Staphylococcus aureus (Sevag and Green, 1944). A member of the vitamin B, group, pyridoxal phosphate, is the coenzyme for the decarboxylation of

Transcription

1 BIOTIN AND THE SYNTHESIS OF ASPARTIC ACID BY MICROORGANISMS J. L. STOKES, ALMA LARSEN, AND MARION GUNNESS Rwearch Laboratories, Merck and Company, Inc., Ralhway, New Jersey Received for publication May 15, 1947 Considerable evidence is accumulating concerning the role of growth factors in the metabolism of amino acids by microorganisms. Koser, Wright, and Dorfman (1942) demonstrated a relationship between aspartic acid and biotin in that aspartic acid can serve as a partial substitute for biotin in the growth of Torula cremoris. Pantothenic acid influences the synthesis of tryptophane by Staphylococcus aureus (Sevag and Green, 1944). A member of the vitamin B, group, pyridoxal phosphate, is the coenzyme for the decarboxylation of tyrosine, lysine, arginine, and other amino acids (Gale and Epps, 1944; Gunsalus, Bellamy, and Umbreit, 1944; Baddiley and Gale, 1945; Umbreit and Gunsalus, 1945). Pyridoxamine and pyridoxal are involved in the synthesis of lysine, threonine, and alanine by lactic acid bacteria (Stokes and Gunness, 1945). A combination of vitamin B. and CO2 apparently promotes synthesis of arginine, phenylalanine, and tyrosine by Lactobacillus arabinosus (Lyman et al., 1947). Few data have been available concerning the specific role of biotin in the growth of microorganisms. That biotin must play an important metabolic role is indicated by the need for biotin by many microorganisms for growth, its wide distribution in cells, and its great activity per unit weight. The data presented below demonstrate that biotin is involved in the synthesis of aspartic acid by microorganisms. A preliminary report of this work has been published (Stokes, Larsen, and Gunness, 1947). METHODS Stab cultures of the bacteria were carried in a medium of the following composition: 1 g of glucose, 0.5 g of Difoo peptone, 0.6 g of anhydrous sodium acetate, salts A and B in half the concentration given in table 1, and 1.5 g of agar per 100 ml of medium, at ph 6.8. Inocula for the experiments were prepared by subculturing from stab cultures into a liquid medium of the same composition as that given above. After incubation for 16 to 24 hours at 37 C, the cells of the broth cultures were centrifuged, washed with water, and suspended in 100 ml of water. One drop of this suspension served to inoculate each tube in an experiment. The basal medium (table 1) was prepared as described previously (Stokes and Gunness, 1945) and distributed in 5-ml quantities in 22-by-150-mm tubes. After addition of the experimental compounds, the volume in the tubes was brought to 10 ml with water prior to sterilization by autoclaving. Unless indicated otherwise, cultures of Streptococcus faeccalis R were incubated for 40 hours and the remaining organisms for 64 hours at 37 C, at which timies maximum 219

