Purification and characterization of a heterogeneous glycosylated invertase from the rumen holotrich ciliate Isotricha prostoma.

The invertase (beta-fructofuranosidase, EC 3.2.1.26) of the rumen holotrich ciliate Isotricha prostoma has been purified. This is the first report of an enzyme purification from a known species of rumen protozoon. Cells were disrupted by ultrasonic treatment and the enzyme was purified from the cell-free extract by three successive liquid column chromatographies (Sepharose CL4B/octyl-Sepharose CL4B, DE52 DEAE-cellulose and concanavalin A-Sepharose 4B). This resulted in a 160-fold purification and a 15% yield. The major form of the purified enzyme was a tetramer with Mr about 350,000 that was readily dissociated by electrophoresis. The invertase was heterogeneous, as five types of monomers were shown by SDS/polyacrylamide-gel electrophoresis after denaturation. Part of this heterogeneity was due to different glycosylated forms of one of the polypeptides present in the purified enzyme. Isotricha prostoma invertase exhibited maximum activity at pH 5.5-6.0 and 50 degrees C. The kinetic properties of the purified enzyme were very similar to those of invertases from other sources such as yeast or plants (substrate and product inhibition, transferase activity).     

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species,the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied.Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for K_m, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent K_m and V_max values for NADH of the total LDH activity in MRM were followed.It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.The investigation were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Some factors controlling the attachment of the rumen holotrich protozoa Isotricha intestinalis and I. prostoma to plant particles in vitro.

The rumen holotrich protozoa Isotricha intestinalis and I. prostoma showed chemotaxis to sucrose, glucose and fructose. They attached themselves, by means of an organelle on the anterior cell surface, to particulate sources of these carbohydrates provided soluble protein was present in the medium. The concentration of protein eliciting attachment varied with the species and the state of nutrition of the cell, but was between 20 and 150 microgram ml-1. Attachment occurred only if the concentration of carbohydrate, at its source, exceeded the chemotaxis threshold concentration (50 micrometer for sucrose) and if it was less than 1 mM. At concentrations exceeding 1 mM, indiscriminate attachment to gas-liquid and solid-liquid interfaces occurred, provided the protein concentration was high enough to elicit attachment. In the rumen, soluble carbohydrates diffusing from food particles may attract the protozoa which attach themselves to the particles in the presence of soluble plant protein at less than 20 microgram ml-1; these conditions exist in the host animal soon after feeding when fed infrequently. The attachment mechanism may confer an ecological advantage on the Isotricha spp. over other rumen organisms dependent on soluble carbohydrates as energy and carbon sources.     

Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid

From the simultaneous accumulation of hydrogenation intermediates and the disappearance of Isotricha prostoma after algae supplementation, we suggested a role of this ciliate and/or its associated bacteria in rumen biohydrogenation of unsaturated fatty acids. The experiments described here evaluated the role of I. prostoma and/or its associated endogenous and exogenous bacteria in rumen biohydrogenation of C18:2n-6 and its main intermediates CLA c9t11 and C18:1t11. Fractions of I. prostoma and associated bacteria, obtained by sedimentation of rumen fluid sampled from a monofaunated sheep, were used untreated, treated with antibiotics or sonicated to discriminate between the activity of I. prostoma and its associated bacteria, the protozoan or the bacteria, respectively. Incubations were performed in triplicate during 6 h with unesterified C18:2n-6, CLA c9t11 or C18:1t11 (400 μg/ml) and 0.1 g glucose/cellobiose (1/1, w/w). I. prostoma did not hydrogenate C18:2n-6 or its intermediates whereas bacteria associated with I. prostoma converted a limited amount of C18:2n-6 and CLA c9t11 to trans monoenes. C18:1t11 was not hydrogenated by either I. prostoma or its associated bacteria but was isomerized to C18:1c9. A phylogenetic analysis of clones originating from Butyrivibrio-specific PCR product was performed. This indicated that 71% of the clones from the endogenous and exogenous community clustered in close relationship with Lachnospira pectinoschiza. Additionally, the biohydrogenation activity of solid-associated bacteria (SAB) and liquid-associated bacteria (LAB) was examined and compared with the activity of the non-fractioned I. prostoma monofaunated rumen fluid (LAB + SAB). Both SAB and LAB were involved in rumen biohydrogenation of C18:2n-6. SAB fractions performed the full hydrogenation reaction to C18:0 while C18:1 fatty acids, predominantly C18:1t10 and C18:1t11, accumulated in the LAB fractions. SAB and LAB sequence analyses were mainly related to the genera Butyrivibrio and Pseudobutyrivibrio with 12% of the SAB clones closely related to the C18:0 producing B. proteoclasticus branch. In conclusion, this work suggests that I. prostoma and its associated bacteria play no role in C18:2n-6 biohydrogenation, while LAB convert C18:2n-6 to a wide range of C18:1 fatty acids and SAB produce C18:0, the end product of rumen lipid metabolism.     

