UTILIZATION OF AMINO ACIDS AS CARBON SOURCES BY STREPTOMYCESFRADIAE1

ANTONIO H. ROMANO2 AND WALTER J. NICKERSONInstitute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication August 8, 1957

There have been several investigations on theamino acid nutrition of Streptomyces. Early workon this subject by Waksman (1920) showed thatcertain single amino acids satisfied the nitrogenrequirements of representative species of thegenus Streptomyces. Considerable specificity in theability to deaminate amino acids is exhibited byStreptomyces, as Woodruff and Foster (1943)have shown with Streptomyces lavendulae, whileGottlieb and Ciferri (1956) showed that the onlyamino acids deaminated by Streptomyces vene-zuelae were those capable of supporting growthwhen supplied as the sole source of nitrogen.There have been relatively few studies on theuse of amino acids as carbon sources by Strepto-myces. Waksman and Lomanitz (1925) showedthat glutamic acid and alanine as sole carbonsources supported good growth of Streptomycesviridochromogenes, whereas glycine and asparaginewere poor carbon sources, but supported goodgrowth in the presence of glucose. Nickerson andMohan (1953a, 1953b) showed that certainamino acids were utilized by Streptomyces fradiaeas sole carbon and nitrogen sources; included inthis group were glutamic acid, proline, andarginine. Other amino acids, such as asparticacid, leucine, isoleucine, methionine, phenylala-nine, and threonine, could not serve as carbonsources. It has been the object of the presentinvestigation to extend these findings, and toelucidate the enzymatic basis of the specificityexhibited by this organism toward amino acidsas carbon sources.

MATERIALS AND METHODS

Organism and culture media. The organismemployed was strain 3535 of Streptomyces fradiae

1 Supported in part by a grant from the Na-tional Institutes of Health, United States PublicHealth Service.

2 Present address: Robert A. Taft SanitaryEngineering Center, United States Public HealthService, Cincinnati 26, Ohio.

from the collection in the Institute of Micro-biology. The organism was carried on potato agarslants; inoculation of test media was made bywashing spores from the surface of the agar withsterile distilled water and transferring 0.1 ml ofthe spore suspension to the test media.The basal medium (A) employed for growth

studies had the following composition: K2HPO4,1.5 g; MgSO4 . 7H20, 0.025 g; CaCl2 - 2H20, 0.025 g;FeSO4 7H20, 0.015 g; ZnSO4.7H20, 0.005 g; anddistilled water to make 1000 ml. This mediumwas distributed in 100 ml amounts to 250-mlErlenmeyer flasks and was sterilized by autoclav-ing at 120 C for 20 min. All additions to thisbasal medium were neutralized if acidic or basic,sterilized separately, and added aseptically. Afterinoculation, the cultures were incubated at 28C on a rotary shaker which was set at 250 rpm.Measurement of growth. Mycelial dry weights

were determined by filtering the cultures throughWhatman no. 2 filter paper which had beenpreviously dried at 90 C for 18 to 24 hr andweighed. The mycelia and filter papers were thendried under the same conditions and the differ-ences in weight were determined.

Reagents. Amino acids used in nutrition experi-ments were obtained from commercial sourcesand were used in the forms stated in tables 1 to3. Glutamic acid and aspartic acid used in experi-ments with cell free extracts were obtained fromMatheson, Coleman, and Bell, and were in theDL form. Purity of the amino acids was checkedby paper chromatography, using a phenol systemto which was added 0.04 per cent 8-hydroxy-quinoline.

Cell free preparations. The cell free extractsused in the transaminase and dehydrogenase ex-periments were prepared in the following manner.Cells were grown on a basal medium (B) similarin composition to that given above but withdifferent salt concentrations: K2HPO4, 0.5 g;MgSO4-7H20, 0.2 g; CaCl2-2H20, 0.25 g;FeSO4-7H20, 0.025 g; ZnSO4-7H20, 0.025 g;

