Tetrahymena pyriformisl

Transcription

1 JOURNAL OF BACTERIOLOGY, Sept., 1965 Copyright 1965 American Society for Microbiology Vol. 90, No. 3 Printed in U.S.A. Free Amino Acids in Serine-Antagonized Cells of Tetrahymena pyriformisl JUNE B. WRAGG, HOWARD REYNOLDS, AND MICHAEL J. PELCZAR, JR. Human Nutrition Research Division, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland, and Department of Microbiology, University of Maryland, College Park, Maryland Received for publication 10 June 1965 ABSTRACT WRAGG, JUNE B. (Agricultural Research Service, Beltsville, Md.), HOWARD REY- NOLDS, AND MICHAEL J. PELCZAR, JR. Free amino acids in serine-antagonized cells of Tetrahymena pyriformis. J. Bacteriol. 90: Growth inhibition of Tetrahymena pyriformis by L-serine in a chemically defined medium was reversed by L-arginine in a manner which resembled competitive antagonism. Composition of the free amino acid pools from cells grown in either a balanced amino acid mixture or a mixture with serine concentrations which inhibited growth suggested an antagonism by serine with energy-yielding reactions. Growth in media with excess serine resulted in the accumulation of higher concentrations of free cellular amino acids and an apparent increase in the rate of conversion of arginine to ornithine, as compared with growth in the balanced medium. The results suggested that serine or a metabolic product of serine interferes with the formation of pyruvic acid. In the presence of high levels of serine, arginine appeared to be metabolized more rapidly and to be spared when alanine, aspartic acid, or glutamic acid was added to the unbalanced medium. Experiments that involve the effect of nutrient balance on growth of microorganisms have often revealed antagonisms between naturally occurring amino acids (Meister, 1957). The antagonizing effects of serine have been reported for a number of microorganisms (McCoy et al., 1954; O'Barr, Levin, and Reynolds, 1958; Schwartz, Maas, and Simon, 1959; Smith and Higuchi, 1960; Durham and Milligan, 1962; Nicolle and Walle, 1963; Grula and Grula, 1962, 1963). Several of these studies revealed growth inhibition by D-serine, whereas few noted growth inhibition by L-serine. Davis and Maas (1949) concluded that the mechanism of inhibition by D-serine had no close relation to the metabolism Of L-serine. Dewey and Kidder (1958) reported growth inhibition of Tetrahymena pyriformis by high concentrations of all required amino acids with the exception of lysine. L-Serine only antagonized the utilization of L-threonine. In general, antagonism by one amino acid of the utilization of another involved structurally related com- I Presented in part at the Annual Meeting of the American Society for Microbiology, Washington, D.C., 3 to 7 May A portion of this material was submitted by June B. Wragg in partial fulfillment of the requirements for the M.S. degree, University of Maryland, College Park. pounds. Singer (1961) observed that inhibition of growth of T. pyriformis H by high DL-serine levels was most pronounced at high concentrations of folic acid, and was relieved by raising the level of methionine or by adding pyrimethamine to decrease available folate. Research on the subject of free amino acid pools in microorganisms was summarized recently (Holden, 1962). Reports included the effect of amino acid antagonisms on transport and accumulation of specific amino acids by bacteria, but none dealt with the effect of antagonisms on the pattern of free amino acids. However, in animal studies, investigators (Rogers, Spolter, and Harper, 1962; Tannous, Rogers, and Harper, 1963; Sanahuja and Harper, 1963) examined leucine-isoleucine antagonism in rats by analysis of plasma amino acids. The present study is concerned with the examination of an apparent interaction between serine and arginine. In an attempt to gain information on the nature of this antagonism, the patterns of free amino acids in cells of T. pyriformis were examined after cultivation in media with serine at balanced or at growth-limiting levels. MATERIALS AND METHODS Cultures and medium composition. T. pyriformis strain W was maintained and cells for experi- 748

