Sheila I. McCrae

Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon

The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.

Cellulosomal scaffoldin-like proteins from Ruminococcus flavefaciens.

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium, Ruminococcus flavefaciens. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The scaB gene was completely sequenced, and its upstream neighbor scaA was partially sequenced. The sequenced portion of scaA contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in Escherichia coli, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several R. flavefaciens enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.

Purification and Some Properties of the Extracellular β-d-Glucosidase of the Cellulolytic Fungus Trichoderma koningii

SUMMARY: The β-d-glucosidase which was associated with the cellobiohydrolase and endo-(1·4)-β-d-glucanase activities in the cellulase of the fungus Trichoderma koningii was purified by gel filtration on a column of Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose and sulphoethyl-Sephadex (SE-Sephadex), and finally by isoelectric focusing in a pH gradient supported in a sucrose density gradient. The separation of β-glucosidase and endo-(l·4)-β-glucanase, which was effected with some difficulty on SE-Sephadex, could be achieved readily by chromatography on a column of concanavalin A-Sepharose. Isoelectric focusing yielded two β-glucosidase components (pI 5·53 and 5·85) of identical molecular weight (39800), but with different affinities for o-nitrophenyl-β-glucoside (K m 0·37 · 0·04 and 0·85 · 0·3 mm), cellobiose (K m 1·18 · 0·09 and 0·86 · 0·02mm) cellotriose (K m 5·10 · 0·22 and 7·10 · 0·33 mm), cellotetraose (K m 2·38 · 0·14 and 4·0 · 0·4mm) and cellopentaose (K m 1·51 · 0·04 and 2·19 · 0·06 mm). One of the β-glucosidases was devoid of carbohydrate, the other contained approximately 2% carbohydrate. Gluconolactone was a powerful inhibitor of the action of both enzymes on o-nitrophenyl-β-glucoside (K i 1·8 · 0·08 and 1·17 · 0·05 μm); glucose was less effective (K i 1·05 · 0·07 and 0·66 · 0·04 mm). Inhibition was of the competitive type in each case. Both β-glucosidases showed the same capacity for acting in synergism with a mixture of the endo-(l·4)-β-glucanase and cellobiohydrolase in solubilizing the cellulose in cotton fibre. They differed markedly, however, in the degree of synergism they showed when acting in concert with the cellobiohydrolase on partially degraded H3PO4-swollen cellulose.

Distribution of β-glucosidase and β-glucuronidase activity and of β-glucuronidase gene gus in human colonic bacteria

beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.

Novel Organization and Divergent Dockerin Specificities in the Cellulosome System of Ruminococcus flavefaciens

The DNA sequence coding for putative cellulosomal scaffolding protein ScaA from the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 was completed. The mature protein exhibits a calculated molecular mass of 90,198 Da and comprises three cohesin domains, a C-terminal dockerin, and a unique N-terminal X domain of unknown function. A novel feature of ScaA is the absence of an identifiable cellulose-binding module. Nevertheless, native ScaA was detected among proteins that attach to cellulose and appeared as a glycosylated band migrating at around 130 kDa. The ScaA dockerin was previously shown to interact with the cohesin-containing putative surface-anchoring protein ScaB. Here, six of the seven cohesins from ScaB were overexpressed as histidine-tagged products in E. coli; despite their considerable sequence differences, each ScaB cohesin specifically recognized the native 130-kDa ScaA protein. The binding specificities of dockerins found in R. flavefaciens plant cell wall-degrading enzymes were examined next. The dockerin sequences of the enzymes EndA, EndB, XynB, and XynD are all closely related but differ from those of XynE and CesA. A recombinant ScaA cohesin bound selectively to dockerin-containing fragments of EndB, but not to those of XynE or CesA. Furthermore, dockerin-containing EndB and XynB, but not XynE or CesA, constructs bound specifically to native ScaA. XynE- and CesA-derived probes did however bind a number of alternative R. flavefaciens bands, including an approximately 110-kDa supernatant protein expressed selectively in cultures grown on xylan. Our findings indicate that in addition to the ScaA dockerin-ScaB cohesin interaction, at least two distinct dockerin-binding specificities are involved in the novel organization of plant cell wall-degrading enzymes in this species and suggest that different scaffoldins and perhaps multiple enzyme complexes may exist in R. flavefaciens.