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and distilled water to make 1000 ml. It was

found that transaminase activity in cell freeextracts was greater when cells were grown on a

medium with this basal formulation than whengrown in the medium with higher phosphate andlower concentrations of zinc and iron. This find-ing was not investigated in further detail. Tobasal medium B there was added sodium gluta-mate 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, andwere washed twice with distilled water and twicewith M/40 phosphate buffer. In the transaminasestudies, a buffer at pH 7.2 was used; in the dehy-drogenase experiments, a buffer of pH 7.0 was

used. The cells were resuspended in buffer to givea suspension containing approximately 10 mg

cells per ml. This cell suspension was subjectedto disintegration for 5 min in a Raytheon 50 watt,9-kc Magnetostriction oscillator at a plate voltageof 130 v and an output voltage of 100 v. Thesuspension was then centrifuged at 2500 X G for5 min to remove whole cells. The supernatantsolution was used as the enzyme preparation.

Enzymatic studies. Transaminase activity was

determined by a modification of the method usedby Feldman and Gunsalus (1950), whereby theappearance of specific amino acid spots on paper

chromatograms was noted. Whatman no. 1 filterpaper was spotted with 5 .AL of the reactionmixtures after suitable incubation periods. Thepapers were irrigated with water-saturated phenolcontaining 0.04 per cent 8-hydroxyquinoline for24 hr. The papers were dried at room temperaturein a current of air for 3 hr, and were then sprayedwith 0.25 per cent ninhydrin in water-saturatedbutanol. It was found necessary to dialyze thecell free extract against buffer at 5 C for 24 hr inorder to remove free amino acids which were

present in the enzyme preparation in amountssufficient to give spots on the chromatograms, andthus make detection of activity difficult. Pyri-doxal phosphate (obtained from California Bio-chemical Research Foundation), was added to thereaction mixtures to supply the coenzyme re-

quirement. Solutions of oxalacetic acid anda-ketoglutaric acid (obtained from KrishellLaboratories) were prepared immediately beforeuse and were neutralized with equivalent amountsof NaOH. Dehydrogenase activity was deter-mined by the method of Fahmy and Walsh(1952), using 2,3, 5-triphenyltetrazolium chlo-

ride (obtained from Arapahoe Laboratories) asthe hydrogen acceptor. Coenzyme I (DPN) (90per cent purity) was obtained from SchwarzLaboratories, and coenzyme II (TPN) (65 percent purity) was obtained from Sigma ChemicalCompany. Optical density readings were madewith a Klett-Summerson photoelectric color-imeter.

RESULTS

Growth with amino acids as sole sources of carbonand of nitrogen. As shown in table 1, glutamicacid, proline, arginine, alanine, and histidine sup-

TABLE 1Growth of Streptomyces fradiae with single amino

acids as sole sources of carbon and of nitrogen

GrowthRelative to

Addition to Basal Medium* Growth Glutamate(on MolarBasis)

mg dry WI!100 ml %culturet

L-Glutamate, monosodium.... 166.7 100L-Proline ..................... 101.2 41.4L-Arginine * HCl .............. 57.4 42.9DL-Alanine ................... 122.0 38.5

(77.0)tL-Histidine*HCl ........ 129.0 96.1L-Lysine*HCl ................ 38.1 24.6

Hydroxy-L-proline ............ 12.0 5.6Glycine ...................... 10.2 2.7Glycine anhydride ........... 13.3 5.4L-Aspartate, monosodium .... 4.1 1.9L-Asparagine....... 3.0 1.6L-Leucine .................... 0.0 0.0DL-Methionine................ O. 0.0DL-Threonine ............... 0.0 0.0Creatine H20................ 1.9 1.0DL-a-Amino-n-butyrate 0.......O. O .ODL-a-Amino-n-isobutyrate .... 0.0 0.0DL-ca-Amino-n-valerate ....... 0.0 0.0

* All substances sterilized separately in aque-ous solution, and incorporated aseptically to givefinal concentration of 1 g per 100 ml medium.

t Cultures incubated with continuous agitationfor 5 days at 28 C. All values are the average ofduplicate flasks from at the least two differentexperiments; the values for glutamate and aspar-tate are based on determinations from 5 differentexperiments, all in close agreement.

t With DL-alanine the growth of S. fradiae is77 per cent of that with glutamate, if only thej,-form of alanine is utilized.

