An adhesion-defective mutant of Ruminococcus albus SY3 is impaired in its capability to degrade cellulose

An adhesion-defective mutant of Ruminococcus albus SY3 was isolated by a subtractive enrichment procedure, which involved repetitive adsorption of cellobiose-grown cells to cellulose. The growth characteristics of the mutant were compared with those of the wild type. Like the wild-type cells, the mutant was capable of growing on soluble substrates, i.e. cellobiose and xylan. However, in contrast to the wild type strain, the mutant was impaired in its capacity to utilize insoluble substrates, e.g. crystalline cellulose, acid-swollen cellulose or alfalfa cell walls. Scanning electron microscopy revealed protuberance-like surface structures on the wild-type strain which were absent on the mutant. The levels of endoglucanase and xylanase enzymatic activities released into the extracellular culture fluid were higher in the wild type compared to the mutant. However, Avicelase activity was not detected in the extracellular culture fluid of either strains when grown on cellobiose.     

Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda

The formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.     

Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85

Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new insights into the mechanism of cellulose digestion. The genome has a single family 45 beta-glucanase gene, and some of the enzymes in this family have good activity against native cellulose. The gene encoding the family 45 glycosyl hydrolase from F. succinogenes S85 was cloned into Escherichia coli JM109(DE3) using pMAL-c2 as a vector. Recombinant E. coli cells produced a soluble fusion protein (MAL-F45) that was purified on a maltose affinity column and characterized. MAL-F45 was most active on carboxymethylcellulose between pH 6 and 7 and it hydrolyzed cellopentaose and cellohexaose but not cellotetraose. It also cleaved p-nitrophenyl-cellopentose into cellotriose and p-nitrophenyl-cellobiose. MAL-F45 produced cellobiose, cellotriose and cellotetraose from acid swollen cellulose and bacterial cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 genome lacks dockerin and cohesin sequences, does not encode any known processive cellulases, and most of its endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

Features of Fibrobacter intestinalis DR7 mutant which is impaired with its ability to adhere to cellulose

A spontaneous adhesion-defective mutant (DR7-M) of Fibrobacter intestinalis DR7 was isolated which was capable of growing on glucose and cellobiose, but impaired in its capacity to degrade cellulose. Levels of enzyme activities were determined in solubilized fractions of DR7 and DR7-M. Total endoglucanases and xylanase activity values of parent DR7 fractions were 2.84 and 1.85 folds higher than those of the mutant, and were distributed mainly in the bacterial envelope fractions, with some activity also found in the extracellular fluid. In a separate assay, measurement of the enzymatic activity bound to cellulose showed that a portion of the endoglucanase activity bound to cellulose while most xylanase activity did not bind. Notwithstanding, the wild type DR7 cells had 26-fold higher total activities of cellulose-degrading enzymes than the mutant, and 96% of its activity was exclusively located in outer membrane and periplasm fractions. In the mutant, the lower cellulose degrading enzymes activity was located only in the extracellular fluid. Most of the cellulose degrading enzymes activity of DR7 had the capability to bind to cellulose. SDS-page electrophoresis of outer membrane and periplasm cell fractions showed that DR7 and DR7-M possess similar molecular weight (MW) profiles but different quantities of 16 cellulose-binding-proteins (CBPs) in the MW range of 36 up to 225 kDa. Zymogram analysis with soluble substrates, either carboxymethylcellulose or soluble xylan, following SDS-page of DR7 and DR7-M fractions, suggested that CBPs of approximate MW 120, 110, 100, 90, 70 and 40 kDa have endoglucanase activity, and that CBPs of all fractions lack any xylanase activity.     

Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase.

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85.

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Formation, Location, and Regulation of Endo-1,4-β-Glucanases and β-Glucosidases from Cellulomonas uda

The formation and location of endo-1,4-β-glucanases and β-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-β-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-β-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. β-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. β-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and β-glucosidases and the end products of cellulose degradation were determined.     

Regulation and distribution of Fibrobacter succinogenes subsp. succinogenes S85 endoglucanases.

The distribution of endoglucanase activities in cultures of Fibrobacter succinogenes subsp. succinogenes S85 grown on different carbon sources was examined by a variety of biochemical and immunological techniques. Total culture endoglucanase activity was primarily cell associated and was expressed constitutively, although synthesis of endoglucanase 1 (EG1) was repressed by cellobiose. Western immunoblotting showed that EG1 and EG3 were released into the culture fluid during growth, while EG2 remained largely associated with the cell. Subcellular localization showed low endoglucanase activity in the periplasmic fraction and similar, high levels in the cytoplasmic and membrane fractions. Western immunoblotting showed that EG2 was absent from the periplasmic fraction. Data from immunoelectron microscopy with either polyclonal or monoclonal antibody to EG2 revealed a high density of gold labeling at sites where there was a disruption in the regular features of the cell surface, such as in blebbing or physical tearing of the membrane. When cells were grown on cellulose, there was a high density of labeling on the cellulose but not on the cells, indicating that EG2 has limited exposure at the cell surface. On the basis of these data, export of enzymes from their intracellular locations appears to occur via three different mechanisms: a specific secretory pathway independent of cellulose, a secretory mechanism which is mediated by contact with cellulose, and a generalized blebbing process that occurs irrespective of the carbon source.     

