Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide.

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.

Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase.

The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.

Seasonal changes in the adherent microflora of the rumen in high-arctic Svalbard reindeer.

Seasonal changes in bacterial colonization of the epithelial tissue were examined in the rumen of high-arctic Svalbard reindeer. Samples of tissue were collected from eight sites in the rumen of reindeer during summer and winter and bacterial colonization was examined using scanning and transmission electron microscopy. At two of these sites, colonization by adherent bacteria was estimated to cover approximately 30% of the ruminal epithelium in specimens collected from reindeer during summer. Bacteria at these sites resembled Ruminococcus sp. and were surrounded by large amounts of glycocalyx. In winter specimens, less than 10% of the epithelial surface was covered by adherent bacteria. Those bacteria that did colonize the epithelial surface were smaller and had virtually no glycocalyx on their surface. Bacteria attached to plant cell wall material in summer samples of reindeer ingesta contained large intracellular glycogen deposits, whereas feed particle-associated bacteria in ingesta collected in winter contained no intracellular glycogen. These data demonstrate that the ruminal bacterial population responds to seasonal changes in feed intake and quality. It is yet to be determined if these bacterial changes enhance the ability of Svalbard reindeer to survive in the hostile environment of the high Arctic.

A novel polysaccharide hydrolase cDNA (celD) from Neocallimastix patriciarum encoding three multi-functional catalytic domains with high endoglucanase, cellobiohydrolase and xylanase activities.

A plant polysaccharide hydrolase cDNA, designated celD, was isolated from a cDNA library of the rumen fungus Neocallimastix patriciarum. The enzyme encoded by celD had endoglucanase, cellobiohydrolase and xylanase activities. Deletion analysis revealed that celD cDNA can be truncated to code for three catalytically active domains. Each domain had the same substrate specificity as the enzyme produced by the untruncated celD and also possessed cellulose-binding capacity. Substrate competition studies showed that carboxymethylcellulose and xylan appear to compete with methylumbelliferyl cellobioside for the same active site within each domain. Expression of celD transcript in the rumen fungus was constitutive and was not affected by the presence of cellulose in the culture medium.

Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin.

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli.

A cDNA expression library of the rumen fungus Neocallimastix patriciarum was made in Escherichia coli. Cellulolytic clones were identified by screening on a medium containing carboxymethylcellulose. Restriction mapping and Southern hybridization analysis of selected clones revealed three distinct cellulase cDNAs, designated celA, celB and celC. Studies on the substrate specificity showed that the enzyme encoded by celA had high activity towards amorphous and microcrystalline cellulose, while the celB and celC enzymes had relatively high activity on carboxymethylcellulose, with little activity on microcrystalline cellulose. Analysis of hydrolysis products from defined cellodextrins showed that the celB and celC enzymes hydrolysed beta-1,4-glucosidic linkages randomly, whereas the celA enzyme cleaved cellotetraose to cellobiose, and cellopentaose to cellobiose and cellotriose. Cellobiose was also the only product detectable from hydrolysis of microcrystalline cellulose by the celA enzyme. Based on substrate specificity and catalytic mode, celA appears to encode a cellobiohydrolase, while celB and celC encode endoglucanases. Northern blot hybridization analysis showed that expression of the three cellulase transcripts in N. patriciarum was induced by cellulose.

Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium.

Anaerobic fungi were isolated from rumen fluid of a domestic sheep (Ovis aries; a ruminant) and from faeces of five non-ruminants: African elephant (Loxodonta africana), black rhinoceros (Diceros bicornis), Indian rhinoceros (Rhinoceros unicornis), Indian elephant (Elephas maximus) and mara (Dolichotis patagonum). The anaerobic fungus isolated from the sheep was a Neocallimastix species and the isolates from non-ruminants were all species similar to Piromyces spp. A defined medium is described which supported growth of all the isolates, and was used to examine growth characteristics of the different strains. For each fungus the lipid phosphate content was determined after growth on cellobiose and the resulting values were used to estimate fungal biomass after growth on solid substrates. The ability of isolates from ruminants and non-ruminants to digest both wheat straw and cellulose was comparable. More than 90% and 60%, respectively, of filter paper cellulose and wheat straw were digested by most strains within 60-78 h. Growth of two fungi, isolated from rumen fluid of a sheep (Neocallimastix strain N1) and from faeces of an Indian rhinoceros (Piromyces strain R1), on cellobiose was studied in detail. Fungal growth yields on cellobiose were 64.1 g (mol substrate)-1 for N1 and 34.2 g mol-1 for R1. The major fermentation products of both strains were formate, lactate, acetate, ethanol and hydrogen.