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port relatively good growth of Streptomycesfradiae. Lysine supports modest growth. Glycineand hydroxy proline permit very slight growth,and are ineffective on a molar basis, whereasother amino acids listed do not support growth ofS. fradiae. In addition to the amino acids listedin table 1, the following have been shown previ-ously (Nickerson and Mohan, 1953a) not to per-

mit growth of S. fradiae when supplied as solecarbon sources: phenylalanine, tyrosine, trypto-phan, valine, serine, and isoleucine.

Utitization of aspartate in combination uithanother carbon source. It was demonstrated previ-ously (Nickerson and Mohan, 1953b) that al-though aspartic acid and asparagine were

ineffective when given as the sole source of carbonfor S. fradiae, very much more growth was ob-tained on a "nutrient pair," such as aspartate(or asparagine) + glutamate, than on glutamatealone. This observation indicated that the carbonof aspartate could be used if a suitable "primer"were also present. Since aspartic acid has beenimplicated in the biosynthesis of a variety ofcellular constituents, many areas were exploredby the nutrient pair technique in an attempt todiscover a biochemical "lesion" that would pre-

vent aspartate from being utilized as the solesource of carbon. One such area examined dealtwith the known involvement of aspartic acid inpyrimidine biosynthesis, another area that was

studied dealt with the biosynthesis of asparticand glutamic acids and the interconversionthereof via their respective keto acids.

There is substantial evidence that asparticacid plays a role in the synthesis of pyrimidines.Reichard and Lagerkvist (1953) and Reichard(1954) demonstrated a reaction in rat livermitochondria by which ammonia, carbon diox-ide, and L-aspartate condense to give L-ureido-succinate which is a precursor of orotic acid.Accordingly, the effect of uracil and of oroticacid on the utilization of aspartate by S. fradiaewas examined. No significant growth was ob-tained with either of these substances whenpaired with aspartate. From these results it mightappear that the biochemical lesion responsible forlack of aspartate utilization by S. fradiae does notinvolve a block in the synthesis of pyrimidinesfrom aspartic acid. (That only one "lesion" ap-pears to be involved in aspartate utilization isapparent 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 whichare formed by operation of the Krebs cycle (thiswork has been reviewed critically by Ehrensviird,1955). The reverse reaction, the formation ofKrebs cycle intermediates from these aminoacids, has also been amply demonstrated in manyorganisms. The possibility was considered thatS. fradiae was in some manner unable to convertaspartic acid (when supplied alone) into oxal-acetic acid, thereby lacking any energy-yieldingmechanism through which this amino acid couldbe metabolized. This possibility was tested byadding various intermediates of the Krebs cycleto culture media to form nutrient pairs withaspartic acid.As seen in table 2 good growth of S. fradiae

was obtained in media with aspartate plusfumarate, malate, or succinate. Little or nogrowth was obtained in combinations of aspartateplus acetate, glutarate, itaconate, citrate, oxal-acetate, or butyrolactone. The lack of growth un-der these conditions with citrate has been shownin control experiments to be due to the powerfulmetal-chelating property of citrate (S. fradiae,and other species of Streptomyces examined areextraordinarily sensitive to inhibition by power-ful metal-chelating agents). Lack of growth withoxalacetate is probably due to the instability ofthis substance which decomposes rapidly in

TABLE 2Effect of Krebs cycle intermediates and other carbon

sources on the utilization of aspartic acidby Streptomyces fradiae

GrowthAddition to Basal Medium

3 days I5 days

mg dry wt/100 mlculture

L-Aspartate* ............ .......... 1.0 4.0L-Aspartate + Na succinate....... 43.1 95.8L-Aspartate + Na fumarate....... 67.9 114.9

L-Aspartatet + Na malate........ 55.5L-Aspartate + Na acetate......... _ 4.0L-Aspartate + Na citratet. 1.0

L-Glutamate*..................... 63.2 153.0L-Glutamate + L-aspartate... 295.2L-Glutamate + glucose............ 251.1

* All substrates supplied at 1.0 g per 100 mlmedium.

t All substrates supplied at 0.5 g per 100 mlmedium.