2 VOL. 90, 1965 SERINE ANTAGONISM IN T. PYRIFORMIS 749 mental use were produced as described by Reynolds and Wragg (1962). Cells for inoculum were removed from the medium during the logarithmic phase of growth (72 hr) by centrifugation at 60 X g, at 2 C. After two washes with sterile distilled water, the cell concentrate was resuspended in water to provide initial inoculations of 10,000 cells per milliliter. The defined experimental medium was as follows. The essential amino acid mixture, which was adapted from Elliott, Brownell, and Gross (1954), provided 304,ug/ml of total L-amino acid nitrogen (Table 1). Other ingredients represented a modification from Dewey and Kidder (1958) and were described earlier (Reynolds and Wragg, 1962). Dextrin, 1.8% (w/v; equivalent to 2%o glucose), was sterilized by autoclaving at 15 psi for 15 min with the other ingredients. Conditions of growth. For growth studies, T. pyriformis was grown in triplicate 4-ml quantities of medium dispensed in vials (22 by 50 mm). Turbidity was determined with a Bausch and Lomb Spectronic-20 colorimeter at 650 m,. Cell crops for amino acid pool analyses were grown in 200-ml quantities of medium contained in 2,500-ml culture flasks. For determination of a-amino nitrogen in cell pools, T. pyriformis was cultured in 10-ml quantities of medium dispensed in 50-ml Erlenmeyer flasks. Incubation was at 29 C for 72 hr. The three types of cultures all provided highly aerobic conditions. Free amino acid extraction from T. pyriformis cells and preparation for chromatography. T. pyriformis cells, cultivated as described above, were harvested by centrifugation at 60 X g (at 2 C for 15 min), washed two times in Ringer's solution, and suspended in approximately 5 ml of distilled water. With this washing procedure, the cells remained viable, and there was no detectable amino nitrogen in the final wash. The suspension was heated for 5 min in a boiling-water bath, and the extracted amino acids were decanted from the cell residue after centrifugation. The cell residue was extracted with two additional portions of water, and the combined extracts were dried in vacuo at 50 C. The dried extract was heated in 80% (v/v) ethyl alcohol, and all alcohol-soluble substances were transferred to a separatory funnel and extracted with chloroform-alcohol (3:1) to remove lipid material. The chloroform fraction was extracted with additional alcohol. Combined alcohol fractions were evaporated to near-dryness and made up to volume with 0.1 N HCl for direct application to the chromatographic column. The volume of each extract was adjusted to provide approximately 35,ug of total a-amino nitrogen in 0.5 ml of solution. Total a-amino nitrogen was determined by use of the ninhydrin method of Yemm and Cocking (1955). The Technicon Autoanalyzer (Technicon Chromatography Corp., Chauncey, N.Y.) was employed for complete quantitative separation of 18 or more amino acids within a 24-hr period by ion-exchange chromatography. Two separate amino acid extracts were TABLE 1. Balanced amino acid mixture used for studies with Tetrahymena pyriformis L-Amino acid Amt yg/ml Arginine HCI Histidine HCl*H Isoleucine Leucine Lysine HCl Methionine Phenylalanine Serine Threonine Tryptophan Valine Total nitrogen prepared and analyzed from cells grown in each experimental medium, and each extract was chromatographed twice. Calculation of amino acid concentration and test of significance. Concentrations of individual amino acids in extracts were calculated from curves obtained by chromatographing a standard solution containing 0.25,pmole of each amino acid. Comparisons of amino acid patterns are based on 18 amino acids, selected because of their definite identification and presence in significant quantities. Data on individual amino acids are reported as moles per cent of the total 18 amino acids. The significance of differences in relative concentrations of specific amino acids was tested by analysis of variance and mean separation by use of Duncan's multiple range test (LeClerg, 1957). The coefficient of