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).     

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.     

2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.     

2-Aminoethylphosphonic acid concentrations in some rumen ciliate protozoa.

The concentration of 2-aminoethylphosphonic acid has been measured in seven genera of rumen ciliate protozoa. Expressed as milligrams per gram of total nitrogen, 2-aminoethylphosphonic acid concentrations ranged from 17.2 in Ophryoscolex spp. to 72.4 in Eremoplastron spp.     

Proteinase activity in rumen ciliate protozoa

Azocasein-degrading proteinase activity was detected in all rumen ciliate protozoa that were examined from four entodiniomorphid and two holotrich genera. All of the activities were optimal in the range pH 4.0-5.0 and were inhibited by cysteine proteinase inhibitors, notably leupeptin. The inhibition profiles and extent of inhibition observed with the different groups of inhibitors were organism-specific. Gelatin-SDS-polyacrylamide gel electrophoresis of protozoal lysates revealed multiple forms of the proteinases in the species examined. The number of enzymes detected, their molecular masses, the level of activity and inhibitor susceptibility was genus-dependent. The proteinase profiles of the two holotrich species differed and inter-species differences were also apparent among species of the genus Entodinium. The characteristics and molecular size distribution of rumen bacterial proteinases were different to the protozoal proteinases. Low levels of proteinase activity, of apparently bacterial origin, were detected by gelatin-SDS-PAGE analysis of cell-free rumen liquor.     

Hemicellulose-degrading enzymes in rumen ciliate protozoa

Hemicellulose-degrading enzymes were detected in cell-free extracts of protozoa representing ten genera of rumen entodiniomorphid and holotrich ciliates. The enzyme preparations released monosaccharides, disaccharides, and oligomers fromLolium perenne hemicellulose B and oat spelt xylan; the activity was present both in cells isolated directly from rumen contents and in those cultured in vitro. The specific activities were higher in the cellulolytic entodiniomorphid genera (Polyplastron, Diploplastron, Eremoplastron, Epidinium, Ophryoscolex, Eudiplodinium) than in the holotrich ciliates (Dasytrichia ruminantium, Isotricha intestinalis/I. prostoma) and the entodinia examined (Entodinium bursa, E. simplex, E. caudatum). The rate of hemicellulose-B degradation to alcohol-soluble products was approximately 5–10 times higher than the rate of reducing sugar accumulation; this indicates an initial depolymerization to intermediate oligosaccharide fragments. Examination of the hemicellulose degradation products by thin-layer and gas-liquid chromatography confirmed oligosaccharide formation, revealed markedly different rates of arabinose and xylose release, and indicated that the mode of polysaccharide degradation was similar in the protozoal preparations examined.     

Hydrolysis of Fraction 1 leaf protein and casein by rumen entodiniomorphid protozoa

Cell-free extracts of fourteen individual species of rumen ciliate protozoa and of mixed rumen ciliates degraded Fraction 1 leaf protein. For Entodinium caudatum and Eudiplodinium maggii the optimum pH was 3·2. The maximum rates of proteolysis (in µmol acid soluble-tyrosine formed/mg protein/h) were 0·16 to 5·7 with protozoa grown in vivo and 0·38 to 6·4 with protozoa grown in vitro. The highest rates were obtained with Entodinium caudatum and E. simplex and the lowest with the cellulolytic species grown in vivo. K _m values (mg/ml) ranged from 0·42 to 19 with protozoa grown in vivo and 0·35 to 13·3 with protozoa grown in vitro. All single species (with one exception) whether grown in vivo or in vitro degraded Fraction 1 leaf protein faster (1·4 to 21 times) than casein. Partial inhibition of the activity of Entodinium caudatum was obtained with pepstatin and N-ethylmaleimide and almost complete inhibition with leupeptin suggesting the presence of 'carboxyl' and 'thiol' enzymes.     

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro ) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 μg/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 μg/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2–7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 μg/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum , but not Entodinium spp. or Dip. affine , synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 μg/mg protein/min. If these reactions occur in the rumen in vivo , up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.     

The rate of uptake and metabolism of starch grains and cellulose particles by Entodinium species, Eudiplodinium maggii, some other entodiniomorphid protozoa and natural protozoal populations taken from the ovine rumen.

The rates of engulfment and breakdown of starch grains and cellulose particles and of the rate of synthesis of amylopectin from cellulose by individual species of entodiniomorphid protozoa (grown in vivo and in vitro) and incubated anaerobically in vitro were studied. Rates of starch uptake varied from 2.3 to 770 micrograms/mg protozoal protein/min; the lowest was found with Diploplastron affine and the highest with Entodinium spp. on initial incubation with starch grains. The rate of starch breakdown varied from 0.49 to 8.6 micrograms/mg protein/min; the rate was dependent on the initial starch concentration inside the protozoa. Eudiplodinium maggii engulfed cellulose particles more rapidly (2-7 times) than rice starch grains and digested the cellulose at rates of 10 to 16.5 micrograms/mg protein/min. In a mixture of starch grains and cellulose particles, it engulfed the latter at 1.35 to 25 times the rate of the former. Eudiplodinium maggii and Epidinium caudatum, but not Entodinium spp. or Dip. affine, synthesized an amylopectin-like material from cellulose at rates of 0.4 to 4.75 micrograms/mg protein/min. If these reactions occur in the rumen in vivo, up to 9 g of amylopectin could be synthesized from cellulose each day by the entodiniomorphid protozoa.     