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aqueous solution into acetate and oxalate ions.Experiments in which freshly prepared solutionsof oxalacetate were added at intervals of 4 hr toculture medium containing 1.0 per cent aspartatewere also unsuccessful. The permeability of wholecells to these substances may also be a limitingfactor. The data in table 2 indicate, however,that growth of S. fradiae with aspartate is pos-sible if intermediates of the Krebs cycle are madeavailable. It is most probable that in these ex-periments aspartate is serving primarily as asource of nitrogen, and that its amino group istransferred to a suitable acceptor through theagency of a transaminase. As is shown in a fol-lowing section, an active aspartate-glutamatetransaminase system is present in S. fradiae. Thedata of table 2 confirm that aspartate is usableby this organism as a source of nitrogen, andindicate that the unsuitability of aspartate as asole substrate rests on the evident inability ofthe organism to transform aspartate (except bytransamination) so as to provide an amino groupacceptor.Comparison of the amount of growth (table 2)

made on glutamate alone with that on glutamate+ aspartate and glutamate + glucose, indicatesthat the carbon of aspartate can also be utilizedprovided a suitable primary carbon source issupplied.

Dehydrogenase studies. We next tested to seewhether or not this organism had a mechanism todeaminate aspartate when this substance wassupplied alone; if not, aspartate could not enterinto the tricarboxylic acid cycle or other pathwayof carbon metabolism. The fact that fumarate isa suitable carbon source for S. fradiae (table 2)would appear to exclude the presence of aspartase,an enzyme which has been shown by Virtanenand Tarnanen (1932) to catalyze the followingreaction: aspartic acid :. fumaric acid + am-monia. This enzyme system is the only firmlyestablished mechanism for the deamination ofaspartic acid (exclusive of transamination).The utilization of glutamate as an energy

source would presumably take place through theagency of glutamic dehydrogenase, which cata-lyzes the oxidative deamination of glutamate.This enzyme has been found by other workers tobe widely distributed in animal, plant, andmicrobial cells. Further oxidation of a-keto-glutarate would occur via the tricarboxylic acidcycle.The presence of glutamic dehydrogenase in

Streptomyces fradiae is indicated by experimentswith cell free extracts, as shown in figure 1. It isseen that there is a rapid reduction of 2,3,5-triphenyltetrazolium chloride (TTC) in thepresence of the enzyme preparation, coenzyme I(DPN), and sodium glutamate. This enzymesystem in Streptomyces fradiae appears to operatewith coenzyme I; a marked lag in TTC reductionin presence of coenzyme II (TPN) is seen infigure 1. The onset of activity after the lag maybe due to an enzymatic conversion of coenzymeII to coenzyme I by a mechanism similar to thatfound in yeasts, and first described by von Eulerand co-workers in 1937. It is also seen in figure 1that there was no dehydrogenase activity in thepresence of aspartate, cell free extract, and co-enzyme I or coenzyme II. This confirms the

1.01

i-zIda

0

0.8-

0.6

0Extract +DPN+ glutamate

0

0

Extract + DPt-.. . , Extract 4 DP?

0.41

021N.N9

II* a- + asparfa<te

0A - Extract + TPNI + glutamate

0I Extract + TPN,A-A Extract + TPN

O 1 1 4-I aspartate0 10 20 30

MINUTES

Figure 1. (At top) Rate of reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by a cellfree extract of Streptomyces fradiae in the pres-ence of DPN and sodium glutamate or sodiumaspartate. Reaction mixtures: cell free extract,2.0 ml; DPN, 0.5 Amole; sodium glutamate orsodium aspartate, 50,umoles; TTC, 0.5 ml of 0.5per cent solution; and M/40 phosphate buffer, pH7.0 to make a total of 4.0 ml. (At bottom) Rate ofreduction of 2,3,5-triphenyltetrazolium chlorideby a cell free extract of Streptomyces fradiae inthe presence of TPN and sodium glutamate orsodium aspartate. Reaction mixtures as given,except that TPN was substituted for DPN.

