisms ferment glucose, arabinose, maltose, mannose, rhamnose, sucrose, trehalose, xylose, cellobiose, raffinose, melibiose and melezitose. The major end products of fermentation with glucose are ethanol and CO2, with lesser amounts of acetate, formate, lactate and hydrogen. The DNA G+C contents of Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov. are 46 and 42 mol%, respectively. The type strains are KMTHCJT (= ATCC BAA-17T = DSM 13641T) and mel2T (= ATCC BAA-16T = DSM 13642T), respectively.