Reagents. Amino acids used in nutrition experiments

Transcription

1 UTILIZATION OF AMINO ACIDS AS CARBON SOURCES BY STREPTOMYCES FRADIAE1 ANTONIO H. ROMANO2 AND WALTER J. NICKERSON Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey Received for publication August 8, 1957 There have been several investigations on the amino acid nutrition of Streptomyces. Early work on this subject by Waksman (1920) showed that certain single amino acids satisfied the nitrogen requirements of representative species of the genus Streptomyces. Considerable specificity in the ability to deaminate amino acids is exhibited by Streptomyces, as Woodruff and Foster (1943) have shown with Streptomyces lavendulae, while Gottlieb and Ciferri (1956) showed that the only amino acids deaminated by Streptomyces venezuelae were those capable of supporting growth when supplied as the sole source of nitrogen. There have been relatively few studies on the use of amino acids as carbon sources by Streptomyces. Waksman and Lomanitz (1925) showed that glutamic acid and alanine as sole carbon sources supported good growth of Streptomyces viridochromogenes, whereas glycine and asparagine were poor carbon sources, but supported good growth in the presence of glucose. Nickerson and Mohan (1953a, 1953b) showed that certain amino acids were utilized by Streptomyces fradiae as sole carbon and nitrogen sources; included in this group were glutamic acid, proline, and arginine. Other amino acids, such as aspartic acid, leucine, isoleucine, methionine, phenylalanine, and threonine, could not serve as carbon sources. It has been the object of the present investigation to extend these findings, and to elucidate the enzymatic basis of the specificity exhibited by this organism toward amino acids as carbon sources. MATERIALS AND METHODS Organism and culture media. The organism employed was strain 3535 of Streptomyces fradiae 1 Supported in part by a grant from the National Institutes of Health, United States Public Health Service. 2 Present address: Robert A. Taft Sanitary Engineering Center, United States Public Health Service, Cincinnati 26, Ohio. from the collection in the Institute of Microbiology. The organism was carried on potato agar slants; inoculation of test media was made by washing spores from the surface of the agar with sterile distilled water and transferring 0.1 ml of the spore suspension to the test media. The basal medium (A) employed for growth studies had the following composition: K2HPO4, 1.5 g; MgSO4. - 7H20, g; CaCl2 2H20, g; FeSO4 7H20, g; ZnSO4.7H20, g; and distilled water to make 1000 ml. This medium was distributed in 100 ml amounts to 250-ml Erlenmeyer flasks and was sterilized by autoclaving at 120 C for 20 min. All additions to this basal medium were neutralized if acidic or basic, sterilized separately, and added aseptically. After inoculation, the cultures were incubated at 28 C on a rotary shaker which was set at 250 rpm. Measurement of growth. Mycelial dry weights were determined by filtering the cultures through Whatman no. 2 filter paper which had been previously dried at 90 C for 18 to 24 hr and weighed. The mycelia and filter papers were then dried under the same conditions and the differences in weight were determined. Reagents. Amino acids used in nutrition experiments were obtained from commercial sources and were used in the forms stated in tables 1 to 3. Glutamic acid and aspartic acid used in experiments with cell free extracts were obtained from Matheson, Coleman, and Bell, and were in the DL form. Purity of the amino acids was checked by paper chromatography, using a phenol system to which was added 0.04 per cent 8-hydroxyquinoline. Cell free preparations. The cell free extracts used in the transaminase and dehydrogenase experiments were prepared in the following manner. Cells were grown on a basal medium (B) similar in composition to that given above but with different salt concentrations: K2HPO4, 0.5 g; MgSO4-7H20, 0.2 g; CaCl2-2H20, 0.25 g; FeSO4-7H20, g; ZnSO4-7H20, g; 161

