Stimulation of growth and polyamine biosynthesis of the ciliated protozoan Tetrahymena thermophila. Regulation by L-arginine

Tetrahymena thermophila cells grown in a synthetic nutrient medium for 9 h removed 97% of the free L-arginine but less than 50% of any of the other essential amino acids. The major portion of the arginine was degraded rapidly (76-92%) whereas 5-15% was conserved as intact and only 2.5-10% were incorporated into protein. However, if bovine serum albumin (BSA) was present in the medium as a macromolecular arginine source the incorporation of free arginine into protein was reduced to less than 1% but the degraded fraction was increased. Apparently, the uptake mode of arginine determines its fate: arginine taken up by phagocytosis is bound for protein biosynthesis, arginine taken up by membrane receptors is chanelled to degradation. Media without arginine did not support growth of Tetrahymena. Citrulline and ornithine, the precursors of arginine biosynthesis in yeast and vertebrates, were not able to substitute for arginine. Pronounced morphological changes, e.g. greatly reduced ribosome content, were observed in Tetrahymena cells after 24 h of arginine starvation in otherwise complete medium, but not in cells starved in water, salt solution, or buffer. Thus, arginine is an essential nutrient component for Tetrahymena and the rapid degradation of this compound involving the enzymes arginine deiminase (ADI) and citrulline hydrolase (CH) might be of regulatory importance for the unicellular, as it is the case with acetylcholine and catecholamines in mammalian organisms. Since the product of these enzymes, L-ornithine, is the substrate for the regulatory key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), the effects of the presence of absence of arginine on the activities of each particular enzyme of the pathway were studied, including ODC and the enzyme ornithine-oxo-acid aminotransferase (O delta T), which is a competitor of ODC for the common substrate. The arginine-degradative pathway was stimulated by extracellular free but not by peptide-bound arginine and was modulated by extracellular protein which induced phagocytosis; O delta T was stimulated with a time lag. The stimulation of ODC was in a reciprocal relation to the arginine concentration and enhanced by phagocytosis and previous arginine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A pseudo-phytochelatin synthase in the ciliated protozoan Tetrahymena thermophila

Phytochelatins (PCs) and metallothioneins (MTs) are the two major heavy metal chelating peptides in eukaryotes. We report here on the identification of a biosynthetically inactive pseudo-phytochelatin synthase enzyme (TtψPCS) in the ciliate Tetrahymena thermophila, the first of this kind (pseudo-PCS) to be described in eukaryotes. TtψPCS which resembles a true PCS at the N-terminal region, while it is most divergent in its Cys-poor C-terminal region, was found to be up-regulated under cadmium stress conditions. However, only glutathione (GSH) hydrolysis products, but not PCs, could be detected in extracts from Cd-treated cells. The latter feature is reminiscent of pseudo-PCS enzymes recently identified in cyanobacteria, which are also biosynthetically inactive, but capable to hydrolyze GSH.     

Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila.

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.     

Activation of prostacyclin synthesis by mechanical stimulation in the ciliated protozoan Tetrahymena thermophila

We detected a HPLC peak corresponding to 6-keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI(2)), in [1-(14)C]arachidonate metabolites from the ciliated protozoan Tetrahymena thermophila. Quantitative analysis of 6-keto-PGF(1alpha) by enzyme immunoassay revealed that the synthesis and release were rapidly activated by the mechanical stimulation of a short centrifugation. The activation was suppressed significantly by a cyclooxygenase inhibitor, ibuprofen, and was independent of the extracellular Ca(2+). External addition of PGI(2) and its stable analogue, beraprost, caused a transient increase in the tumbling frequency of swimming. Other prostanoids, PGE(2) and PGF(2alpha), have no effect on the swimming. These results indicate that a free-living ciliate, T. thermophila, synthesizes and has a specific sensitivity to PGI(2).     

Analysis of expressed sequence tags (ESTs) in the ciliated protozoan Tetrahymena thermophila

To assess the utility of expressed sequence tag (EST) sequencing as a method of gene discovery in the ciliated protozoan Tetrahymena thermophila, we have sequenced either the 5' or 3' ends of 157 clones chosen at random from two cDNA libraries constructed from the mRNA of vegetatively growing cultures. Of 116 total non-redundant clones, 8.6% represented genes previously cloned in Tetrahymena. Fifty-two percent had significant identity to genes from other organisms represented in GenBank, of which 92% matched human proteins. Intriguing matches include an opioid-regulated protein, a glutamate-binding protein for an NMDA-receptor, and a stem-cell maintenance protein. Eleven-percent of the non-Tetrahymena specific matches were to genes present in humans and other mammals but not found in other model unicellular eukaryotes, including the completely sequenced Saccharomyces cerevisiae. Our data reinforce the fact that Tetrahymena is an excellent unicellular model system for studying many aspects of animal biology and is poised to become an important model system for genome-scale gene discovery and functional analysis.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

Inhibition of L-arginine iminohydrolase (EC 3.5.3.6) from Tetrahymena thermophila by putrescine and spermidine: feedback control of polyamine biosynthesis

L-Arginine iminohydrolase (arginine deiminase, ADI) from Tetrahymena thermophila was purified approx. 75-fold by means of gel permeation chromatography. The Km of the purified enzyme for L-arginine was 412 +/- 25 microM and L-ornithine inhibited the reaction competitively with a Ki of 985 +/- 105 microM. D-Ornithine was a weak inhibitor with a Ki of greater than 10mM. The polyamines putrescine and spermidine inhibited ADI incompetitively with a Kii of 2.8mM for putrescine and 4.3mM for spermidine. Since the concentrations required for inhibition were within the range of the normal intracellular polyamine concentrations in Tetrahymena (maximally 14mM putrescine and 4mM spermidine), it is suggested that the polyamine effects on ADI are of regulatory nature. Thus, polyamine biosynthesis in Tetrahymena thermophila is regulated not only on the level of ornithine decarboxylase activity, but also on an earlier step, the supply of ODC with substrates.     