2 220 J. STOKES, A. LARSEN, AND M. GUNNESS [VOL. 54 acid production has occurred. Lactic acid was determined by titration with alkali using bromthymol blue as the indicator. S. faecalis R cultures were titrated with.05 N NaOH and the other bacteria with 0.1 N NaOH. S. faecalis forms less acid than the lactobacilli in the basal medium employed. The titrations were made directly in the culture tubes. Growth is usually expressed in terms of the amount of acid formed in the cultures since the latter can be easily measured quantitatively. Synthetic dl-aspartic acid was used in all experiments. The biotin was d-biotin obtained from synthetic dl-biotin. Additional details of methods are described later. dl-leucine... dl-isoleucine... dl-valine... I(-)-Cystine... dl-methionine... dl-tryptophane... I(-)-Tyrosine... dl-phenylalanine... dl-glutamic acid... dl-threonine... dl-alanine... dl-aspartic acid... 1(+)-Lysine... I(+)-Arginine... l(+)-histidine... dl-serine... 1l(-)-Proline... I(-)-Hydroxyproline... dl-norleucine... Glycine... Glucose... TABLE 1 Basal medium 200 mg 100 rfg 50 mg 5g Sodium acetate (anhydrous)... 3 g Adenine mg Guanine... 5 mg Uracil... 5 mg Pantothenic acid pg Riboflavin ;g Thiamine HCI pag Nicotinic acid pg Pyridoxamine pg p-aminobenzoic acid...20 pag Biotin pg Folic acid*...1..o1 g Salts A K2HPO mg KH2PO mg Salts B MgSO4.7HtO... NaCI... 5 mg FeSO4 7Hs mg MnSO4.4H mg Adjust to ph 6.8 Add distilled H20 to cc * Obtainable from Dr. R. J. Williams, University of Texas, Austin, Texas; pteroyl glutamic acid may also be used. Equivalent to 1.0,pg of material of "potency 40,000" or 1.0 pg of pteroyl glutamic acid. EXPERIMENTS In preliminary experiments designed to extend the basic microbiological assay method for the ten essential amino acids (Stokes, Gunness, Dwyer, and Caswell, 1945) to include the assay of aspartic acid, poor agreement of values at different levels of impure proteins was noted. The test organism was Streptococcusfaecalis R, which in the usual synthetic media (table 1) requires aspartic acid for growth. An attempt was made to improve the basal medium by increasing the content of vitamins and the purine and pyrimidine bases fivefold. Surprisingly, this change caused almost maximum growth and lactic acid formation of S.

3 19471 SYNTHESIS OF ASPARTIC ACID 221 faecalis in the blank tubes which contained no aspartic acid. It appeared, therefore, that the increase in growthfactor supplements stimulated synthesis of aspartic acid by S.faecalis. Fractionation of the growth factor mixture demonstrated that the increase in biotin alone was responsible for the growth of S. faecalis in the absence of aspartic acid (table 2). Increases in adenine, guanine, uracil, ribo-' flavin, pantothenic acid, thiamine, nicotinic acid, p-aminobenzoic acid, pyridoxamine, and folic acid were ineffective in supporting appreciable growth in the absence of aspartic acid. The ability of biotin to substitute for aspartic acid is not confined to S. faecalis R. A survey of eight additional aspartic-acid-requiring bacteria revealed that, with the exception of the heterofermentative Leuconostoc mesenteroides P-60, TABLE 2 Effect of increased concentrations of growth factors on development of Streptococcus faecalis R in the ab-sene of aspartic acd XL 0.05 N LACTIC ACID ADDENDUXM GRoWiEt FORMED PER 10 ML OF MEDIIJXt Nil Aspartic acid, 0.5 mg Fivefold increase in All vitamins + adenine, guanine, uracil Adenine, guanine, uracil Riboflavin Pantothenic acid Thiamine Nicotinic Biotin acid p-aminobenzoic acid Pyridoxamine Folic acid * Added to the basal medium (table 1) from which aspartic acid was omitted. t After incubation at 37 C for 40 hr. addition of excess biotin to the basal mediuim resulted in full or almost full growth, as measured by acid production, of all strains of streptococci and lactobacilli tested in the absence of aspartic acid (table 3). For Streptococcus faecalis loc1 and F24 and for Streptococcus zymogenes 5C1, 0.5 millimicrograms of biotin were sufficient to permit considerable growth in the absence of aspartic acid, although the stimulatory effect of additional biotin is clearly evident. In figure 1 it can be seen that if a production of 6 ml of acid is used as a point of reference, it is necessary to supply the Lactobacillus casei strains with 3 to 5 times, and L. arabitnosus with 2.7 times, as much biotin for growth in the absence of aspartic acid as when aspartic acid is present. Similar ratios were obtained for the other bacteria listed in table 3. It is also evident from the graph that the lactobacilli require biotin for growth even when liberally supplied with aspartic acid, a fact which indicates that biotin is required for metabolic func-