2 162 ROMANO AND NICKERSON [VOL. 75 and distilled water to make 1000 ml. It was found that transaminase activity in cell free extracts was greater when cells were grown on a medium with this basal formulation than when grown in the medium with higher phosphate and lower concentrations of zinc and iron. This finding was not investigated in further detail. To basal medium B there was added sodium glutamate and sodium aspartate, 0.5 per cent each, and glucose, 1.0 per cent. The cultures were incubated for 72 hr at 28 C on the rotary shaker. The cells were harvested by centrifugation, and were washed twice with distilled water and twice with M/40 phosphate buffer. In the transaminase studies, a buffer at ph 7.2 was used; in the dehydrogenase experiments, a buffer of ph 7.0 was used. The cells were resuspended in buffer to give a suspension containing approximately 10 mg cells per ml. This cell suspension was subjected to disintegration for 5 min in a Raytheon 50 watt, 9-kc Magnetostriction oscillator at a plate voltage of 130 v and an output voltage of 100 v. The suspension was then centrifuged at 2500 X G for 5 min to remove whole cells. The supernatant solution was used as the enzyme preparation. Enzymatic studies. Transaminase activity was determined by a modification of the method used by Feldman and Gunsalus (1950), whereby the appearance of specific amino acid spots on paper chromatograms was noted. Whatman no. 1 filter paper was spotted with 5.AL of the reaction mixtures after suitable incubation periods. The papers were irrigated with water-saturated phenol containing 0.04 per cent 8-hydroxyquinoline for 24 hr. The papers were dried at room temperature in a current of air for 3 hr, and were then sprayed with 0.25 per cent ninhydrin in water-saturated butanol. It was found necessary to dialyze the cell free extract against buffer at 5 C for 24 hr in order to remove free amino acids which were present in the enzyme preparation in amounts sufficient to give spots on the chromatograms, and thus make detection of activity difficult. Pyridoxal phosphate (obtained from California Biochemical Research Foundation), was added to the reaction mixtures to supply the coenzyme requirement. Solutions of oxalacetic acid and a-ketoglutaric acid (obtained from Krishell Laboratories) were prepared immediately before use and were neutralized with equivalent amounts of NaOH. Dehydrogenase activity was determined by the method of Fahmy and Walsh (1952), using 2,3, 5-triphenyltetrazolium chloride (obtained from Arapahoe Laboratories) as the hydrogen acceptor. Coenzyme I (DPN) (90 per cent purity) was obtained from Schwarz Laboratories, and coenzyme II (TPN) (65 per cent purity) was obtained from Sigma Chemical Company. Optical density readings were made with a Klett-Summerson photoelectric colorimeter. RESULTS Growth with amino acids as sole sources of carbon and of nitrogen. As shown in table 1, glutamic acid, proline, arginine, alanine, and histidine sup- TABLE 1 Growth of Streptomyces fradiae with single amino acids as sole sources of carbon and of nitrogen Growth Relative to Addition to Basal Medium* Growth Glutamate (on Molar Basis) mg dry WI! 100 ml culturet % L-Glutamate, monosodium L-Proline L-Arginine * HCl DL-Alanine (77.0)t L-Histidine*HCl L-Lysine*HCl Hydroxy-L-proline Glycine Glycine anhydride L-Aspartate, monosodium L-Asparagine L-Leucine DL-Methionine... O. 0.0 DL-Threonine Creatine H DL-a-Amino-n-butyrate 0... O. O.O DL-a-Amino-n-isobutyrate DL-ca-Amino-n-valerate * All substances sterilized separately in aqueous solution, and incorporated aseptically to give final concentration of 1 g per 100 ml medium. t Cultures incubated with continuous agitation for 5 days at 28 C. All values are the average of duplicate flasks from at the least two different experiments; the values for glutamate and aspartate are based on determinations from 5 different experiments, all in close agreement. t With DL-alanine the growth of S. fradiae is 77 per cent of that with glutamate, if only the j,-form of alanine is utilized.