Ribosome biosynthesis in Tetrahymena thermophila. III. Regulation of ribosomal RNA degradation in growing and growth arrested cells

We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient-deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one-half hours lasting for six hours is followed by a slower exponential degradation with a half-life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.     

Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila.

The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA. In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3'. Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei. The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.     

Changes in ornithine decarboxylase activity and polyamine levels during the growth of Tetrahymena thermophila cultures

Ornithine decarboxylase activity and polyamine levels were determined at various growth phases of Tetrahymena thermophila cultures. Enzyme activity and intracellular polyamines increased in exponentially growing cells and peaked just before the stationary phase. Putrescine was the predominant polyamine and spermidine and spermine concentrations were low throughout. The increase in putrescine level can be totally accounted for by the enzyme activity detected, provided that there is an ample supply of the precursor, L-ornithine.     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Multiple tubulin forms in ciliated protozoan Tetrahymena and Paramecium species

Summary. Tetrahymena and Paramecium species are widely used representatives of the phylum Ciliata. Ciliates are particularly suitable model organisms for studying the functional heterogeneity of tubulins, since they provide a wide range of different microtubular structures in a single cell. Sequencing projects of the genomes of members of these two genera are in progress. Nearly all members of the tubulin superfamily (α-, β-, γ-, δ-, ɛ-, η-, θ-, ι-, and κ-tubulins) have been identified in Paramecium tetraurelia. In Tetrahymena spp., the functional consequences of different posttranslational tubulin modifications (acetylation, tyrosination and detyrosination, phosphorylation, glutamylation, and glycylation) have been studied by different approaches. These model organisms provide the opportunity to determine the function of tubulins found in ciliates, as well as in humans, but absent in some other model organisms. They also give us an opportunity to explore the mechanisms underlying microtubule diversity. Here we review current knowledge concerning the diversity of microtubular structures, tubulin genes, and posttranslational modifications in Tetrahymena and Paramecium species. Correspondence and reprints: Institute of Molecular Genetics, Czech Academy of Sciences, Vídeňská 1083, 14220 Prague, Czech Republic.     

Regulation of polyamine transport by polyamines and polyamine analogs

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in V_max to levels ～4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N¹, N¹²-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 μM BESPM, V_max values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400–500 pmol/10⁶ cells—about 15–20% of the total polyamine pool (～2.8 nmol/10⁶ cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 μM produced an increase in SPD and SPM V_max values, whereas concentrations 3 μM and higher produced a marked suppression of these values. Cells treated with 3 μM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15–20%) changes in intracellular polyamine pools.     

Structural components of sphingophosphonolipids from the ciliated protozoan, Tetrahymena pyriformis WH-14.

1. Two sphingophosphonolipids were isolated from the lipids of the ciliated protozoan, Tetrahymena pyriformis WH-14. They were ceramide N-methyl-2-aminoethylphosphonate (CMAEP) and ceramide 2-aminoethylphosphonate (CAEP), in yields of 0.05 mg/g and 1.74 mg/g dry cells, respectively. 2. Two chromatographically distinguishable CAEP species were found, a slow-moving major component and a minor component which moved faster; the slow-moving one contained primarily hydroxy fatty acids, while in the other one nonhydroxy fatty acids were predominant. However, their long-chain base constituents were similar. 3. The major fatty acids of CAEP were 2-hydroxy acids with carbon numbers of 16 to 19, which were almost exclusively iso-types. The fatty acids of CMAEP consisted mainly of palmitic, iso-octadecanoic, and 2-hydroxy iso-heptadecanoic acids. 4. The long-chain bases were dominated by C16, C17, and C19 iso-4-sphingenine homologs.     

Environmental noise and population dynamics of the ciliated protozoa Tetrahymena thermophila in aquatic microcosms

Population theory predicts that the reddened environmental noise, especially in combination with high population growth rate, reddens population dynamics, increases population variability and strengthens environment–population correlation. We tested these predictions with axenic populations of ciliated protozoa Tetrahymena thermophila. Populations with low and high growth rate were cultured in a stable environment, and in environments with sublethal temperature fluctuations that had blue, white and red spectra (i.e. negatively autocorrelated, uncorrelated, or positively autocorrelated, respectively). Population size and biomass of individuals were determined at 3-h intervals for 18 days.
Dynamics of all populations were reddened, suggesting that internal mechanisms can redden the population spectra. However, population dynamics were reddest, variability highest, and environment–population correlation strongest in the red environment as predicted. Contrary to theoretical predictions and previous empirical findings, population growth rate (r_max being equal to 0.05 and 0.3 h⁻¹) had no effect on population dynamics.
Mean cell size and variability of cell size were affected by the presence and type of environmental noise suggesting that the physiological consequences of variability depend on colour. Environmental variability decreased mean population size and biomass and the decrease was strongest in rapidly fluctuating blue and white environments. The latter finding implies that rapid fluctuations are physiologically stressful, an effect that is not accounted for in the basic population models.