4 1222 J. STOKES, A. LARSEN, AND M. GUNNESS [vol. 54 tions other than those concerned with synthesis of aspartic acid. From the quantitative biotin ratios given above, it appears that much more biotin is neces- TABLE 3 Substituton of biotin for aspartic acid in the growth (acid formation) of various aspartic-acid-requiring bacteria MICROORGANISM 0.5 MIMLIMICOGRAMS BIOTIN 20 VLMICROoxAMS DIoIN PEN 10 ML MEDIUM PER 10 ML MEDIU No aspartic 2 mg No aspartic 2 mf acid dl-aspartic acid acid dl-aspartic acid ml acid formed per 100 ml nedium" Streptococcus faecalis R Streptococcw faecalis 10C... l Streptococcus faecalis F Streptococcus durans 98A Streptococcus zymogenes 5C Lactobacillus casl i LD5t Lactobacillus casei Lactobacillus arabinosus Leuconostoc mesenteroides P * The lactobacillus cultures were titrated with 0.1 N NaOH and the remaining cultures with 0.05 N NaOH after 8 days' incubation at 37 C. t Formerly known as Lactobacillus delbriickcii LD5 but recently identified as a strain of Lactobacillus casei (Rogosa, 1946). 08 IzLCAE II~~~~~~~~~~~~~~~~~~~~0, LC~i zs 0.4 -J4 MILLIMICROGRAMS Of BIOTIN PER IO ML MEDIUM FIG. 1. QUANTITIES OF BIOTIN REQUIRED FOR GROWTH OF LACTOBACILLI WITH AND WImOUT ASPARTIC ACID sary for synthesis of aspartic acid thanwfor the other function or functions of biotin. The need of the bacteria for biotin in the presence of aspartic acid elimi-

5 19471 SYNTHESIS OF ASPARTIC ACID 223 nates the possibility that all of the foregoing results could be explained by assuming that the bacteria do not require aspartic acid but that growth with aspartic acid is due to biotin present as an impurity in the aspartic acid. The biotin-aspartic-acid relationship is very specific. As previously indicated (table 2) only biotin of the vitamins tested stimulated growth in the absence of aspartic acid. Also, although S. faecalis R, L. arabinrosus, and L. casei require leucine, isoleucine, valine, cystine, methionine, tryptophane, tyrosine, TABLE 4 Influence of biotin on the amino acid requirements of lactic acid bacteria AMINO AI OMUTMD STREPTOCOCCUS LACTOLACIBaLUS ACTOBACILLUS FACAL RARAXBINOSuS 17-5 CASEI pg 0.lpg pg 0.1ga g 0.lpg Bhtin' Biotin Biotn Biotin Biotin Biotin ml acid formed per 10 ml medium None Leucine Isoleucine Valine Cystine Methionine Tryptophane Tyrosine Phenylalanine Glutamic acid Threonine Alanine Aspartic acid Lysine Arginine Histidine Serine Proline Hydroxyproline Norleucine Glycine phenylalanine, glutamic acid, threonine, lysine, arginine, histidine, and serine for growth in addition to aspartic acid, the requirement for only aspartic acid is eliminated by the use of excess biotin in the medium (table 4). So far it has been assumed that the ability of biotin to substitute for aspartic acid in the nutrition of the bacteria indicates that biotin is involved in the synthesis of that amino acid. However, since it has been shown, apparently, that the proteins of certain algae are lacking in lysine, tyrosine, arginine, and methionine (Mazur and Clarke, 1938, 1942), it seemed necessary to prove that the bacterial cells grown with excess biotin in place of aspartic acid actually