3 19581 AMINO ACID AS CARBON SOURCE IN S. FRADIAE 163 port relatively good growth of Streptomyces fradiae. Lysine supports modest growth. Glycine and hydroxy proline permit very slight growth, and are ineffective on a molar basis, whereas other amino acids listed do not support growth of S. fradiae. In addition to the amino acids listed in table 1, the following have been shown previously (Nickerson and Mohan, 1953a) not to permit growth of S. fradiae when supplied as sole carbon sources: phenylalanine, tyrosine, tryptophan, valine, serine, and isoleucine. Utitization of aspartate in combination uith another carbon source. It was demonstrated previously (Nickerson and Mohan, 1953b) that although aspartic acid and asparagine were ineffective when given as the sole source of carbon for S. fradiae, very much more growth was obtained on a "nutrient pair," such as aspartate (or asparagine) + glutamate, than on glutamate alone. This observation indicated that the carbon of aspartate could be used if a suitable "primer" were also present. Since aspartic acid has been implicated in the biosynthesis of a variety of cellular constituents, many areas were explored by the nutrient pair technique in an attempt to discover a biochemical "lesion" that would prevent aspartate from being utilized as the sole source of carbon. One such area examined dealt with the known involvement of aspartic acid in pyrimidine biosynthesis, another area that was studied dealt with the biosynthesis of aspartic and glutamic acids and the interconversion thereof via their respective keto acids. There is substantial evidence that aspartic acid plays a role in the synthesis of pyrimidines. Reichard and Lagerkvist (1953) and Reichard (1954) demonstrated a reaction in rat liver mitochondria by which ammonia, carbon dioxide, and L-aspartate condense to give L-ureidosuccinate which is a precursor of orotic acid. Accordingly, the effect of uracil and of orotic acid on the utilization of aspartate by S. fradiae was examined. No significant growth was obtained with either of these substances when paired with aspartate. From these results it might appear that the biochemical lesion responsible for lack of aspartate utilization by S. fradiae does not involve a block in the synthesis of pyrimidines from aspartic acid. (That only one "lesion" appears to be involved in aspartate utilization is apparent from the work that follows.) For a number of microorganisms it has been demonstrated that aspartic acid and glutamic acid arise from the respective a-keto acids which are formed by operation of the Krebs cycle (this work has been reviewed critically by Ehrensviird, 1955). The reverse reaction, the formation of Krebs cycle intermediates from these amino acids, has also been amply demonstrated in many organisms. The possibility was considered that S. fradiae was in some manner unable to convert aspartic acid (when supplied alone) into oxalacetic acid, thereby lacking any energy-yielding mechanism through which this amino acid could be metabolized. This possibility was tested by adding various intermediates of the Krebs cycle to culture media to form nutrient pairs with aspartic acid. As seen in table 2 good growth of S. fradiae was obtained in media with aspartate plus fumarate, malate, or succinate. Little or no growth was obtained in combinations of aspartate plus acetate, glutarate, itaconate, citrate, oxalacetate, or butyrolactone. The lack of growth under these conditions with citrate has been shown in control experiments to be due to the powerful metal-chelating property of citrate (S. fradiae, and other species of Streptomyces examined are extraordinarily sensitive to inhibition by powerful metal-chelating agents). Lack of growth with oxalacetate is probably due to the instability of this substance which decomposes rapidly in TABLE 2 Effect of Krebs cycle intermediates and other carbon sources on the utilization of aspartic acid by Streptomyces fradiae Addition to Basal Medium Growth 3 days I5 days mg dry wt/100 ml culture L-Aspartate* L-Aspartate + Na succinate L-Aspartate + Na fumarate L-Aspartatet + Na malate L-Aspartate + Na acetate... _ 4.0 L-Aspartate + Na citratet. 1.0 L-Glutamate* L-Glutamate + L-aspartate L-Glutamate + glucose * All substrates supplied at 1.0 g per 100 ml medium. t All substrates supplied at 0.5 g per 100 ml medium.