The ultrastructure and possible relationships of four obligate anaerobic chytridiomycete fungi from the rumen of sheep.

Zoospores and vegetative growth phases of three cellulolytic rumen chytridiiomycetes, Piromonas, Sphaeromonas and NF1 have been examined by electron microscopy and compared with published and new data on Neocallimastix. The four genera have some 16 distinctive ultrastructural features in common, which collectively may be used to define the group. Some of the common features may individually be sufficient to distinguish these obligate anaerobes from facultative and aerobic chytridiomycetes. These features are the presence of hydrogenosomes at all stages of the life cycle, the presence in rhizoids and sporangia of characteristic crystals coated with hexagonal arrays of particles, and in zoospores the presence of distinct surface layers on the motility organelles and cell body respectively, the organization of the ribosomes into helical and globular arrays and the structures associated with the kinetosomes.

Nutrition and biochemistry of anaerobic Chytridiomycetes.

The nutrition and biochemistry of anaerobic Chytridiomycetes is at present poorly understood. Data has been obtained principally from studies of rumen isolates of Neocallimastix spp. grown in vitro. Our knowledge of the nutrition of Neocallimastix is summarised. Current information on glycolysis and fermentation product generation via cystosolic and hydrogenosomal systems, production of enzymes involved in plant cell wall hydrolysis, lipid metabolism and the role of Chytridiomycetes in ruminal proteolysis is discussed. At present this is insufficient to provide useful phylogenetic information.

Polysaccharide-degrading enzymes formed by three species of anaerobic rumen fungi grown on a range of carbohydrate substrates.

The range of polysaccharide-degrading enzymes formed by three anaerobic rumen fungi (Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate (F] was monitored following growth on seven mono-, di-, and poly-saccharide carbohydrate substrates. Enzymes capable of degrading a variety of alpha- and beta-glucans, beta-galactans, galactomannan, and hemicellulosic arabinoxylans were present in all three isolates. Although reducing saccharides were released from pectin, polygalacturonic acid was not degraded by the preparations. Enzyme activity was present in both the zoospore and vegetative stages of the life cycle and was also detected extracellularly in culture supernatants after vegetative growth. The specific activities of the polysaccharidases were affected by the growth substrate, being lowest in preparations grown on mono- and di-saccharides, whereas polysaccharidic growth substrates resulted in increased activity of the corresponding polysaccharidases. The enzymes were, however, formed after growth on all substrates. Oligomers and monosaccharides were produced as a result of polysaccharide breakdown by the unfractionated enzyme preparations. Studies on hemicellulose (arabinoxylan) breakdown by unfractionated vegetative preparations of the three isolates indicated that their modes of action, pH optima, substrate affinities, and response to potential inhibitors were similar.

Glycoside hydrolase enzymes present in the zoospore and vegetative growth stages of the rumen fungi Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate, grown on a range of carbohydrates.

The rumen fungi Neocallimastix patriciarum, Piromonas communis, and a morphologically distinct but unidentified isolate were cultivated on the polysaccharides starch, cellulose, xylan, and their principal component monosaccharides and disaccharides, and the range and specific activities of the glycoside hydrolases formed were monitored using gluco-oligosaccharide and p-nitrophenyl glycoside substrates. A wide range of enzyme activities was detected in preparations from vegetative growth and zoospores of all three isolates. Enzyme activity was also present in the culture medium. The specific activities were affected by the carbohydrate source available in the growth medium, although the more active hydrolases involved in the degradation of plant structural and storage polysaccharides were formed on all seven carbohydrate sources evaluated. Enzyme activities were increased in the zoospore, vegetative, and extracellular preparations after growth on the appropriate structurally related disaccharide or polysaccharide. The hemicellulolytic glycosidases (alpha-L-arabinofuranosidase, beta-D-xylosidase) were most active after growth on xylan, whereas alpha-/beta-glucosidase activity was increased with the corresponding glucan as growth substrate. However, whereas wide-ranging beta-glucosidase activity was detected following growth on maltose or starch, the alpha-glucosidase activities of P. communis were lower or undetectable in vegetative preparations grown on glucose or the beta-glucans cellobiose and cellulose.