6 224 J. STOKES, A. LARSEN, AND M. GUNNESS [VOL. 54 contain aspartic acid. S. faecalis R, L. arabinosus, and L. casei LD5 were grown in the usual basal medium with 50 millimicrograms of biotin per 10 ml of medium and no aspartic acid, and also, as controls, in media containing 1 millimicrogram of biotin and 2 mg of dl-aspartic acid per 10 ml of medium. Each organism was grown in four 250-ml Erlenmeyer flasks, each containing 100 ml of medium. As customary, S. faecalis cultures were incubated for 2 days and the lactobacilli for 3 days at 37 C. As expected, controls consisting of media with 1 millimicrogram of biotin and no aspartic acid did not support growth. Macroscropically and microscopically there were no significant differences between cultures grown with and without aspartic acid. Also, the turbidity, acid production, and dry weight of the cell crops were determined. Fifty-milligram quantities of dry cells from each medium were hydrolyzed by autoclaving them for 5 hours at 15 pounds' pressure with 2 ml of 10 per cent HC1 in sealed ampules. TABLE 5 Comparison of cultures grown with and without aspartic acid, with special reference to the aspartic acid content of the harested cells DETERMINATION S. JARCAL K L. AXABINOSUS L. CASE! LDS No aspartic Aspartic No aspartic Aspartic No aspartic Aartic acid acid acd acid acd acid Turbidity... 50* M acid formed Mg dry cells % aspartic acid in cells * Per cent transmissible light (Evelyn colorimeter); uninoculated medium 100. The hydrolyzates were neutralized, filtered, diluted to 50-ml volume, and assayed for aspartic acid content with Leuconostoc mesenteroides (Hac and Snell, 1945). It will be recalled that for L. mesenteroides, biotin cannot substitute for aspartic acid. As shown in table 5, the bacterial cultures with and without aspartic acid are quite similar in turbidity, acid production, crop yield, and aspartic acid content of the harvested cells. There is no doubt that aspartic acid is synthesized, and in normal quantities, by the bacterial cells grown without aspartic acid in the presence of excess biotin. It seems reasonable to conclude, therefore, that biotin is involved in the synthesis of aspartic acid by those microorganisms. The sulfur-free derivative of biotin, d-desthiobiotin, and the biotin stereoisomers, dl-allobiotin and dl-epiallobiotin (Stokes and Gunness, 1945), failed to support growth of S. faecalis, L. casei, L. casei LD5, and L. arabinomus when used in place of biotin in aspartic-acid-free media. The oxygen analog of biotin dl-o-heterobiotin' (Duschinsky et al., 1945) is an effective substitute for biotin for S. faecalis and L. arabinosus, but not for the L. casei strains. However, 100 to 500 times as much o-heterobiotin as biotin must be used. The diamino- I Kindly supplied by Dr. Saul Rubin of Hoffman-La Roche, Inc.

7 19471 SYNTHESIS OF ASPARTIC ACID 225K carboxylic acid derivative of biotin obtained synthetically as dl-diamino acid sulfate (Stokes and Gunness, 1945) can completely replace biotin in asparticacid-free media for the four organisms mentioned above but 10,000 times as much of this compound as compared to biotin must be used for full growth. The ability of S. faecalis R to grow without aspartic acid in the presence of excess biotin appears to be a characteristic of at least the majority of the cells in the parent culture, since 22 single colony isolates obtained by plating all showed the same phenomenon. Also, the four organisms listed above developed fully through seven serial loop subcultures in the basal aspartic-acid-free medium containing 20 millimicrograms of biotin, a result which indicates that the biotinaspartic-acid phenomenon is independent of any factor that might have been carried over from the original peptone glucose sodium-acetate.inoculum medium. The ability of biotin to substitute for aspartic acid is independent of the ph of the medium between ph 6 to ph 8; at ph 5 growth is submaximum even when aspartic acid is supplied to the medium. Varying the ph of the growth medium between ph 5 and ph 8 did not alter the need of Leuconostoc mesenteroides for aspartic acid, although excess biotin, 20 millimicrograms per 10 ml, was in the medium. Also, almost invariably initiation of growth of the bacteria in aspartic-acid-free medium lags by 10 to 20 hours behind simultaneously inoculated media containing aspartic acid. Apparently, forcing the organisms to synthesise their required quota of aspartic acid greatly increases the lag phase. Although biotin can replace aspartic acid in the nutrition of many bacteria that under some conditions at least appear to require both compounds for growth, the reverse is not true. Saccharomyces cerevioiae F6.4 (Snell, Eakin, and Williams, 1940) and Neurospora sitophila (Stokes, Larsen, Woodward, and Foster, 1943) both require biotin for growth. With the media and procedures described in the literature cited, aspartic acid at a concentration of 2 mg of the dl form per 10 ml of medium failed to eliminate the need of the yeast and the mold for biotin. This inability of aspartic acid to substitute for biotin is understandable in view of the results given above, which demonstrate that biotin is also required by the bacterial cells for metabolic functions other than those concerned with the synthesis of aspartic acid. The multiple function of biotin probably also explains the inability of Koser et al. (1942) completely to replace biotin by aspartic acid in the case of Torula cremoris. Experiments with Resting Cell Suspeonws A number of experiments were made with resting cell suspensions to determine the specific aspartic-acid-forming reaction catalyzed by biotin. The following reactions which are known to lead to or might be expected to give rise to aspartic acid in biological systems were investigated: 1. Glutamic acid + oxalacetic acid -+ aspartic acid + a-keto-glutaric acid (Cohen and Hekhius, 1941). 2. Alanine + oxalacetic acid -+ aspartic acid + pyruvic acid (Cohen and Hekhius, 1941).