variation was calculated from successive standard amino acid mixtures for each of the reported 18 amino acids. Coefficients of variation were less than 5%, with the exception of proline, leucine, and threonine which were 7.3, 5.2, and 5.4%, respectively. Preparation of T. pyriformis cell extract for determination of a-amino nitrogen in cell pools. T. pyriformis cells were counted and measured, and amino acids were extracted. The cell suspensions were treated with 10% (w/v) trichloroacetic acid, heated, and extracted with 80% (v/v) ethyl alcohol. The total a-amino nitrogen was determined directly in the ethyl alcohol fraction by use of the method of Yemm and Cocking (1955). Cell counts were made with a haemocytometer on diluted cell suspensions that were inactivated with phosphate-buffered formaldehyde. Cell volume was calculated from the measurements of cell length and width on the basis of cell shape as approximating that of a prolate spheroid (Corbett, 1958). Average values were derived from measurements of 20 cells from each sample. RESULTS Amino acid composition of media for growth studies. In 't synthetic medium containing the L

3 750 WRAGG, REYNOLDS, AND PELCZAR J. BACTERIOL. TABLE 2. Growth of Tetrahymena pyriformis with 80 or 452 mg/ml of arginine and various levels of serine L-Serine Growth* with L-arginine hydrochloride at 80 jg/ml 452 pg/ml pg/ml , , , * Growth of T. pyriformis expressed as percentage of that obtained when the serine concentration was 200,ug/ml. forms of 11 amino acids in the proportions shown in Table 1, the growth response of T. pyriformis was constant at nitrogen concentrations ranging from approximately 300 to 1,500,g/ml. In a medium containing the DL-isomers of these 11 amino acids, or the 16 amino acids used by Dewey and Kidder (1958), less growth of T. pyriformis resulted. Interaction between L-serine and L-arginine. While examining amino acid interactions in T. pyriformis, a growth-limiting effect of elevated L-serine concentrations was observed. In the experimental medium with L-arginine hydrochloride at concentrations between 80 and 452 jig/ml, growth of T. pyriformis was progressively inhibited as the serine concentration was raised. The results in Table 2 show the effect of elevated serine levels on growth. In media with similar background amino acids, T. pyriformis growth with 1,000 and 2,000,ug/ml of serine was approximately 70 and 40%, respectively, as compared with that in medium with 200 Utg/ml of serine. The percentage of inhibition of growth by serine was the same whether the medium contained 80 or 452,ug/ml of arginine, although growth at the higher level averaged approximately twice that at the low level of arginine. Figure 1 shows the effect of increasing arginine concentration on growth of T. pyriformis at two serine levels with various background concentrations of amino acid nitrogen. The background amino acid nitrogen of 156,ug/ml is the mixture of Table 1, minus the serine and arginine. Variations of this background nitrogen concentration included 79 and 394 jg/ml, with the ratio of one amino acid to the other remaining constant. L-Serine at 2,000,ug/ml was inhibitory, as compared with 200 Aug/ml of serine, at all three levels of background amino acid nitrogen. Increased levels of arginine counteracted this serine inhibition, but reversal was essentially complete only in the medium with the highest level (394,ug/ml) of amino acid nitrogen. In the media with the lower background levels of nitrogen, decreased growth at high levels of arginine may be assumed to indicate inhibition by arginine of one or more of the other amino acids in the basal medium. Effect of other amino acids on growth inhibition of T. pyriformis by serine. Observations pre L-ARGININE HCL 103 jg/ml FIG. 1. Growth response of Tetrahymena pyriformis to increasing levels of L-arginine hydrochloride in chemically defined medium as determined spectrophotometrically at 650 m,. Two levels of L-serine, 200 (0) and 2,000 (0) gg/ml, were used with different background amino acid concentrations at 79 (A), 156 (B), and 394 (C),ug/ml of amino acid nitrogen.