Seasonal changes in the cecal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus).

The dominant cecal bacteria in the high-arctic Svalbard reindeer were characterized, their population densities were estimated, and cecal pH was determined in summer, when food quality and availability is good, and in winter, when it is very poor. In summer the total culturable viable bacterial population was (8.9 +/- 5.3) X 10(8) cells ml-1, whereas in winter it was (1.5 +/- 0.7) X 10(8) cells ml-1, representing a decrease to 17% of the summer population density. Of the dominant species of cultured bacteria, Butyrivibrio fibrisolvens represented 23% in summer and 18% in winter. Streptococcus bovis represented 17% in summer and 5% in winter. Bacteroides ruminicola represented 10% in summer and 26% in winter. In summer and winter, respectively, the proportion of the viable population showing the following activities was as follows: fiber digestion, 36 and 48%; cellulolysis, 10 and 6%; xylanolysis, 33 and 48%; and starch utilization, 77 and 71%. The most abundant cellulolytic species in summer was Butyrivibrio fibrisolvens, representing 62% of the total cellulolytic population, and in winter it was Ruminococcus albus, representing 80% of the total cellulolytic population. The most abundant xylanolytic species in summer was Butyrivibrio fibrisolvens, and in winter it was Bacteroides ruminicola, representing 59 and 54% of the xylanolytic isolates in summer and winter, respectively. The cecal bacterial of the Svalbard reindeer have the ability to digest starch and the major structural carbohydrates of the diet that are not digested in the rumen. The cecum in these animals has the potential to contribute very substantially to the digestion of the available plant material in both summer and winter.

Microcetus lappus gen. nov., sp. nov.: new species of ciliated protozoon from the bovine rumen.

A new species of small, ciliated protozoon, Microcetus lappus gen. nov., sp. nov., from the rumen of Norwegian Red cattle is described. M. lappus possesses a novel cytopharyngeal apparatus of two rod-shaped structures, one situated on the dorsal side of the buccal cavity and one on the ventral side, suggesting that it belongs to a previously undescribed taxon.

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.

Seasonal changes in the ruminal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus).

The dominant rumen bacteria in high-arctic Svalbard reindeer were characterized, their population densities were estimated, and ruminal pH was determined in summer, when food quality and availability are good, and in winter, when they are poor. In summer the total cultured viable population density was (2.09 +/- 1.26) X 10(10) cells ml-1, whereas in winter it was (0.36 +/- 0.29) X 10(10) cells ml-1, representing a decrease to 17% of the summer population density. On culture, Butyrivibrio fibrisolvens represented 22% of the bacterial population in summer and 30% in winter. Streptococcus bovis represented 17% of the bacterial population in summer but only 4% in winter. Methanogenic bacteria were present at 10(4) cells ml-1 in summer and 10(7) cells ml-1 in winter. In summer and winter, respectively, the proportions of the viable population showing the following activities were as follows: starch utilization, 68 and 63%; fiber digestion, 31 and 74%; cellulolysis, 15 and 35%; xylanolysis, 30 and 58%; proteolysis, 51 and 28%; ureolysis, 40 and 54%; and lactate utilization, 13 and 4%. The principal cellulolytic bacterium was B. fibrisolvens, which represented 66 and 52% of the cellulolytic population in summer and winter, respectively. The results indicate that the microflora of the rumen of Svalbard reindeer is highly effective in fiber digestion and nitrogen metabolism, allowing the animals to survive under the austere nutritional conditions typical of their high-arctic habitat.

The rumen microbiology of seaweed digestion in Orkney sheep.

The microbial populations of the rumens of seaweed-fed and pasture-fed Orkney sheep were examined. The populations in the pasture-fed sheep were similar to those of other domestic ruminants fed on land plants, but those of the seaweed-fed animals showed major differences in the dominant species. Total ciliate populations were quantitatively similar, but in the seaweed-fed animals Dasytricha ruminantium was one of the most dominant species. No phycomycete fungi or cellulolytic bacteria were found in the seaweed-fed animals, and the bacterial population was dominated by Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens and lactate-utilizing species. Electron microscopy revealed that spirochaetes and an unidentified filamentous bacterium were probably of major significance in seaweed digestion. The ability of bacterial strains from both groups of animals to metabolize plant and algal constituents was examined.

