Lipid Metabolism of Rumen Ciliates and Bacteria: II. Uptake of Fatty Acids and Lipid Analysis of Isotrichia intestinalis and Rumen Bacteria with Further Information on Entodinium simplex

The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C¹⁴-labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells.     

Bacterial Ingestion by the Rumen Ciliates Entodinium and Diplodinium

SYNOPSIS. In cattle fed a high-starch diet, species of Entodinium and Diplodinium ingested associated ruminal bacteria. Stained preparations of diluted rumen contents showed Entodinium caudatum, E. minimum, E. dubardi , (syn. E. simplex ), E. longinucleatum, E. bursa, E. nanellum, E. exiguum , and E. vorax contained gram-positive diplococci. Starch grains with adherent gram-positive diplococci were observed within Entodinium spp. Diplodinium ecaudatum forma ecaudatum, D. ecaudatum forma caudatum, D. neglectum and an unidentified species of Diplodinium also ingested ruminal diplococci. Bacteria were isolated from mixed species of Entodinium by washing and culturing the protozoa in a starch feed-extract agar medium. The strains isolated from the ciliates were gram-positive diplococci, 0.8 times 1–1.5 μm, which attached themselves to starch granules and were able to digest the starch. Conclusive evidence of bacterial ingestion by the oligotrichs was obtained by providing the bacterial cultures to Entodinium species ( E. dubardi and E. minimum ) which had been starved 24 hr. Gram-stained preparations showed the ciliates readily ingested the bacteria. The amylolytic cocci utilized by Entodinium spp. were identified as Streptococcus bovis.     

Competition between Food Particles and Rumen Bacteria in the Uptake of Long-chain Fatty Acids and Triglycerides

S ummary . Suspensions of mixed rumen bacteria were incubated with long-chain fatty acids and their corresponding triglycerides in the presence and absence of the food particles present in the rumen. The uptake of free fatty acids and triglycerides by the bacteria was due largely to physical adsorption. With increasing unsaturation of the free fatty acids, less adsorption occurred and in all instances the presence of food particles reduced greatly the extent to which the fatty acids were adsorbed on the bacteria. The triglycerides were adsorbed to the bacteria to a much less extent than their corresponding free fatty acids, more of the substrate remaining in the supernatant fraction. The presence of food particles had little effect on the extent to which the triglycerides became adsorbed to the bacteria, but reduced greatly the amount of triglyceride present in the supernatant fraction. The decrease in amount of free fatty acid associated with the bacteria and the decrease in the amount of triglyceride in the supernatant liquid when food particles were present, was wholly accounted for by the increase in amount of these substrates associated with the food particles. The implications of these findings with respect to the metabolism of lipids in the rumen is discussed.     

Glycogen Phosphorylase and Synthase Activities of Rumen Ciliates of the Genus Entodinium

Glycogen phosphorylase and synthase activities were detected in the sonic lysate of rumen ciliates of the genus Entodinium. The ciliate phosphorylase had the following properties. The pH optimum was narrow and centered at pH 5.9. The activity was maximum at 30°C; above 40°C a rapid inactivation occurred. The K_m value for glucose-1-phosphate (G-1-P) and for glycogen was 15 mM and 0.069% (w/v), respectively. NaF and ethylenediamine tetraacetic acid had no stimulative effect on the enzyme activity, though adenosine 3′,5′-monophosphate and theophylline activated it. NaHSO₃ inhibited the enzyme activity at a concentration of 1 mM. The inhibition of glucose was noncompetitive for G-1-P. Glycolytic intermediates and nucleotides had a minor effect on phosphorylase activity. Glycogen synthase existed in two forms, glucose-6-phosphate dependent and independent forms: the proportion of the latter form increased with the decrease of reserve polysaccharide levels in the ciliates. Correlations between glycolytic enzyme activities included phosphorylase and synthase activities and reserve polysaccharide contents in the ciliates were determined, and a possible regulatory mechanism of polysaccharide synthesis and degradation was discussed.     

The Uptake and Utilization of Bacteria, Amino Acids and Carbohydrate by the Rumen Ciliate Entodinium longinucleatum in Relation to the Sources of Amino Acids for Protein Synthesis

Entodinium longinucleatum grown in vitro in the presence of bacteria engulfed a wide range of bacterial species at rates of 130–3400 bacteria/h/protozoon (from suspensions of 10 bacteria/ml), but showed a preference for Klebsiella aerogenes and Proteus mirabilis which occurred in the growth medium. Some of the bacteria were digested with release of soluble material into the medium. Free amino acids were incorporated by the protozoa in the presence of chloramphenicol at rates of 5·4–15·1 nmol/h/10⁶ protozoa and approximately 40% of the amino acid-carbon was incorporated into protein. There was no appreciable synthesis of protozoal protein from carbohydrate. Evidence was obtained that the protozoa obtained the amino acids required for growth largely from engulfed bacteria.     