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1 2 3 4 5 6 7

Figure 2. Transamination reactions carried outby a cell free extract of Streptomyces fradiae. Con-tents 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) dialyzedenzyme, 0.3 ml; pyridoxal phosphate, 20,g; so-

dium glutamate, 10 ,moles; sodium oxalacetate,10,moles (6) dialyzed enzyme, 0.3 ml; pyridoxalphosphate, 20 Mg; sodium aspartate, 10,umoles (7)dialyzed enzyme, 0.3 ml; pyridoxal phosphate,20,g; sodium aspartate, 10 MAmoles; sodium a-keto-glutarate, 10 ,moles. M/15 phosphate buffer, pH7.2 in all tubes to give total volume of 1.0 ml.Tubes incubated at 37 C for 60 min.

absence of an aspartic dehydrogenase. Thesefindings indicate that Streptomyces fradiae can

deaminate glutamate through the agency ofglutamic dehydrogenase, but has no mechanismto deaminate aspartic acid directly.

Transamination studies. The fact that aspar-

tate was utilized by living cells when certainKrebs cycle intermediates were added indicatedthat this amino acid could be deaminated if a

suitable ammonia acceptor, such as a-keto-glutarate, were present. Transaminase experi-ments with cell free extracts were thereforecarried out. The presence of aspartic-a-keto-glutaric transaminase and glutamic-oxalacetictransaminase were readily demonstrated. In thepresence of a dialyzed cell free extract, pyridoxalphosphate, glutamate, and oxalacetate, the for-mation of aspartate was detected, and in thepresence of cell free extract, pyridoxal phosphate,aspartate, and a-ketoglutarate, the formation ofglutamate occurred (figure 2). It is thus clearthat oxalacetate can be formed from aspartateprovided a suitable amino acceptor is present.

DISCUSSION

Studies on utilization of various amino acidsas sole carbon sources by Streptomyces fradiaehave revealed some striking parallelisms in themetabolism of this organism and in the metabo-lism of bacteria, and at the same time, some sig-nificant differences. The amino acids belonging tothe glutamic acid series (glutamic acid, proline,and arginine), as defined in Escherichia coli byRoberts, Abelson, and co-workers (1953), are allutilized as sole carbon sources by Streptomycesfradiae. Members of the aspartic acid series(aspartic acid, threonine, leucine, isoleucine, andmethionine) do not support growth as carbonsources. The only exception to this generalizationis lysine, which is a member of the aspartic acidseries in E. coli, but which supports growth ofS. fradiae as a sole source of carbon and nitrogen.It has been pointed out by Work (1955) and byRoberts and co-workers (1955) that the metabo-lism of this amino acid appears to be different invarious organisms.

These amino acids are probably utilized by S.fradiae as sources of energy through the agencyof the tricarboxylic cycle. The extensive opera-tion of this cycle in Streptomyces has beendemonstrated by Cochrane and Peck (1953), and

METHIONINE ISOLEUCINE THREONINE

ASPARTATE

ASPARTASE TRANSAMINASEPRESENT IN E.COLI PRESENT INABSENT FROM E. COLI AND

S. FRADIAE S. FRADIAE

FUMARATE OXALACETATE(.t KREBS

TCACYCLE

a-KETOGLUTARATE

+NH3 GLUTAMICDEHYDROGENASE

PRESENT INE. COLI AND S. FRADIAE

GLUTAMATE

ARGININE PROLINEFigure S. Diagram comparing interrelationships

among a-keto acids of TCA cycle and origins ofaspartate and glutamate in Streptomyces fradiaeand Escherichia coli.

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Gilmour et al. (1955). The latter authors havefurther shown that the carbon skeletons for thesynthesis of these amino acids arise from thetricarboxylic acid cycle. In S. fradiae, asparticacid can, after transamination, enter into thecycle at the oxalacetate stage provided a suitableammonia acceptor (such as a-ketoglutarate) ispresent to permit transamination. In the case ofE. coli, however, through the agency of aspartase,aspartic acid can be deaminated to form fuma-rate; hence this organism has two direct linksbetween the amino acids and the TCA cycle,exclusive of transamination. The relationshipsare outlined in figure 3.

SUMMARY

A number of amino acids will support thegrowth of Streptomyces fradiae as sole sources ofcarbon and nitrogen. These are alanine, histidine,lysine, glutamic acid, proline, and arginine.Aspartic acid, threonine, leucine, isoleucine, andmethionine 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 supportgrowth 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 bywhich members of the glutamic acid series areutilized via the tricarboxylic acid cycle. We havebeen unable to demonstrate the existence of anenzyme system in S. fradiae by which asparticacid can be directly deaminated when suppliedalone; hence, for members of this series there isno direct entrance into the TCA cycle.