Association of rumen ciliate populations with plant particles in vitro.

Seven known species of rumen ciliates and mixedEntodinium spp. showed association with plant particles in rumen fluid in vitro. Association was greater with fresh particles than with hay, and substantially decreased when the water-soluble components of the particles were removed, suggesting that the water-soluble components may be responsible for the association. The association was rapid and maximal between 5 and 35 min (depending on the ciliate species) after exposure to the particles, and involved major transfers of ciliate populations and biomass from the liquid phase to the solid phase of the system. The most rapid and largest population transfers to the particles from the rumen fluid were shown by the holotrich ciliates, where transfers of up to 97% of the population were recorded. Association with plant particles by all species examined occurred within the pH range 5.5-7.5, and decreased with time when the particles were incubated in rumen contents in vivo. The ciliate biomass transferring from the liquid to the solid phase varied with the composition of the ciliate population.

The lipids of the rumen fungus Piromonas communis.

The major phospholipids of the anaerobic rumen phycomycete Piromonas communis were phosphatidylethanolamine (38%), phosphatidylcholine (26%) and phosphatidylinositol (13%); no sphingolipids, glycolipids, plasmalogens or phosphonyl lipids were detected. Free fatty acids, triacylglycerols, 1:2 diacylglycerols and a variable amount of 1:3 diacylglycerol were identified, as were minor amounts of squalene and a triterpenol which is probably tetrahymanol. Approximately half the fatty acids were straight chain, even 12 to 24 carbon, saturated acids, the remainder being even 16 to 24 carbon, mono-unsaturated fatty acids. The double bonds in all except the 16 carbon acid were in the omega 9 position. The unsaturation is introduced by a delta 9 desaturase which uses stearic acid as substrate and which does not use oxygen as a terminal electron acceptor. 14C from acetate and glucose was incorporated into the fatty acids of all complex lipids, as were lauric, myristic, palmitic, stearic and oleic acids. [14C]Choline was incorporated into phosphatidylcholine and [14C]ethanolamine into phosphatidylethanolamine and phosphatidylcholine. Label from [14C]serine was recovered in phosphatidylserine and phosphatidylethanolamine, but was not detected in phosphatidylcholine.

Isolation of proteolytic rumen bacteria by use of selective medium containing leaf fraction 1 protein (ribulosebisphosphate carboxylase).

The principle proteolytic bacteria isolated from bovine rumen contents by virtue of the ability to obtain nitrogen from the proteolysis of leaf fraction 1 protein (ribulosebisphosphate carboxylase, EC 4.1.1.39) were identified as Streptococcus bovis and Butyrivibrio spp. Substitution of fresh fodder, rich in soluble protein, for a hay-concentrates diet resulted in enhanced ruminal proteolytic activity and a significant increase in the number of bacteria able to use fraction 1 protein as the sole nitrogen source. Isolated proteolytic bacteria degraded fraction 1 protein and casein readily. Bovine serum albumin was attacked by Butyrivibrio spp. but was resistant to proteolysis by the streptococci.

Ultrastructural studies of the free zoospore of the rumen phycomycete Neocallimastix frontalis.

The structure of the free zoospores of Neocallimastix frontalis has been examined by electron microscopy of thin-sectioned and negatively stained preparations. There are up to 15 flagella arranged in two rows. The free end of each flagellum is narrow and its tip does not contain microtubules. The flagella and the cell body are coated with distinct surface layers composed of regular arrays of particles and fibrils, respectively. The cell body contains a variety of inclusions. Near to the flagellar pole there are numerous membrane-bound electron-dense globules about 0.2 to 0.7 mum in diameter, between which are microtubules, particles and small vesicles. In the region of the centrally placed nucleus are arrays of helices of ribosome-like particles. These particles also occur in the form of globular aggregates, each partially enclosed within a membrane. The remainder of the cytoplasm is filled with material resembling glycogen. The zoospores stain positively for glycogen and contain ribonuclease-sensitive particulate material which is stained by toluidine blue. Scanning electron microscopy shows that the zoospores attach to the substrate by the flagellar pole.