8 226 J. STOKES, A. LARSEN, AND M. GUNNESS [VOLJ Cysteic acid + oxalacetic acid -- aspartic acid + sulfapyruvic acid (Cohen and Hekhius, 1941). 4. Succinic acid 2H fumaric acid +H20 malic acid 2H oxalacetic acid (Harrow, 1940); followed by reaction (1) to give aspartic acid. 5. Fumaric acid + NH3, aspartic acid (Quastel and Woolf, 1926). 6. Pyruvic acid + CO2 oxalacetic acid (Krampitz, Wood, and Werkman, 1943); followed by reaction (1) to give aspartic acid. It is evident that with the exception of reaction (5), transamination is directly or indirectly involved in, all of them. In a typical experiment L. arabinwsus was grown in 250-ml Erlenmeyer flasks containing 100 ml of the basal medium (table 1). The biotin content, however, was reduced to the very small quantity of.05 millimicrogram per 10 ml of medium, and 200,ug of oleic acid per 10 ml were added as a substitute for the remainder of the required biotin (Williams and Fieger, 1946). The medium was adjusted to ph 5.6. Cells harvested from such a medium are essentially free from biotin. After incubation for 3 days at 37 C, the cells were collected by centrifugation, washed once with M/15 phosphate buffer at ph 7, and resuspended in sufficient buffer of the same type to give a galvanometer deflection of 5 on the Evelyn photoelectric colorimeter at 520 millimicrons wave length. Four-ml aliquots of cell suspension were mixed in test tubes (22 by 150 mm) with 10 mg each of the compounds shown in table 6 except that 20 mg of dl-alanine were used. To one of duplicate sets, 5,ug of biotin were added. Where necessary, the ph of the suspensions was adjusted to ph 7 and the volume to 5 ml. The thoroughly shaken tubes were stoppered and incubated overnight, for approximately 18 hours at 37 C. After incubation, the aspartic acid in the suspensions, cells plus fluid, was determined by quantitative assay with Leuconostoc mesenteroides (Hac and Snell, 1945) employing the medium shown in table 1 and a total assay volume of 1.0 ml. Titrations were made with 0.01 N NaOH. In this way as little as 2 MAg to 10 MAg of aspartic acid per ml of suspension could be readily measured (figure 2). It is evident from table 6 that resting cells of L. arabinosus form aspartic acid from glutamic acid, alanine, or cysteic acid plus oxalacetic acid. Malic and fumaric acids and to a lesser extent succinic acid can substitute for oxalacetic acid, presumably because they are converted to oxalacetic acid by the resting cells. However, all of these reactions proceed as well without biotin as with it. This clearly indicates that biotin is not involved in any of these reactions. The cells in this particular experiment contained less than.04 millimicrograms of biotin per ml of suspension as measured microbiologically (Wright and Skeggs, 1944) on acid-hydrolyzed cells. Therefore, the possibility of carry-over of significant amounts of biotin by the cells is eliminated. Glutamine can replace glutamic acid to give aspartic acid with either malic, fumaric, or succinic acid. Similar results were obtained with L. arabinosus cells grown in media containing excess biotin and no aspartic acid, and also in media with vitamin-free casein hydrolyzate, as a substitute for all of the amino acids except cystine and trypto-