4 VOL. 90X 1965 SERINE ANTAGONISM IN T. PYRIFORMIS 751 TABLE 3. Growth of Tetrahymena pyriformis in media with 200, 1,000, or 2,000,ug/ml of serine, and with 2,000,ug/ml of serine plus supplemental amino acids Growth* with L-arginine hydro- Serine Supplemental amino acid chloride at (1,000 lag/ml) 266 jag/ml 452,ug/mI,ug/mil , , ,000 L-Arginine HC ,000 L-Aspartic acid ,000 L-Alanine ,000 L-Glutamic acid ,000 L-Valine ,000 L-Leucine ,000 L-Histidine HCI-H ,000 L-Proline ,000 L-Phenylalanine ,000 L-Isoleucine ,000 L-Lysine HCl ,000 L-Threonine ,000 L-Methionine ,000 Glycine * Growth expressed as percentage of that obtained with serine at 200,Ug/mi. sented later of the effect of serine inhibition on the free amino acids in T. pyriformis cells led to experiments testing the effect of supplements of other amino acids on growth of the protozoan in media with 2,000,ug/ml of serine. Data showing the effects of 1,000,ug/ml supplements of other amino acids are presented in Table 3 as per cent of growth obtained in the unsupplemented medium with 200,ug/ml of serine. Several levels of L-arginine, ranging from 45 to 452,ug/ml, were tested. However, only the data obtained at 266 and 452 Ag/ml of arginine are reported, because the results at other levels were similar. Additions of L-alanine, L-aspartic acid, or L-glutamic acid resulted in complete reversal of serine inhibition. L-Valine, L-leucine, and L-histidine partially reversed the inhibition of 2,000,ug/ml of L-serine, permitting growth comparable to that in medium with 1,000 jig/ml of L-serine. L-Proline, L-phenylalanine, L-isoleucine, L-lysine, L-threonine, and L-methionine had little or no effect in reversing serine inhibition, whereas glycine further enhanced depression of growth. Amino acid patterns in extractable pools of T. pyriformis in media with balanced or growthinhibiting serine levels. The patterns of free amino acids in cells grown with 200, 1,000, or 2,000 Mg/ml of L-serine are presented in Table 4. Since there was little cysteic acid and cystine, the data for these two compounds were combined, as were those for methionine and methionine sulfoxide. With the exception of ornithine, the free amino acids in the pools of T. pyriformis were in agreement with those reported by others (Wu and Hogg, 1956; Scherbaum, 1959; Schleicher, 1959; Wells, 1960; Loefer and Scherbaum, 1961). Wu and Hogg (1956) identified ornithine but suggested that ornithine could have been derived from arginine during desalting. Since the free amino acids in this investigation were obtained from boiling-water extracts, the destruction of free arginine is improbable. Hill and van Eys (1964) have also reported the appearance of small amounts of free ornithine in T. pyriformis grown in a defined medium. The proportion of serine was more than double in cells from media with 1,000 or 2,000,ug/ml of serine than in cells from medium with 200 Atg/ml of serine. These cells also contained significantly higher proportions of ornithine and lower proportions of leucine than cells from the balanced medium. The free amino acid pool from cells grown with the highest level of serine contained significantly reduced levels of arginine and aspartic acid. Also recorded in Table 4 is the total a-amino nitrogen per unit volume of cells grown in media with various serine concentrations. Total a-amino acid nitrogen in cells from media with 1,000 and 2,000,g/ml of serine was approximately 1.5 and 3.5 times as high, respectively, as that in cells from the medium with 200,ug/ml of serine. Chromatograms indicated the presence of small amounts of several additional compounds in the pools, which are not recorded in Table 4. Peaks corresponding with the elution time for urea appeared on the chromatograms of all pools, but the levels indicated were too low and variable to provide useful correlations with media composition. Although several investigators (Wu and Hogg, 1956; Dewey, Heinrich, and Kidder, 1957; Elliott, 1959) found no evidence of the urea cycle in T. pyriformis, Seaman (1954) reported that extracts of this protozoan contained all the enzymes of the Krebs-Henseleit urea cycle and later (Seaman, 1959) presented cytochemical evidence of urease activity in intact cells. Data on tryptophan were also inadequate because of the low concentrations of this amino acid. Taurine, as well as several unknown nitrogen compounds, was present in all cell pools. Ammonia nitrogen was approximately three times as high in cells from the medium with