Detection of methanogens and proteobacteria from a single cell of rumen ciliate protozoa

Rumen ciliate-associated bacteria and methanogenic archaea were analyzed by a 16S rRNA gene retrieved from a single cell of Polyplastron multivesiculatum, Isotricha intestinalis, and Ophryoscolex purkynjei. Rumen fluid was taken from a ruminally fistulated goat to prepare a ciliate fraction. Ciliate mixtures were incubated under mixtures of antibiotics for 48 h to eliminate extracellular bacteria. Individual cells of rumen ciliates were selected under microscopic observation after fixation with ethanol. Bacterial and archaeal 16S rRNA gene sequences were retrieved from each cell of three genera of ciliate. Two archaeal sequences related to Methanobrevibacter smithii were distributed to nearly all ciliate cells tested. These two methanogenic archaea were likely to be endosymbiotic methanogens commonly carried by the rumen ciliate, although some other sequences similar to the other genera were detected. A range of proteobacteria was retrieved from cells of P. multivesiculatum. Some sequences showed similarities to the previously known endosymbiotic proteobacteria. However, there were no proteobacteria that were carried by all the ciliate cells tested.     

Polypeptides of hydrogenosome-enriched fractions from rumen ciliate protozoa and trichomonads: Immunological studies

Abstract The evolution of hydrogenosomes, energy-generating organelles of rumen ciliate protozoa and the flagellate trichomonads has been the subject of much speculation. Polypeptides of the hydrogenosome-enriched fractions from the rumen ciliates, Dasytricha ruminantium, Isostricha spp., Polyplastron multivesiculatum and Eudiplodinium maggii were separated by SDS-PAGE and compared to analogous polypeptide preparations from Tritrichomonas foetus . Immunoblotting with antisera specific to the hydrogenosomes of T. foetus identified common immunoreactive polypeptides present at estimated molecular masses of 28, 35, 38, 44, 48, 58, 100 and 120 kDa. That at 120 kDa corresponds to a single subunit of the purified pyruvate: ferredoxin oxidoreductase from the hydrogenosome of Trichomonas vaginalis .     

The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid

Predation by ciliate protozoa can account for 90% of the eubacterial protein turnover in the rumen. However, little is known about the factors affecting the lysis of archaea in rumen fluid. Bacterial lysis was followed from the release of acid-soluble ¹⁴C from ¹⁴C leucine-labelled bacteria. The rumen methanogen Methanobrevibacter MF1 was broken down more rapidly than other non-ruminal archaea in rumen fluid withdrawn from sheep harbouring either a mixed protozoal population or monofaunated with Polyplastron multivesiculatum or Entodinium spp. The removal of protozoa from the rumen fluid had little effect on the breakdown of Methanobrevibacter , while lysis of the non-methanogenic ruminal bacterium Selenomonas ruminantium decreased by over 70%. Substantial lysis of Methanobrevibacter occurred in cell-free rumen fluid and thzis effect could be abolished by autoclaving. In view of the high number of bacteriophages in rumen fluid and susceptibility of ruminal bacteria to phage-induced lysis it is tempting to suggest that phages have a role in the lysis of archaea in rumen fluid.     

The effect of sterols and haemin on the growth of the rumen ciliate Ophryoscolex caudatus and some other Entodiniomorphid protozoa

Seven isolates of Ophryoscolex caudatus have been cultured anaerobically in vitro (at a population density of 56/ml) for an average of 18 months each in the presence of bacteria on a reduced buffered salts medium containing prepared fresh rumen fluid with the daily addition of ground wheat and dried grass and with twice weekly dilution of the culture with an equal volume of fresh medium. The ground wheat and dried grass could be replaced by ground wheat coated with β-sitosterol, stigmasterol, ergosterol or α-spinasterol and with β-sitosterol the population density increased to 110/ml. Haemin further increased the population density obtained in the presence of sterol by 9–160%. The population density of cultures of Epidinium ecaudatum caudatum was also increased by sterols and haemin, that of Polyplastron multivesiculatum by sterols only, and some sterols and haemin, under certain conditions, increased that of Entodinium caudatum.     

The role of the rumen ciliate protozoa for methane suppression caused by coconut oil

Diets containing either coconut oil or rumen-protected fat (54 g kg⁻¹ dry matter each) were supplied to Rumen Simulation Technique fermenters filled with faunated and defaunated rumen fluid in a 2 × 2 factorial design. Defaunation immediately reduced methane formation by about 40% with each diet. With coconut oil, methane gradually declined in faunated and defaunated rumen fluid. Finally, the extent of methane suppression was similar, both with coconut oil and with defaunation. Independently of the status of protozoa, the population of methanogens in rumen fluid was significantly reduced by coconut oil. The results suggest that defaunation and coconut oil independently and additively suppress rumen methanogenesis.