Effects of Long-Chain Fatty Acids on Growth of Rumen Bacteria

The effects of low concentrations of long-chain fatty acids (palmitic, stearic, oleic, and vaccenic) on the growth of seven species (13 strains) of rumen bacteria were investigated. Except for Bacteroides ruminicola and several strains of Butyrivibrio fibrisolvens, bacterial growth was not greatly affected by either palmitic or stearic acids. In contrast, growth of Selenomonas ruminantium, B. ruminicola, and one strain of B. fibrisolvens was stimulated by oleic acid, whereas the cellulolytic species were markedly inhibited by this acid. Vaccenic acid (trans Δ11 18:1) had far less inhibitory effect on the cellulolytic species than oleic acid (cis Δ9 18:1). Inclusion of powdered cellulose in the medium appeared to reverse both inhibitory and stimulatory effects of added fatty acids. However, there was little carry-over effect observed when cells were transferred from a medium with fatty acids to one without. Considerable variation in response to added fatty acids was noted among five strains of B. fibrisolvens. In general, exogenous long-chain fatty acids appear to have little, if any, energy-sparing effect on the growth of rumen bacteria.     

The Cytology of Sheep Rumen Ciliates. III. Ultrastructure of the Genus Entodinium (Stein)1

This study examines previously undescribed general and cytopharyngeal features of the genus Entodinium. The cytopharynx contains three types of microtubular ribbons underlying the cytostomal membrane as well as a loose palisade of nematodesmata. A protoesophagus composed of microtubular bundles associated with a fibrous wall lies internally to one side of an extrusible peristome on which the adoral zone of syncilia (AZS) is mounted. Macronuclear structures are very similar to those of other ophryoscolecids. The micronucleus has chromatin bodies forming a compact mass but lacks the thick wall found in other species. A tubular spongiome surrounds the contractile vacuole and the cytoproct is relatively undifferentiated. Cortical structure follows the usual five-layered ophryoscolecid pattern with subcortical barren kinetosomes arranged into indistinct kineties. The infraciliature of the AZS has kinetosomes set upon a subkinetal rod and with associated bifurcated kinetodesmata and transverse microtubules, some of which extend into the cytopharynx. Components newly described for Entodinium are the one to three postciliary microtubules and the interkinetosomal centro-lateral strand, all of which are present in other species of ophryoscolecid ciliates. The infraciliature of the paralabial ciliary tuft shows similar components to that of the main AZS, but lacks the subkinetal rod. The microtubular components of the cytopharynx are discussed in relation to the “alimentary” structures in other ophryoscolecids, and a relationship of these structures to dietary differences is suggested.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

Cellular Fatty Acid Composition and Identification of Rumen Bacteria

The fatty acid compositions of 21 pure cultures of rumen bacteria, representing 12 genera and 14 species, were compared as methyl esters. Each organism possessed a consistent and reproducible fatty acid profile. Overlapping similarities and differences in composition did not allow differentiation between families or genera. Although species differentiation was possible, fatty acid composition appeared to be only an aid in the identification of bacteria.     

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.     

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).     

Lipid Composition and Localization of 10-Methyl Branched-chain Fatty Acids in Corynebacterium simplex Grown on n-Alkanes

1. Corynebacterium simplex ATCC 6946 grown on a n-alkane mixture (C₁₄-C₁₆) contained 13% of total lipids, which composed of approximately equal quantities of neutral lipids and compound lipids. Phospholipids, corresponding to about 60% of compound lipids, comprised phosphatidylinositol mannoside and cardiolipin as the major components, and phosphatidylglycerol and phosphatidylethanolamine as the minor components.

2. When this bacterium was cultivated on n-octadecane in the presence of cobaltous ion, a large amount of tuberculostearic acid (10-methylstearic acid) was synthesized, which was exclusively localized in phosphatidylinositol mannoside and was not detected in neutral lipids.     

Volatile Fatty Acids and the Inhibition of Escherichia coli Growth by Rumen Fluid

Concentrations of volatile fatty acids (VFA) normally found in bovine rumen fluid inhibited growth of Escherichia coli in Antibiotic Medium 3. Acetic, propionic, and butyric acids each produced growth inhibition which was markedly pH-dependent. Little inhibition was observed at pH 7.0, and inhibition increased with decreasing pH. A combination of 60 mumoles of acetate, 20 mumoles of propionate, and 15 mumoles of butyrate per ml gave 96, 69, and 2% inhibition at pH 6.0, 6.5, and 7.0, respectively. Rumen fluid (50%) gave 89 and 48% inhibition at pH 6.0 and 6.5, respectively, and growth stimulation (22%) at pH 7.0. Rumen fluid inhibitory activity was heat-stable, was not precipitated by 63% ethyl alcohol, and was lost by dialysis and by treatment with anion-exchange resins but not with cation-exchange resins. These results are consistent with the idea that VFA are the inhibitory substances in rumen fluid. Previous results which indicated that rumen fluid VFA did not inhibit E. coli growth were due to lack of careful control of the final pH of the growth medium. The E. coli strain used does not grow in rumen fluid alone at pH 7.0.     