5 752 WRAGG, REYNOLDS, AND PELCZAR J. BACTERIOL. TABLE 4. Extractable amino acids in Tetrahymena pyriformts after 72 hr of incubation in several amino acid mixtures Amino acid Amt (ug/ml) of L-serine in medium 2, ,000 2, ,000 1,000 2,000 MAg/mI of L-aspartic pg/ml of L- acid glutamic acid Alanine a Arginine * b Aspartic acid * Cystine + cysteic acid Glutamic acid Glycine Histidine Isoleucine Leucine * 6.10* * Lysine Methione + methionine sulfoxide Ornithine ** Phenylalanine Proline Serine ** 21.64** 27.38** 29.58** Threonine Tyrosine Valine Total i.ag of cz-amino nitrogen/ mm3 of cells ,447 3,581 1, a Results for each amino acid expressed as moles per cent of total. b Significantly different than in medium with 200,ug/ml of L-serine at 5% level (*) or 1% level (**), determined by Duncan's Multiple Range Test (LeClerg, 1957). 2,000,ug/ml of serine as compared with cells from the medium with 200,ug/ml of serine. Amino acid patterns in extractable pools of T. pyriformis in media with growth-inhibiting serine levels plus aspartic acid or glutamic acid. Table 4 also presents data on the free amino acids of T. pyriformis cells grown in medium with 2,000,Lg/ml of serine plus 1,000 jg/ml additions of L-aspartic acid or L-glutamic acid. These are two of the three previously noted amino acids which completely reversed the inhibition caused by 2,000,ug/ml of serine. Their addition to the growth-inhibitory medium also reduced cellular accumulation of amino acids to levels similar to those observed in cells cultured in the medium with 200 Aug/ml of serine. Examination of the individual amino acids revealed that the high proportion of serine in the cell pool was not the immediate cause of growth inhibition in media with high serine content. The proportion of serine was higher in the pools of cells grown in medium with the above amino acid additions than in those from cells grown with 2,000,g/ml of serine alone, whereas arginine increased to the level observed in cells grown with 200,ug/ml of serine, and ornithine decreased. Although high levels of serine in the medium caused an increase of this amino acid in cell pools, additions of L-aspartic acid or L-glutamic acid at a concentration of 1,000,ug/ml to the high serine medium did not increase the pool levels of either of the latter amino acids. DISCUSSION The apparent antagonism of arginine by serine, as evidenced by the growth inhibition of T. pyriformis, was counteracted by increasing arginine concentrations. Alanine, aspartic acid, and glutamic acid completely reversed the antagonizing effects of serine. However, free amino acid analyses of cells grown in media with serine at balanced or at growth-limiting levels indicated an interference by serine with energyyielding reactions. The results demonstrated that serine was not toxic per se. In the unbalanced medium, 1,000,ug/ml of serine caused growth inhibition of T. pyriformis. When the other 10 amino acids in the medium were increased uniformly with this high serine concentration, growth inhibition was

6 VOL. 90, 1965 SERINE ANTAGONISM IN T. PYRIFORMIS not observed. Also, the high pool concentration of serine in cells grown in media with high levels of serine and either aspartic acid or glutamic acid was tolerated and did not impair growth. Dewey and Kidder (1958) also excluded the nonspecific toxicity of high amino acid concentrations as an explanation of their observed antagonisms in T. pyriformis. The reduced proportion of arginine and the relatively increased accumulations of ornithine and ammonia in the pool of cells grown with 2,000,Lg/ml of serine indicate that the high level of serine stimulated intracellular metabolism of arginine. It seems probable that the arginineto-ornithine conversion plays a significant role in energy production in T. pyriformis, and that the serine inhibition imposes extra demands on this pathway. Dewey