REFERENCESABELSON, P. H., BOLTON, E., BRITTEN, R., COWIE,

D. B., AND ROBERTS, R. B. 1953 Synthesisof the aspartic and glutamic families of aminoacids in Escherichia coli. Proc. Natl. Acad.Sci., U. S., 39, 1020-1026.

COCHRANE, V. W. AND PECK, H. D., JR. 1953The metabolism of species of Streptomyces.VI. Tricarboxylic acid cycle reactions inStreptomyces coelicolor. J. Bacteriol., 65,37-44.

EHRENSVARD, G. 1955 Metabolism of aminoacids and proteins. Ann. Rev. Biochem.24, 275-310.

FAHMY, A. R. AND WALSH, E. O'F. 1952 Thequantitative determination of dehydrogenaseactivity in cell suspensions. Biochem. J.(London), 51, 55-56.

FELDMAN, L. I. AND GUNSALUS, I. C. 1950 Theoccurrence of a wide variety of transaminasesin bacteria. J. Biol. Chem., 187, 821-830.

GILMOUR, C. M., BUTTERWORTH, E. M., NOBLE,E. P., AND WANG, C. H. 1955 Studies onthe biochemistry of the Streptomyces. I.Terminal oxidative metabolism in Strepto-myces griseus. J. Bacteriol., 69, 719-724.

GOTTLIEB, D. AND CIFERRI, 0. 1956 Deamina-tion and degradation of amino acids by Strep-tomycetes. Mycologia, 48, 253-263.

NICKERSON, W. J. AND MOHAN, R. R. 1953aNutrition of Streptomyces fradiae, Chapter 4in Neomycin, S. A. Waksman. Rutgers Uni-versity Press, New Brunswick, N. J.

NICKERSON, W. J. AND MOHAN, R. R. 1953bStudies on the nutrition and metabolism ofStreptomyces. In Symposium on Actinomyce-tales, morphology, bioloqy, and systematics,pp. 137-146. Istituto Superiore di Sanita,Rome.

REICHARD, P. 1954 The enzymatic synthesis ofureidosuccinic acid in rat liver mitochondria.Acta Chem. Scand., 8, 795-805.

REICHARD, P. AND LAGERKVIST, U. 1953 Thebiogenesis of orotic acid in liver slices. ActaChem. Scand., 7, 1207-1217.

ROBERTS, R. B., COWIE, D. B., BRITTEN, R.,BOLTON, E., AND ABELSON, P. H. 1953 Therole of the tricarboxylic acid cycle in aminoacid synthesis in Escherichia coli. Proc.Natl. Acad. Sci., U. S., 39, 1013-1019.

ROBERTS, R. B., ABELSON, P. H., COWIE, D. B.,BOLTON, E. T., AND BRITTEN, R. J. 1955Studies of biosynthesis in Escherichia coli.Carnegie Institution of Washington, Publi-cation 607, Washington, D. C.

VIRTANEN, A. I. AND TARNANEN, J. 1932 Dieenzymatische Spaltung und Synthese derAsparaginsaure. Biochem. Z., 250, 193-211.

WAKSMAN, S. A. 1920 Studies in the metabo-lism of the actinomycetes. III. Nitrogenmetabolism. J. Bacteriol., 5, 1-30.

WAKSMAN, S. A. AND LOMANITZ, S. 1925 Con-tribution to the chemistry of decompositionof proteins and amino acids by various groupsof microorganisms. J. Agr. Research, 30,263-281.

WOODRUFF, H. B. AND FOSTER, J. W. 1943 Mi-crobiological aspects of streptothricin. III.Metabolism and streptothricin fermentationin stationary and submerged cultures of A.lavendulae. Arch. Biochem., 2, 301-315.

WORK, E. 1955 Some comparative aspects oflysine metabolism. In A symposium on aminoacid metabolism, pp. 462-492. Edited byW. D. McElroy and H. B. Glass. The JohnsHopkins Press, Baltimore.

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