9 19471 SYNTHESIS OF ASPARTIC ACID 2E27 phane, plus either oleic acid or sufficient biotin for half-maximum growth (0.2 millimicrograms per 10 ml) to reduce carry-over of biotin from the medium by the cells. In general, resting cells of S. faecalis R and L. casei gavre results similar TABLE 6 Formation of aspartic acid by cell suspensions of Lactobacillus arabinosus CELS PLUS ADDENDA NO DIOTI %US NIOTIN Aspartic acid Nsiacogros Per ml of suspeusi Nil... 3 Glutamic acid + oxalacetic acid* Glutamic acid + malic acid Glutamic acid + fumaric acid Glutamic acid + succinic acid Alanine + oxalacetic acid Cysteic acid + oxalacetic acid * Ninety-two per cent pure; kindly supplied by Dr. L. 0. Krampitz. a w 0 Q U z 0 ci JI 7 4- FIG X a le MICROGRAMS 0f ASPARTIC ~~~~~~~~~~~~~~~~~2 I A IA * *J cv 1% ACID RESPONSE OF LEUCONOSTOC MESENTEROIDES TO I-ASPARTIc ACID to those obtained with L. arabinos-us. Indirect evidence that biotin does not catalyze any of the reactions in table 6 is the fact that resting cells of L. mesenteroides, whose requirement for aspartic acid is not influenced by biotin, also produce aspartic acid under these conditions.

10 228 J. STOKES, A. LARSEN, AND M. GUNNESS [VOL. 54 No evidence could be obtained with L. arabinosus for the formation of aspartic acid by the direct amination of fumaric acid. Similarly negative results were obtained with malic or succinic acid and (NH4)2SO4. In this connection, however, it may be significant that biotin-deficient yeast cells are markedly stimulated by biotin to take up ammonia (Winzler, Burk, and duvigneaud, 1944). Our negative results may merely indicate that the proper physiological conditions were not provided in the resting cell suspension experiments. Also, no aspartic acid was formed in cell suspensions of L. arabinosus supplied with glutamic acid, plus pyruvic acid and either NaHCO3 or C02 gas as a source of carbon dioxide. These negative results were not altered by the addition of thiamine, pyridoxamine, p-aminobenzoic acid, riboflavin, pantothenic acid, nicotinic acid, folic acid, glucose, and adenosine triphosphate to the suspensions; TABLE 7 Stimulation of growth (acid formation) of lactic acid bacteria by oxalacetic acid in aspartic-acid-free medium COMPOUND ADDED L. CASE! S. FAZCALIS L. AJABINOSUS EEEDES Per 10 ml medium* m 0.1 N acid farmed per 10. medism Nil dl-aspartic acid, 2 mg Biotin, 0.1pg Oxalacetic acid,t 1 mg Oxalacetic acid, 5 mg Oxalacetic acid, 25 mg * Basal medium contained 0.8 millimicrograms of biotin and no aspartic acid. t Sterilized by filtration. by varying the ph of the suspension from ph 6 to ph 8; nor by the use of acetonedried cells possibly to increase permeability of the cells to adenosine triphosphate. The acetone-dried cells readily formed aspartic acid when mixed with glutamic and oxalacetic acids. A suggestion that biotin may be concerned with the formation of oxalacetate was obtained from growth experiments in which for L. casei and L. arabinosus but not for S. daecalis R oxalacetic acid partially replaced biotin in asparticacid-deficient media (table 7). The possibility that the activity of the oxalacetic acid was due to impurities of biotin or aspartic acid was ruled out by assay of the preparation for these two components. SUMMARY Biotin can completely substitute for aspartic acid in the growth of Lactobacillus arabinosus, Streptococcus faecalis, and related organisms. The biotinaspartic-acid relationship is specific; riboflavin, pantothenic acid, thiamine, p-aminobenzoic acid, and pyridoxamine cannot replace biotin, nor can biotin substitute for 14 amino acids other than aspartic acid which are required for growth. Cells grown with biotin contain as much aspartic acid as those grown