Regulation and distribution of Fibrobacter succinogenes subsp. succinogenes S85 endoglucanases

The distribution of endoglucanase activities in cultures of Fibrobacter succinogenes subsp. succinogenes S85 grown on different carbon sources was examined by a variety of biochemical and immunological techniques. Total culture endoglucanase activity was primarily cell associated and was expressed constitutively, although synthesis of endoglucanase 1 (EG1) was repressed by cellobiose. Western immunoblotting showed that EG1 and EG3 were released into the culture fluid during growth, while EG2 remained largely associated with the cell. Subcellular localization showed low endoglucanase activity in the periplasmic fraction and similar, high levels in the cytoplasmic and membrane fractions. Western immunoblotting showed that EG2 was absent from the periplasmic fraction. Data from immunoelectron microscopy with either polyclonal or monoclonal antibody to EG2 revealed a high density of gold labeling at sites where there was a disruption in the regular features of the cell surface, such as in blebbing or physical tearing of the membrane. When cells were grown on cellulose, there was a high density of labeling on the cellulose but not on the cells, indicating that EG2 has limited exposure at the cell surface. On the basis of these data, export of enzymes from their intracellular locations appears to occur via three different mechanisms: a specific secretory pathway independent of cellulose, a secretory mechanism which is mediated by contact with cellulose, and a generalized blebbing process that occurs irrespective of the carbon source.     

Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Pseudomonas fluorescens subsp. cellulosa: an alternative model for bacterial cellulase.

Pseudomonas fluorescens subsp. cellulosa, a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy. Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose. These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface. Cells of Ps. fluorescens subsp. cellulosa do not adhere to cellulose. In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose. Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps. fluorescens subsp. cellulosa and expressed in Escherichia coli, are synthesized by the pseudomonad under a variety of conditions. These enzymes appear to be post-translationally modified, probably through glycosylation. Overall, it appears that the cellulase/hemicellulase system of Ps. fluorescens subsp. cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum.     

Heterologous co-production of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> Cel9A with other cellulases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.     

Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

The Effect of Cellobiose,Glucose, and Cellulose on the Survival of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> A3C Cultures Grown Under Ammonia Limitation

The ruminal, cellulolytic bacterium, Fibrobacter succinogenes A3C, grew rapidly on cellulose, cellobiose, or glucose, but it could not withstand long periods of energy source starvation. If ammonia was limiting and either cellobiose or glucose was in excess, the viability declined even faster. The carbohydrate-excess, ammonia-limited cultures did not spill energy, but they accumulated large amounts of cellular polysaccharide. Cultures that were carbohydrate-limited had approximately 4 nmol ATP mg cell protein^–1, but ATP could not be detected in cultures that had an excess of soluble carbohydrates. However, if F. succinogenes A3C was provided with excess cellulose and ammonia was limiting, ATP did not decline, and the cultures digested the cellulose soon after additional nitrogen sources were added. From these results, it appears that excess soluble carbohydrates can promote the death of F. succinogenes, but cellulose does not.     

Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose

Fibrobacter succinogenes subsp. succinogenes S85, formerly Bacteroides succinogenes, adheres to crystalline cellulose present in the culture medium. When the cells are suspended in buffer, adhesion is enhanced by increasing the ionic strength. Heat, glutaraldehyde, trypsin, and pronase treatments markedly reduce the extent of adhesion. Treatment with dextrinase, modification of amino and carboxyl groups with Formalin or other chemical agents, and inclusion of either albumin (1%) or Tween 80 (0.5%) do not decrease the degree of adhesion. Adherence-defective mutants isolated by their inability to bind to cellulose exhibited different growth characteristics. Class 1 mutants grew on glucose, cellobiose, amorphous cellulose, and crystalline cellulose. Class 3 mutants grew on glucose and cellobiose but not on amorphous or crystalline cellulose. No substantial changes were detected in the endoglucanase, cellobiosidase, and cellobiase activities of the wild type and the mutants. These data suggest that adhesion to crystalline cellulose is specific and that it involves surface proteins.