Biochemical and immunological characterization of the microfibrillar ecto-endoplasmic boundary in the ciliate Isotricha prostoma

The cytoplasm of the ciliated protozoan Isotricha prostoma is compartmented by a continuous fibrillar system made up of a double layer of 4 nm-diameter filaments: the microfibrillar ecto-endoplasmic boundary (EEB). Isolation of this structure after treatment of the cells in a buffer of low ionic strength in the presence of the detergent Triton X-100 evidenced connections linking the two filamentous layers. One dimensional electrophoresis on SDS-polyacrylamide gel of EEB fractions revealed several major proteins with apparent molecular weights between 11 and 23 K. Of these, two neighboring bands of MW22 and 23 K were removed from gels and used as antigens to obtain rabbit antibodies. The antiserum obtained reacted specifically with injected proteins as shown by the technique of immunological detection on nitrocellulose sheets using the peroxidase reaction product. Electron microscopy localization of the antigens with anti-IgG coupled with colloidal gold showed significant labeling of the EEB within the cortex of Isotricha permeabilized with Triton X-100. We hope that the 22-23 K antiserum will prove to be a useful tool for the comparative study of other non-actin filament systems in Protozoa.     

Biochemical Study of Proteins of Cortical Cytoskeleton in the Ciliate Isotricha prostoma1

In Isotricha prostoma, proteins of isolated cortices retaining the ecto-endoplasmic fibrillar boundary (EEB) have been separated by one- and two-dimensional gel electrophoresis. In addition to that of tubulin, four main bands corresponding to polypeptide molecular weights of 185, 35, 23, and 22 kd have been observed on 9% SDS-polyacrylamide gels. Supernumerary major bands within a low molecular weight range (11-19 kd) are evident after separation on 7.5-15% polyacrylamide gradient gels. Two-dimensional analysis discloses - 150 polypeptide spots, most of them focusing within an acidic pl range 4.6-5.3. The cortical fibrillar structures are still distinguishable after incubation in solutions containing 1.0 M K. Cl (12 h) or 0.6 M Kl (8 h). A prolonged KI treatment (52 h), however, results in the removal of EEB filaments (?4 nm in diameter) and of a large part of the kinetid elements, so that the insoluble material is mainly represented by a reticulum of 6-nm filaments interconnecting the proximal regions of kinetosomes. Two proteins with MW of 58 and 63 kd appear to be constituents of this kinetosome-associated filamentous reticulum. Some of the cortical proteins with MW ≤23 kd are the most likely candidates for components of EEB filaments. The absence of decoration with heavy meromyosin confirms that actin is not a major component of Isotricha cortical cytoskeleton.     

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.     

