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Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum.

ABSTRACT: Choline accumulation and phosphatidylcholine biosynthesis were investigated in the choline-requiring anaerobic protozoon Entodinium caudatum by incubating whole cells or subcellular fractions with [14C] choline, phosphoryl [14C] choline and CDP-[14C] choline. 2. All membrane fractions contained choline kinase (EC 2.7.1.32) and CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), although the specific activities were less in the cell-envelope fraction. Choline phosphate cytidylyltransferase (EC 2.7.7.15) was limited to the supernatant, and this enzyme was rate-limiting for phosphatidylcholine synthesis in the whole cell. 3. Synthesis of phosphatidylcholine from free choline by membranes was only possible in the presence of supernatant. Such reconstituted systems required ATP (2.5 mM), CTP (1 mM) and Mg2+ (5 mM) for maximum synthesis of the phospholipid. CTP and Mg2+ were absolute requirements. 4. Hemicholinium-3 prevented choline uptake by the cells and was strongly inhibitory towards choline kinase; the other enzymes involved in phosphatidylcholine synthesis were minimally affected. 5. Ca2+ ions (0.5 mM) substantially inhibited CDP-choline-1,2-diacylglycerol cholinephosphotransferase in the presence of 15 mM-Mg2+, but choline phosphate cytidylyltransferase and choline kinase were less affected. 6. No free choline could be detected intact cells even after short (10-180s) incubations or at temperatures down to 10 degrees C. The [14C] choline entering was mainly present as phosphorylcholine and to a lesser extent as phosphatidylcholine. 7. It is suggested that choline kinase effectively traps any choline within the cell, thus ensuring a supply of the base for future growth. At low choline concentrations the activity of choline kinase is rate-limiting for choline uptake, and the enzyme might possibly play an active role in the transport phenomenon. Thus the choline uptake by intact cells and choline kinase have similar Km values and show similar responses to temperature and hemicholinium-3.

SUBMITTER: Bygrave FL

PROVIDER: S-EPMC1164264 | biostudies-other | 1976 Dec

REPOSITORIES: biostudies-other

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Project description:Axenic cultures of free-living aerobic ciliates, such as Tetrahymena thermophila and Paramecium aurelia, have been established and routinely used in laboratory research, greatly facilitating, or enabling characterization of their metabolism, physiology, and ecology. Ruminal protozoa are anaerobic ciliates, and they play important roles in feed digestion and fermentation. Although, repeatedly attempted, no laboratory-maintainable axenic culture of ruminal ciliates has been established. When axenic ciliate cultures are developed, antibiotics are required to eliminate the accompanying bacteria. Ruminal ciliates gradually lose viability upon antibiotic treatments, and the resultant axenic cultures can only last for short periods of time. The objective of this study was to evaluate eight antibiotics that have been evaluated in developing axenic cultures of ruminal ciliates, for their toxicity to Entodinium caudatum, which is the most predominant ruminal ciliate species. Scanning and transmission electron microscopy (TEM) showed that the antibiotics damaged both the cell surface and nuclei of E. caudatum and increased accumulation of intracellular glycogen. Combinations of the three least toxic antibiotics failed to eliminate the bacteria that are present in the E. caudatum culture. The combination of ampicillin, carbenicillin, streptomycin, and oxytetracycline was able to eliminate all the bacteria, but the resultant axenic E. caudatum culture gradually lost viability. Adding the bacterial fraction (live) separated from an untreated E. caudatum culture reversed the viability decline and recovered the growth of the treated E. caudatum culture, whereas feeding nine strains of live bacteria isolated from E. caudatum cells, either individually or in combination, could not. Nutritional and metabolic dependence on its associated bacteria, accompanied with direct and indirect inhibition by antibiotics, makes it difficult to establish an axenic culture of E. caudatum. Monoxenic or polyxenic cultures of E. caudatum could be developed if the essential symbiotic partner(s) can be identified.

Project description:BACKGROUND:Rumen ciliates play important roles in rumen function by digesting and fermenting feed and shaping the rumen microbiome. However, they remain poorly understood due to the lack of definitive direct evidence without influence by prokaryotes (including symbionts) in co-cultures or the rumen. In this study, we used RNA-Seq to characterize the transcriptome of Entodinium caudatum, the most predominant and representative rumen ciliate species. RESULTS:Of a large number of transcripts, >?12,000 were annotated to the curated genes in the NR, UniProt, and GO databases. Numerous CAZymes (including lysozyme and chitinase) and peptidases were represented in the transcriptome. This study revealed the ability of E. caudatum to depolymerize starch, hemicellulose, pectin, and the polysaccharides of the bacterial and fungal cell wall, and to degrade proteins. Many signaling pathways, including the ones that have been shown to function in E. caudatum, were represented by many transcripts. The transcriptome also revealed the expression of the genes involved in symbiosis, detoxification of reactive oxygen species, and the electron-transport chain. Overall, the transcriptomic evidence is consistent with some of the previous premises about E. caudatum. However, the identification of specific genes, such as those encoding lysozyme, peptidases, and other enzymes unique to rumen ciliates might be targeted to develop specific and effective inhibitors to improve nitrogen utilization efficiency by controlling the activity and growth of rumen ciliates. The transcriptomic data will also help the assembly and annotation in future genomic sequencing of E. caudatum. CONCLUSION:As the first transcriptome of a single species of rumen ciliates ever sequenced, it provides direct evidence for the substrate spectrum, fermentation pathways, ability to respond to various biotic and abiotic stimuli, and other physiological and ecological features of E. caudatum. The presence and expression of the genes involved in the lysis and degradation of microbial cells highlight the dependence of E. caudatum on engulfment of other rumen microbes for its survival and growth. These genes may be explored in future research to develop targeted control of Entodinium species in the rumen. The transcriptome can also facilitate future genomic studies of E. caudatum and other related rumen ciliates.

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Phosphatidylcholine biosynthesis and choline transport in the anaerobic protozoon Entodinium caudatum.

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