4 164 ROMANO AND NICKERSON [VOL. 75 aqueous solution into acetate and oxalate ions. Experiments in which freshly prepared solutions of oxalacetate were added at intervals of 4 hr to culture medium containing 1.0 per cent aspartate were also unsuccessful. The permeability of whole cells to these substances may also be a limiting factor. The data in table 2 indicate, however, that growth of S. fradiae with aspartate is possible if intermediates of the Krebs cycle are made available. It is most probable that in these experiments aspartate is serving primarily as a source of nitrogen, and that its amino group is transferred to a suitable acceptor through the agency of a transaminase. As is shown in a following section, an active aspartate-glutamate transaminase system is present in S. fradiae. The data of table 2 confirm that aspartate is usable by this organism as a source of nitrogen, and indicate that the unsuitability of aspartate as a sole substrate rests on the evident inability of the organism to transform aspartate (except by transamination) so as to provide an amino group acceptor. Comparison of the amount of growth (table 2) made on glutamate alone with that on glutamate + aspartate and glutamate + glucose, indicates that the carbon of aspartate can also be utilized provided a suitable primary carbon source is supplied. Dehydrogenase studies. We next tested to see whether or not this organism had a mechanism to deaminate aspartate when this substance was supplied alone; if not, aspartate could not enter into the tricarboxylic acid cycle or other pathway of carbon metabolism. The fact that fumarate is a suitable carbon source for S. fradiae (table 2) would appear to exclude the presence of aspartase, an enzyme which has been shown by Virtanen and Tarnanen (1932) to catalyze the following reaction: aspartic acid :. fumaric acid + ammonia. This enzyme system is the only firmly established mechanism for the deamination of aspartic acid (exclusive of transamination). The utilization of glutamate as an energy source would presumably take place through the agency of glutamic dehydrogenase, which catalyzes the oxidative deamination of glutamate. This enzyme has been found by other workers to be widely distributed in animal, plant, and microbial cells. Further oxidation of a-ketoglutarate would occur via the tricarboxylic acid cycle. The presence of glutamic dehydrogenase in Streptomyces fradiae is indicated by experiments with cell free extracts, as shown in figure 1. It is seen that there is a rapid reduction of 2,3,5- triphenyltetrazolium chloride (TTC) in the presence of the enzyme preparation, coenzyme I (DPN), and sodium glutamate. This enzyme system in Streptomyces fradiae appears to operate with coenzyme I; a marked lag in TTC reduction in presence of coenzyme II (TPN) is seen in figure 1. The onset of activity after the lag may be due to an enzymatic conversion of coenzyme II to coenzyme I by a mechanism similar to that found in yeasts, and first described by von Euler and co-workers in It is also seen in figure 1 that there was no dehydrogenase activity in the presence of aspartate, cell free extract, and coenzyme I or coenzyme II. This confirms the i- z Id a Extract + DPN + glutamate Extract + DPt N. -.., Extract 4 DP? N9 - + asparfa<te II* 0A - Extract + TPN I + glutamate 0I I O MINUTES a Extract + TPN, A-A Extract + TPN aspartate Figure 1. (At top) Rate of reduction of 2,3,5- triphenyltetrazolium chloride (TTC) by a cell free extract of Streptomyces fradiae in the presence of DPN and sodium glutamate or sodium aspartate. Reaction mixtures: cell free extract, 2.0 ml; DPN, 0.5 Amole; sodium glutamate or sodium aspartate, 50,umoles; TTC, 0.5 ml of 0.5 per cent solution; and M/40 phosphate buffer, ph 7.0 to make a total of 4.0 ml. (At bottom) Rate of reduction of 2,3,5-triphenyltetrazolium chloride by a cell free extract of Streptomyces fradiae in the presence of TPN and sodium glutamate or sodium aspartate. Reaction mixtures as given, except that TPN was substituted for DPN.