Phylogeny of the Rumen Ciliates Entodinium, Epidinium and Polyplastron (Litostomatea: Entodiniomorphida) Inferred from Small Subunit Ribosomal RNA Sequences

ABSTRACT. Three complete 18S ribosomal RNA gene sequences from the rumen ciliates, Entodinium coudatum (1,639 bp), Epidinium caudarum (1,638 bp), and Polyplastron multivesiculatum (1,640 bp) were determined and confrimed in the opposite direction. Trees produced using maximum parsimony and distance-matrix methods (lest squares and neighbour-joining). with strong bootstrap support, depict the rumen ciliates as a monophyletic group, Entodinium caudatum is the earliest branching rumen ciliate. However, Entodiniwn simplex does not pair with En. caudatum , but rather with Polyplastron multivesiculatum. Signature sequences for these rumen ciliates reveal that the published SSrRNA gene sequence from En. simplex is in fact a Polyoplastron species. The free-living haptorian ciliates, Loxophyllum, Homalozoon and Spathidium (Subclass Hoptoria), are monophyletic and are the sister group to the rumen cilates. The litostomes (class Litostomatea), consisting of the haptorians and the rumen ciliates, are also a monophyletic group.     

The Cytology of Sheep Rumen Ciliates. I. Ultrastructure of Epidinium caudatum Crawley1

Epidinium caudatum has an anterior vestibulum containing the adoral zone syncilia (AZS) on an extrusible peristome. The cytopharyngeal structures include a funnel-shaped arrangement of nematodesmata, longitudinal and transversely oriented microtubular ribbons all of which are located in the peristome, a structure which also contains filamentous phagoplasm. The origins of the microtubular ribbons indicate affinities to the rhabdos type of cytopharynx. The peristomal base is continuous with the tubular esophagus, the region connecting the two being ensheathed by a fibrous layer and low density cytoplasm. The esophagus has a microtubular/microfilamentous wall. A distinct cytoproct with associated myonemal structures occurs posteriorly. The skeletal plates consist of a large number of interconnected, variably shaped platelets and may have dual skeletal and storage functions. The endoplasm is more vesicular than the ectoplasm, the two separated by a fibrous boundary layer. The five-layered cortex has an external glycocalyx, a plasma membrane with two subtending membranes, homogeneous, microtubular, and microfilamentous layers. The syncilia of the AZS are mounted in a U-shaped band on the peristome with transversely oriented kinetics consisting of kinetosomes linked by a sub-kinetosomal rod. There is a bifurcated kinetodesma, dense support material forming a lateral spur with associated transverse microtubules, and postciliary, interkinetal, and occasional basal microtubules, nematodesmata, and a subciliary reticulum. A barren, possibly vestigial, somatic infraciliature consists of non-ciliated kinetosomes and a basal striated fiber with associated basal and perpendicular (cortical) microtubules.     

Conversion of Unsaturated Fatty Acids by Bacteria Isolated from Compost

A compost mixture amended with soybean oil was enriched in microorganisms that transformed unsaturated fatty acids (UFAs). When oleic acid or 10-ketostearic acid was the selective fatty acid, Sphingobacterium thalpophilum (NRRL B-23206, NRRL B-23208, NRRL B-23209, NRRL B-23210, NRRL B-23211, NRRL B-23212), Acinetobacter spp. (NRRL B-23207, NRRL B-23213), and Enterobacter cloacae (NRRL B-23264, NRRL B-23265, NRRL B-23266) represented isolates that produced either hydroxystearic acid, ketostearic acid, or incomplete decarboxylations. When ricinoleic (12-hydroxy-9-octadecenoic) acid was the selective UFA, Enterobacter cloacae (NRRL B-23257, NRRL B-23267) and Escherichia sp. (NRRL B-23259) produced 12-C and 14-C homologous compounds, and Pseudomonas aeruginosa (NRRL B-23256, NRRL B-23260) converted ricinoleate to a trihydroxyoctadecenoate product. Also, various Enterobacter, Pseudomonas, and Serratia spp. appeared to decarboxylate linoleate substrate incompletely. These saprophytic, compost bacteria were aerobic or facultative anaerobic Gram-negative and decomposed UFAs through decarboxylation, hydroxylation, and hydroperoxidation mechanisms. Received: 3 November 1998 / Accepted: 30 November 1998     

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis: I. Some Characteristics of Fatty Acid Synthesis

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH₄)₂SO₄ fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.