Analysis of Intraspecific Sequence Variation Among Eight Isolates of the Rumen Symbiont, Isotricha prostoma (Ciliophora), from Two Continents

The internally transcribed spacer regions 1 and 2 (ITS-1 and ITS-2) and the 5.8S ribosomal RNA gene of Isotricha prostoma were examined for intraspecific sequence variation. There were no differences in the ITS-1/5.8S/ITS-2 region among cattle and sheep isolates of I. prostoma from Australia, Canada, and the United States, indicating that this region is 100% conserved among eight isolates from two continents.     

Thiomolybdates in rumen contents and rumen cultures

Examination of direct and (Cu)-difference spectra of i) the aqueous supernatants of in vitro cultures of bovine rumen contents incubated with MoO42- and potential sources of S2- and ii) samples drawn directly from the rumen of animals receiving high Mo diets yielded evidence of the presence of thiomolybdates. Only MoS42- was detected in the soluble phase of in vitro cultures. Although intense and variable background absorbance precluded full characterization of thiomolybdate species in samples drawn directly from the rumen, both spectral data and the biochemical and clinical responses of animals given high Mo diets were consistent with the conclusion that MoS42- rather than MoOS32- was the predominant thiomolybdate species present in the aqueous phase. Addition of Ca2+ either to rumen cultures before incubation or as a supplement to diets high in MoO42- content inhibited the appearance of MoS42- in the aqueous phase. Evidence of the sequestration of MoS42- and MoOS32- by particulate or microbial fractions of rumen contents is considered in relation to the inhibitory action of Mo upon Cu absorption by ruminants.     

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.     

Hydration of oleic acid by a Selenomonas strain from the ovine rumen

J.A. HUDSON, C.A.M. MACKENZIE AND K.N. JOBLIN. 1996. A Selenomonas sp., isolated from the ovine rumen, was characterized with regard to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydration occurred only in stationary phase in a medium containing 0.1%, 0.5% (w/v) galactose or 0.5% (w/v) glucose, but not in a medium containing 1% galactose. Growth under a hydrogen headspace did not result in the production of stearic acid, the biohydrogenated product of oleic acid. Linoleic and linolenic acids (0.1% v/v) were not hydrated. It is concluded that the growing bacterium is unlikely to contribute to oleic acid hydration in the rumen.     

Lipid Metabolism of Rumen Ciliates and Bacteria: I. Uptake of Fatty Acids by Isotricha prostoma and Entodinium simplex

Washed suspensions of the ruminal ciliates, Isotricha prostoma and Entodinium simplex, concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Radioautographs demonstrated that oleic acid-1-C¹⁴ was hydrogenated to stearic acid by I. prostoma, and Warburg manometric data showed that the sodium salts of oleic, valeric, caproic, and acetic acids, and methyl myristate, methyl laurate, and the triglyceride tributyrin stimulated fermentation of I. prostoma. The total lipid and free fatty acid contents of I. prostoma were determined.     

Occurrence of plasmid-mediated ampicillin resistance among enterobacteria from the ovine rumen

Seasonal samplings of rumen and fecal populations of Enterobacteriacae from sheep digestive tract were done to elucidate potential occurrence and spreading of antibiotic resistance in the environment. Thus 350 rumen and fecal isolates were tested for ampicillin (Amp) resistance in single sampling. Low frequency of Amp resistance (from 0 to 15%) was observed. The occurrence of tem1 encoded Amp resistance confirmed by PCR was observed among both rumen and fecal isolates. The small tem1 carrying plasmid and its transfer (mobilization) was detected and partially characterized after conjugation to laboratory Escherichia coli strain.     

Gas metabolism in ovine rumen cultures on a nitrogen-deficient medium.

Inocula of rumen fluid from sheep were grown on a medium of low nitrogen content under an oxygen-free atmosphere containing N2. The cultures produced methane and carbon dioxide. Hydrogen gas was utilized and the partial pressure of N2 decreased by 24% in 48 h. Assuming this N2 to have been reduced to metabolically available nitrogenous compounds, these account for about 0.2% of the sheep's daily need.