Purification and properties of a feruloyl/ϱ-coumaroyl esterase from the fungus Penicillium pinophilum

A purified extracellular phenolic acid esterase, produced by the fungus Penicillium pinophilum in solid-state culture, released ferulic and ϱ-coumaric acid from methyl esters of the acids. The esterase was also able to release all the alkali-extractable feruloyl and 80% of the ϱ-coumaroyl groups from a water-soluble wheat straw xylan without degrading the polysaccharide, but it showed no capacity to release ferulic and ϱ-coumaric acid from a grass cell wall preparation in the absence of other xylanolytic enzymes. Synergistic effects were observed between the esterase and other xylan-degrading enzymes in hydrolyzing the ferulic and, to a lesser extent, the ϱ-coumaric acid esters from both wheat straw xylan and the grass cell walls. The esterase had an apparent molecular mass of 57 kDa by SDS-PAGE and an isoelectric pH of 4.6: temperature and pH optima were 55°C and 6.0, respectively. Cu2+ and Fe2+ were inhibitory, but Zn2+, Ca2+, Ag+, and Mg2+ stimulated activity. The enzyme had a higher affinity towards the methyl ester of ϱ-coumaric acid than towards the methyl ester of ferulic acid: the apparent Km values for the ferulic acid and ϱ-coumaric acid esters were 0.14 and 0.08 mM, respectively; the Vmax values were 32.5 and 37.9 μmol min-1 mg-1, respectively.

Xylan-degrading enzyme system produced by the fungus Aspergillus awamori : Isolation and characterization of a feruloyl esterase and a p-coumaroyl esterase

Abstract Feruloyl esterase and p -coumaroyl esterase activities were separated by ion-exchange chromatography on a column of DEAE-Sepharose CL 6B and then purified by hydrophobic interaction chromatography and anion-exchange chromatography, respectively. Feruloyl esterase had an apparent molecular mass of 112 kDa (SDS-PAGE) and an isoelectric pH of 3.7; the p -coumaroyl esterase was much smaller (75 kDa) and had a pI of 4.2. The feruloyl esterase was highly specific for the methyl ester of ferulic acid and for feruloyl esters associated with a water-soluble xylan polysaccharide from wheat straw; p -coumaroyl esterase was also highly specific for the corresponding p -coumaroyl ester substrates. The wheat straw xylan was de-esterified by the esterases without prior degradation of the polysaccharide; however, esterified short-chain xylooligosaccharides which were generated by a purified fungal xylanase were better substrates. The apparent K m values for the feruloyl and p -coumaroyl residues were 0.93 ± 0.05 and 0.031 ± 0.02 mM, respectively, using the methyl ester substrates; the respective V max values were 2.82 ± 0.05 and 0.57 ± 0.03 μ mol min −1 mg −1 protein. The esterases had only a small capacity to release ferulic and p -coumaric acids from cell walls of various plants, including mesophyll cell walls from rye grass, but they were more effective when acting in concert with the commercially available plant cell wall-degrading enzyme Celluclast.

Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences.

Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

Cellulase from Fusarium solani: purification and properties of the C1 component.

The C1 component from Fusarium solani cellulase was purified extensively by molecular-sieve chromatography on Ultrogel AcA-54 and ion-exchange chromatography on DEAE-Sephadex. The purified component showed little capacity for hydrolysing highly ordered substrates (e.g., cotton fibre), but poorly ordered substrates (e.g., H3PO4-swollen cellulose), and the soluble cello-oligosaccharides cellotetraose and cellohexaose, were readily hydrolysed; cellobiose was the principal product in each case. Attack on O(-carboxymethyl)cellulose, a substrate widely used for measuring the activity of the randomly acting enzymes (Cx enzymes) of the cellulase complex, was minimal, and ceased after the removal of a few unsubstituted residues from the end of the chain. These observations, and the fact that the rate of change of degree of polymerisation of H3PO4-swollen cellulose was very slow compared with that effected by the randomly acting endoglucanases (Cx, CM-cellulases), indicate that C1 is a cellobiohydrolase. Fractionation by a variety of methods gave no evidence for the non-identity of the cellobiohydrolase and the component that acted in synergism with the randomly acting Cx enzyme when solubilizing cotton fibre.

ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His(6)-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization-time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex.