A note on the effects of oxygen on hydrogen production by the rumen protozoon Dasytricha ruminantium Schuberg

Hydrogen production by the rumen protozoon, Dasytricha ruminantium was reversibly inhibited at O₂ tensions < 2.8 μmol/1. At higher O₂ concentrations irreversible inactivation of the hydrogenase system was observed. In the rumen, H₂ generation is likely to be determined by O₂ concentration since the physiological levels of O₂ in the rumen are in the range 0–1.5 μmol/1.     

Butyrate formation from glucose by the rumen protozoon Dasytricha ruminantium.

Production of butyrate by the holotrich protozoon Dasytricha ruminantium involves the enzymes of glycolysis, pyruvate:ferredoxin oxidoreductase, acetyl-CoA:acetyl-CoA C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxyacyl-CoA hydro-lyase, 3-hydroxyacyl-CoA reductase, phosphate butyryltransferase and butyrate kinase. Subcellular fractionation by differential and density-gradient centrifugation on sucrose gradients indicated that all those enzymes except pyruvate:ferredoxin oxidoreductase were non-sedimentable at 6 X 10(6) g-min. Butyrate kinase and phosphate butyryltransferase were associated with the large- and small-granule fractions. Thus, although metabolic reactions necessary for butyrate production proceed predominantly in the cytosol, hydrogenosomes play a key role in the conversion of pyruvate into acetyl-CoA.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

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.     

Factors affecting the uptake and metabolism of soluble carbohydrates by the rumen ciliate Dasytricha ruminantium isolated from ovine rumen contents by filtration.

A filtration technique is described whereby metabolically-active suspensions of Dasytricha ruminantium can be isolated from rumen contents with negligible contamination by bacteria or other protozoa. The effects of environmental factors and of the diurnal cycle of the rumen on the uptake and metabolism of soluble carbohydrates by these isolated cells were examined. The principal contribution of the protozoan metabolic end-products to the host ruminant is the supply of lactic, acetic and butyric acids during periods when soluble sugars are in excess.     

Hydrogenosomes in the rumen entodiniomorphid ciliate Polyplastron multivesiculatum

The rumen entodiniomorphid ciliate protozoon Polyplastron multivesiculatum was shown, by biochemical and electron microscopic techniques, to possess hydrogenosomes. After differential centrifugation of whole cell homogenates the hydrogenosomal marker enzymes pyruvate:ferredoxin oxidoreductase and hydrogenase were recovered predominantly (61% and 70% of activity respectively) in the large granular fractions that were sedimented by centrifugation for 10(4) g-min (fraction P1) and 10(5) g-min (fraction P2). These subcellular fractions contained membrane-bound organelles that were approximately 0.4-0.6 microns in diameter and which had a mean equilibrium density of 1.22-1.24 g ml-1 after isopycnic centrifugation in sucrose gradients. Malate dehydrogenase (decarboxylating) activity, however, was predominantly non-sedimentable after centrifugation for 6 x 10(6) g-min. Numerous hydrogenosome-like organelles were present in the ectoplasm and endoplasm of the cell. Hydrogenase activity was demonstrated and localized in the protozoan cell using a novel staining procedure with distyryl nitroblue tetrazolium chloride (DSNBT).     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

Hydrogenosomes in known species of rumen entodiniomorphid protozoa

Abstract Yeasts of the genus Kluyveromyces grew very slowly on methylamine as sole nitrogen source. Methylamine oxidase activity in cell-free extracts was very low. Under conditions known to separate methylamine oxidase from benzylamine oxidase in other yeast genera, only a single enzyme was detected in Kluyveromyces lactis . This enzyme could oxidize benzylamine, n -butylamine and (very poorly) methylamine. The enzyme lost no activity on heating at 45°C and had a high affinity and V _max for benzylamine and 1-aminoalkanes of long-chain length, with a very low affinity and V _max for methylamine. It is concluded that growth of K. lactis on methylamine involves only benzylamine oxidase, and that a methylamine oxidase of the type found in other yeasts does not occur.     