11 1947] SYNTHESIS OF ASPARTIC ACID 229 with aspartic acid. It is concluded that biotin participates in the synthesis of aspartic acid. Although resting cell suspensions of Lactobacillus arabinosus can form aspartic acid by typical transamination reactions, the presence of biotin is not required for such reactions. It has not been possible to determine the specific aspartic-acid-forming reaction catalyzed by biotin. REFERENCES BADDILEY, J., AND GALE, E. F Codecarboxylase function of pyridoxal phosphate. Nature, 155, COHEN, P. P., AND HEKHIUS, G. L Rate of transamination in normal tissues. J. Biol. Chem., 140, DUSCHINSKY, R., DOLAN, L. A., FLOWER, D., AND RUBIN, S. H "O-heterobiotin," a biologically active analog of biotin. Arch. Biochem., 6, GALE, E. F., AND Epps, H. R Studies on bacterial amino acid decarboxylase. I. 1 (+)-Lysine decarboxylase. Biochem. J., 38, GUNSALUS, I. C., BELLAMY, W. D., AND UMBREIT, W. W A phosphorylated derivative of pyridoxal as the coenzyme of tyrosine decarboxylation. J. Biol. Chem., 155, HAC, L. R., AND SNELL, E. E The microbiological determination of amino acids. III. Assay of aspartic acid with Leuconostoc mesenteroides. J. Biol. Chem., 159, HARROW, B Textbook of biochemistry. 2d ed. W. B. Saunders and Co., Philadelphia. Refer to p KOSER, S. A., WRIGHT, M. H., AND DORFMAN, A Aspartic acid as a partial substitute for the growth stimulating effect of biotin on Torula cremoris. Proc. Soc. Exptl. Biol. Med., 51, KRAMPITZ, L. O., WOOD, H. G., AND WERKMAN, C. H Enzymatic fixation of carbon dioxide in oxalacetate. J. Biol. Chem., 147, LYMAN, C. M., MOSELEY, O., WOOD, S., BUTLER, B., AND HALE, F Some chemical factors which influence the amino acid requirements of the lactic acid bacteria. J. Biol. Chem., 167, MAZUR, A., AND CLARKE, H. T The anmino acids of certain algae. J. Biol. Chem., 123, MAZUR, A., AND CLARKE, H. T Chemical components of some autotrophic organisms. J. Biol. Chem., 143, QUTASTEL, J. H., AND WOOLF, B The equilibrium between l-aspartic acid, fumaric acid and ammonia in the presence of resting bacteria. Biochem. J., 20, ROGOSA, M Significance in nutritional research of correct identification of Lactobacillus casei, L. delbrueckii, and L. bulgaricus. J. Bact., 51, 575. SEVAG, M. G., AND GREEN, M The role of pantothenic acid in the synthesis of tryptophane. J. Biol. Chem., 154, SNELL, E. E., EAKIN, R. E., AND WILLIAMS, R. J A quantitative test for biotin and observations regarding its occurrence and properties. J. Am. Chem. Soc., 62, STOKES, J. L., AND GUNNESS, M. 1945a Microbiological activity of synthetic biotin, its optical isomers and related compounds. J. Biol. Chem., 157, STOKES, J. L., AND GUNNESS, M. 1945b Microbiological methods for the determination of amino acids. I. Aspartic acid and serine. J. Biol. Chem., 157, STOKES, J. L., AND GUNNESS, M. 1945c Pyridoxamine and the synthesis of amino acids by lactobacilli. Science, 101, STOKES, J. L., GUNNESS, M., DWYER, I., AND CASWELL, M Microbiological methods for the determination of amino acids. II. A uniform assay method for the ten essential amino acids. J. Biol. Chem., 160,

12 230 J. STOKES, A. LARSEN, AND M. GUNNESS [VOL. 54 STOKES, J. L., LARSEN, A., AND GUNNESS, M Biotin and the synthesis of aspartic acid by microorganisms. J. Biol. Chem., 167, STOKES, J. L., LARSEN, A., WOODWARD, C. R., AND FOSTER, J. W A Neurospora assay for pyridoxine. J. Biol. Chem., 10, UMBREIT, W. W., AND GUNSALUS, I. C The function of pyridoxine derivatives: arginine and glutamic acid decarboxylases. J. Biol. Chem., 169, WILLIAMS, V. R., AND FIEGER, E. A Oleic acid as a growth stimulant for Lactobacillus casei. J. Biol. Chem., 166, WINZLER, R. J., BURK, D., AND DUVIGNEAUD, V Biotin in fermentation, respiration, growth and nitrogen assimilation by yeast. Arch. Biochem., 6, WRIGHT, L. D., AND SKEGGS, H. R Determination of biotin with Lactobacillus arabinosus. Proc. Soc. Exptl. Biol. Med., 56, Downloaded from on March 30, 2019 by guest