5 1958] AMINO ACID AS CARBON SOURCE IN S. FRADIAE Figure 2. Transamination reactions carried out by a cell free extract of Streptomyces fradiae. Contents of tubes: (1) Dialyzed enzyme, 0.3 ml; pyridoxal phosphate, 20 jug (2) sodium glutamate, 10 jimoles (3) sodium aspartate, 10,umoles, (4) dialyzed enzyme, 0.3 ml; pyridoxal phosphate, 20,g; sodium glutamate, 10,moles (5) dialyzed enzyme, 0.3 ml; pyridoxal phosphate, 20,g; sodium glutamate, 10,moles; sodium oxalacetate, 10,moles (6) dialyzed enzyme, 0.3 ml; pyridoxal phosphate, 20 Mg; sodium aspartate, 10,umoles (7) dialyzed enzyme, 0.3 ml; pyridoxal phosphate, 20,g; sodium aspartate, 10 MAmoles; sodium a-ketoglutarate, 10,moles. M/15 phosphate buffer, ph 7.2 in all tubes to give total volume of 1.0 ml. Tubes incubated at 37 C for 60 min. absence of an aspartic dehydrogenase. These findings indicate that Streptomyces fradiae can deaminate glutamate through the agency of glutamic dehydrogenase, but has no mechanism to deaminate aspartic acid directly. Transamination studies. The fact that aspartate was utilized by living cells when certain Krebs cycle intermediates were added indicated that this amino acid could be deaminated if a suitable ammonia acceptor, such as a-ketoglutarate, were present. Transaminase experiments with cell free extracts were therefore carried out. The presence of aspartic-a-ketoglutaric transaminase and glutamic-oxalacetic transaminase were readily demonstrated. In the presence of a dialyzed cell free extract, pyridoxal phosphate, glutamate, and oxalacetate, the formation of aspartate was detected, and in the presence of cell free extract, pyridoxal phosphate, aspartate, and a-ketoglutarate, the formation of glutamate occurred (figure 2). It is thus clear that oxalacetate can be formed from aspartate provided a suitable amino acceptor is present. DISCUSSION Studies on utilization of various amino acids as sole carbon sources by Streptomyces fradiae have revealed some striking parallelisms in the metabolism of this organism and in the metabolism of bacteria, and at the same time, some significant differences. The amino acids belonging to the glutamic acid series (glutamic acid, proline, and arginine), as defined in Escherichia coli by Roberts, Abelson, and co-workers (1953), are all utilized as sole carbon sources by Streptomyces fradiae. Members of the aspartic acid series (aspartic acid, threonine, leucine, isoleucine, and methionine) do not support growth as carbon sources. The only exception to this generalization is lysine, which is a member of the aspartic acid series in E. coli, but which supports growth of S. fradiae as a sole source of carbon and nitrogen. It has been pointed out by Work (1955) and by Roberts and co-workers (1955) that the metabolism of this amino acid appears to be different in various organisms. These amino acids are probably utilized by S. fradiae as sources of energy through the agency of the tricarboxylic cycle. The extensive operation of this cycle in Streptomyces has been demonstrated by Cochrane and Peck (1953), and METHIONINE ISOLEUCINE THREONINE ASPARTATE ASPARTASE TRANSAMINASE PRESENT IN E.COLI PRESENT IN ABSENT FROM E. COLI AND S. FRADIAE S. FRADIAE FUMARATE (ṭ KREBS TCA CYCLE a-ketoglutarate +NH3 GLUTAMATE OXALACETATE GLUTAMIC DEHYDROGENASE PRESENT IN E. COLI AND S. FRADIAE ARGININE PROLINE Figure S. Diagram comparing interrelationships among a-keto acids of TCA cycle and origins of aspartate and glutamate in Streptomyces fradiae and Escherichia coli.