et al. (1957) reported that the main pathway of arginine metabolism in T. pyriformis was through citrulline and ornithine, and Wu and Hogg (1952) observed that a high rate of arginine catabolism was characteristic of this protozoan. The suggestion that arginine metabolism is important for energy production in T. pyriformis has parallels in other organisms, e.g., Streptococcus faecalis (Deibel, 1964). If arginine is used as one of several energy sources, and if serine blocks the production of energy from one of these other sources (see below), there would be an increased demand on other energy sources including arginine. This would be reflected by an increase in the concentration of ornithine and ammonia in the free pool, which was observed previously. Additional evidence pointing to interference with the production of energy was that supplements of aspartic acid and glutamic acid (which readily convert to tricarboxylic acid cycle intermediates), and of alanine (which readily produces pyruvate; Seaman, 1955; Schleicher, 1959), completely reversed growth inhibition of T. pyriformis by serine. Cells grown with either aspartic acid or glutamic acid additions in the medium with 2,000,ug/ml of serine resulted in pool levels of arginine and ornithine comparable to those levels in pools from cells grown in the balanced medium. Because the pool concentration of aspartic acid and glutamic acid did not increase in cells grown in the presence of these amino acids, we suggest that these amino acids were used as sources of energy, thus reducing demands on the arginine pathway as an additional energy source. Although the cells grown in medium with alanine addition were not analyzed, it is suggested that alanine was also used for energy. 753 Fishman and Artom (1945) hypothesized that DL-serine caused a defect in disposal of pyruvic acid in rats. Gado et al. (1961) also found that serine additions to media with Streptomyces rimosus resulted in a pyruvate increase, and reduced growth. However, the present results do not indicate that large quantities of pyruvate accumulated in T. pyriformis as a result of interference with pyruvate metabolism by high serine levels, because alanine, as well as aspartic acid and glutamic acid, reversed the growth inhibition when added to the unbalanced medium. The block by serine must therefore occur prior to pyruvate formation and may involve interference at some point in the three-carbon-compound conversion between 3-phosphoglycerate and pyruvate by either serine or one of its metabolic products. Such a suggestion would be consistent with the results of recent studies with Escherichia coli which have shown that the biosynthesis of serine in this organism proceeds via 3-phosphoglycerate, phosphohydroxypyruvate, and phosphoserine (Umbarger, Umbarger, and Siu, 1963; Pizer and Potochny, 1964), and that L-serine was an effective inhibitor of phosphoglycerate dehydrogenase (Pizer, 1963). LITERATURE CITED CORBETT, J. J Factors influencing substrate utilization by Tetrahymena pyriformis. Exptl. Cell Res. 15: DAVIS, B. D., AND W. K. MAAS Inhibition of Escherichia coli by D-serine and the production of serine-resistant mutants. J. Am. Chem. Soc. 71:1865. DEIBEL, R. H Utilization of arginine as an energy source for the growth of Streptococcus faecalis. J. Bacteriol. 87: DEWEY, V. C., M. R. HEINRICH, AND G. W. KID- DER Evidence for the absence of the urea cycle in Tetrahymena. J. Protozool. 4: DEWEY, V. C., AND G. W. KIDDER Amino acid antagonisms in Tetrahymena. Arch. Biochem. Biophys. 73: DURHAM, N. N., AND R. MILLIGAN A mechanism of growth inhibition by D-serine in a Flavobacterium. Biochem. Biophys. Res. Commun. 7: ELLIOTT, A. M., L. E. BROWNELL, AND J. A. GROSS The use of Tetrahymena to evaluate the effects of gamma radiation on essential nutrilites. J. Protozool. 1: ELLIOTT, A. M Biology of Tetrahymena. Ann. Rev. Microbiol. 13: FISHMAN, W. H., AND C. ARTOM Variations of some constituents in the urine of rats receiving DL-serine and DL-alanine. Proc. Soc. Exptl. Biol. Med. 60: GADO, I., A. SZENTIRMAI, K. STECZEK, AND I. HORVATH Metabolic studies with Strep-

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