The Selectivity of Carbohydrate Assimilation by the Anaerobic Rumen Ciliate Dasytricha ruminantium

The extent of selectivity in the uptake and metabolism of soluble carbohydrates by the rumen ciliate Dasytricha ruminantium has been examined. The protozoon was not selective in the uptake of available carbohydrates and did not appear to regulate sugar entry by preferential or sequential utilization so that there was a concomitant assimilation of all utilizable soluble-sugars regardless of molecular size or metabolic effects. The presence of readily fermentable substrates did not preclude the uptake and subsequent effects of the toxic monomers mannose and glucosamine hydrochloride. The characteristics of galactose co-utilization differed from those of other monosaccharides examined. Although non-metabolizable substrates did not impede the uptake or metabolism of effective substrates, maximum metabolite production rates were not maintained in the presence of slowly utilized substrates.     

Genetic diversity in Selenmonas ruminantium isolated from the rumen

Abstract Non-denaturing polyacrylamide gel electrophoresis was used to detect genetic variation at loci coding for there intracellular enzymes in the obligately anaerobic rumen bacterium Selenomonas ruminantium . Four mobility variants were detected for lactate dehydrigenase, seven for glucokinase and at least five for NADP-dependent glutamate dehydrogenase among 28 newly isolated, and eitght previously isolated strains from sheep and cattle. No evidence was found for an exclusive association of any particular electrophoretic mobility type with variable metabolic traits such as the ability to utilise lactate, to reduce nitrate or to ferment trehalose, sorbitol, rhamnose or glycerol. The most commonly occurring electromorph type was recovered from more than one animal, while most animals examined were show to harbour more than one electromorph type.     

Genetic transfer of lactate-utilizing ability in the rumen bacterium Selenomonas ruminantium

M. GILMOUR, W.J. MITCHELL AND H.J. FLINT. 1996. Matings between the lactate-utilizing, tetracycline-sensitive Selenomonas ruminantium strains 5521Cl and 5934e and the lactate-non-utilizing, tetracycline-resistant strain FB322 resulted in putative recombinant strains capable of growth on lactate. Analysis of total protein by SDS-PAGE and chromosomal DNA by hybridization, indicated that the recombinants were derived from strain FB322. DNA hybridization produced no evidence that plasmid transfer occurred, leaving chromosomal DNA transfer as the most likely mechanism for the altered phenotype. Analysis of strains 5934e, FB322 and the resulting recombinant TC3 indicated that all three strains contained D-nLDH and L-nLDH activities. In addition strains 5934e and TC3 possessed D-iLDH activity when grown on DL-lactate. The ability of strain FB322 to grow on pyruvate but not lactate suggested that the lactate-utilizing recombinant had acquired the ability to synthesize D-iLDH.     

Metabolism of the rumen bacterium Selenomonas ruminantium grown in continuous culture

Selenomonas ruminantium 0078A was grown in a glucose-limited chemostat over a dilution rate range of 0.049-0-137/h. Fermentation products were acetate, propionate, succinate, lactate and C0₂; traces of ethanol were also detected. Succinate accounted for up to 52% of the substrate glucose carbon. When dilution rate was increased without a concomitant increase in glucose supply per unit time there were changes in the fermentation pattern which were not apparent when both dilution rate and glucose supply were simultaneously increased; the molar proportion of acetate increased at the expense of propionate.     

Purification of a nickel-containing urease from the rumen anaerobe Selenomonas ruminantium

Urease was purified 592-fold to homogeneity from the anaerobic rumen bacterium Selenomonas ruminantium. The urease isolation procedure included a heat step and ion-exchange, hydrophobic, gel filtration, and fast protein liquid chromatography. The purified enzyme exhibited a Km for urea of 2.2 +/- 0.5 mM and a Vmax of 1100 mumol of urea min-1 mg-1. The molecular mass estimated for the native enzyme was 360,000 +/- 50,000 daltons, whereas a subunit value of 70,000 +/- 2,000 daltons was determined. These results are in contrast to the findings of Mahadevan et al. (Mahadevan, S., Sauer, F. D., and Erfle, J. D. (1977) Biochem. J. 163, 495-501) in which isolated rumen urease was reported to be one-third this size (Mr 120,000-130,000) and to catalyze urea hydrolysis at a maximum velocity of only 53 mumol min-1 mg-1. S. ruminantium urease contained 2.1 +/- 0.4 nickel ions/subunit, comparable to the nickel content in jack bean urease (Dixon, N.E., Gazzola, C., Blakeley, R.L., and Zerner, B. (1975) J. Am. Chem. Soc. 97, 4131-4133). Thus, the active site of bacterial urease is very similar to that found in the plant enzymes.