6 166 ROMANO AND NICKERSON [VOL. 75 Gilmour et al. (1955). The latter authors have further shown that the carbon skeletons for the synthesis of these amino acids arise from the tricarboxylic acid cycle. In S. fradiae, aspartic acid can, after transamination, enter into the cycle at the oxalacetate stage provided a suitable ammonia acceptor (such as a-ketoglutarate) is present to permit transamination. In the case of E. coli, however, through the agency of aspartase, aspartic acid can be deaminated to form fumarate; hence this organism has two direct links between the amino acids and the TCA cycle, exclusive of transamination. The relationships are outlined in figure 3. SUMMARY A number of amino acids will support the growth of Streptomyces fradiae as sole sources of carbon and nitrogen. These are alanine, histidine, lysine, glutamic acid, proline, and arginine. Aspartic acid, threonine, leucine, isoleucine, and methionine will not support growth. The amino acids that support growth include those of the glutamic acid series (glutamic acid, proline, and arginine); those that do not support growth include the aspartic acid series. A coenzyme I-linked glutamic dehydrogenase has been indicated in a cell free extract of S. fradiae; presumably, this is the mechanism by which members of the glutamic acid series are utilized via the tricarboxylic acid cycle. We have been unable to demonstrate the existence of an enzyme system in S. fradiae by which aspartic acid can be directly deaminated when supplied alone; hence, for members of this series there is no direct entrance into the TCA cycle. REFERENCES ABELSON, P. H., BOLTON, E., BRITTEN, R., COWIE, D. B., AND ROBERTS, R. B Synthesis of the aspartic and glutamic families of amino acids in Escherichia coli. Proc. Natl. Acad. Sci., U. S., 39, COCHRANE, V. W. AND PECK, H. D., JR The metabolism of species of Streptomyces. VI. Tricarboxylic acid cycle reactions in Streptomyces coelicolor. J. Bacteriol., 65, EHRENSVARD, G Metabolism of amino acids and proteins. Ann. Rev. Biochem. 24, FAHMY, A. R. AND WALSH, E. O'F The quantitative determination of dehydrogenase activity in cell suspensions. Biochem. J. (London), 51, FELDMAN, L. I. AND GUNSALUS, I. C The occurrence of a wide variety of transaminases in bacteria. J. Biol. Chem., 187, GILMOUR, C. M., BUTTERWORTH, E. M., NOBLE, E. P., AND WANG, C. H Studies on the biochemistry of the Streptomyces. I. Terminal oxidative metabolism in Streptomyces griseus. J. Bacteriol., 69, GOTTLIEB, D. AND CIFERRI, Deamination and degradation of amino acids by Streptomycetes. Mycologia, 48, NICKERSON, W. J. AND MOHAN, R. R. 1953a Nutrition of Streptomyces fradiae, Chapter 4 in Neomycin, S. A. Waksman. Rutgers University Press, New Brunswick, N. J. NICKERSON, W. J. AND MOHAN, R. R. 1953b Studies on the nutrition and metabolism of Streptomyces. In Symposium on Actinomycetales, morphology, bioloqy, and systematics, pp Istituto Superiore di Sanita, Rome. REICHARD, P The enzymatic synthesis of ureidosuccinic acid in rat liver mitochondria. Acta Chem. Scand., 8, REICHARD, P. AND LAGERKVIST, U The biogenesis of orotic acid in liver slices. Acta Chem. Scand., 7, ROBERTS, R. B., COWIE, D. B., BRITTEN, R., BOLTON, E., AND ABELSON, P. H The role of the tricarboxylic acid cycle in amino acid synthesis in Escherichia coli. Proc. Natl. Acad. Sci., U. S., 39, ROBERTS, R. B., ABELSON, P. H., COWIE, D. B., BOLTON, E. T., AND BRITTEN, R. J Studies of biosynthesis in Escherichia coli. Carnegie Institution of Washington, Publication 607, Washington, D. C. VIRTANEN, A. I. AND TARNANEN, J Die enzymatische Spaltung und Synthese der Asparaginsaure. Biochem. Z., 250, WAKSMAN, S. A Studies in the metabolism of the actinomycetes. III. Nitrogen metabolism. J. Bacteriol., 5, WAKSMAN, S. A. AND LOMANITZ, S Contribution to the chemistry of decomposition of proteins and amino acids by various groups of microorganisms. J. Agr. Research, 30, WOODRUFF, H. B. AND FOSTER, J. W Microbiological aspects of streptothricin. III. Metabolism and streptothricin fermentation in stationary and submerged cultures of A. lavendulae. Arch. Biochem., 2, WORK, E Some comparative aspects of lysine metabolism. In A symposium on amino acid metabolism, pp Edited by W. D. McElroy and H. B. Glass. The Johns Hopkins Press, Baltimore.