EFFECTS OF CERTAIN AMINO ACIDS ON ANTHRANILATE PRODUCTION IN NEUROSPORA CRASSA JOSEPHINE SOBOREN AND JOSEPH F. NYC Department of Physiological Chemistry, University of California at Los Angeles, Los Angeles, California Received for publication 23 November 1962

ABSTRACT to various supplements and the accumulation of SOBOREN, JOSEPHINE (University of California, during growth. This organism Los Angeles) AND JOSEPH F. Nyc. Effects of exhibited the best growth under those environ- certain amino acids on anthranilate production mental conditions which depressed the accumula- in Neurospora crassa. J. Bacteriol. 85:881-888. tion of anthranilic acid in the medium. A study 1963.-The level of anthranilic acid produced in was made on the accumulation of fluorescent cultures of a tryptophanless mutant of Neuro- products by strain C-83 in an effort to clarify spora crassa strain C-83 was closely related to the metabolic significance of this phenomenon. the growth response. A good growth response MIATERIALS AND METHODS was attained only on supplements which greatly repressed the level of anthranilate accumulated. Cultures. The strains of N. crassa employed The improved growth response of strain C-83 to were the normal wild-type strain 1A and a variable levels of and to supplements tryptophan-requiring mutant strain C-83 pre- of the amino acids , , viously described by Mitchell and Lein (1948). , and was correlated with a Strain C-83 cannot form tryptophan from - decreased anthranilate production. The inhibi- 3-glycerol phosphate, its immediate l)recursor, tory effect of these supplements on anthranilate and accumulates large quantities of the fluores- production was ascribed to effects on the mecha- cent precursors of tryptophan in the culture nisms which regulate the formation of anthranilic medium (Haskins and Mitchell, 1949). Tracer acid. studies (Partridge, Bonner, and Yanofsky, 1952) showed that the genetic block concerned with tryptophan in strain C-83 is com- Previous investigations with Neurospora crassa plete. The mutant is thus entirely dependent disclosed that anthranilic acid, indole, trypto- upon exogenous tryptophan for growth. In phan, and inhibit the growth of this addition, the growth response of this organism to organism, and that the growth of normal and tryptophan supplements is known to be depen- mutant strains of N. crassa on media supple- dent upon the presence or absence of certain mented with tryptophan is influenced by the other amino acids (Soboren and Nyc, 1961). presence of other amino acids (Soboren and Nyc, Procedure for growing molds. Strains IA and 1961). Most of the substances known to be C-83 were cultured at 25 C in 125-ml Erlenmeyer metabolically related to tryptophan have been flasks on 20 ml of appropriately supplemented tested for their effect on the growth of N. crassa, Fries minimal medium (Beadle and Tatum, and of these anthranilic acid is the most effective 1945) buffered at pH 5.6. The time of incubation growth inhibitor (Soboren and Nyc, 1961). was varied in the range between 24 and 96 hr. Haskins and Mitchell (1949) showed that exog- The growth was expressed as the dry weight of enous supplements of indole, tryptophan, and mycelium harvested at the end of the incubation kynurenine give rise to fluorescent products in period. The reagents employed as sul)plements the culture medium during growth of N. crassa. and the sterilization procedures used were In these studies, the major fluorescent component described in a previous communication (Soboren in the medium was identified as anthranilic acid. and Nyc, 1961). In the present work, a close correlation was Determination of fluorescent products in the noted between the growth response of a trypto- culture medium. Studies on the accumulation of phan-requiring mutant of N. crassa (strain C-83) fluorescent products were carried out on replicate 881 882 SOBOREN AND NYC J. BACTERIOL. samples of fresh culture medium from which the for each partition. Analyses with paper mycelium had been removed by filtration. The chromatography revealed the presence of three culture medium and water washes of the my- other unidentified fluorescent compounds in these celium were combined, the pH of each sample extracts. To quantitate the anthranilic acid in was adjusted to 4, and the volume was brought the culture media, it was necessary to separate up to 25 ml with acetate buffer (pH 4). Fluori- this component from other interfering substances. metric analysis of extracts of the mycelial pads This was accomplished by partitioning the solutes indicated that, even under conditions of maximal in the culture medium between diethylether and fluorescence output, the amount of fluorescent water, according to the countercurrent-distribu- material retained 'in the mycelium did not tion technique described by Craig (1944). A exceed 10% of the total produced. Thus, analysis separation of anthranilic acid from the other of the culture medium represented a true reflec- fluorescent substances could be achieved by this tion of the production of fluorescence by these procedure with six transfers between equal cultures. The determinations on these solutions volumes of the immiscible phases. For the sake were made using a model 12B photofluorometer, of convenience in the routine analysis of many employing Coleman filters PC-6 and B-2 for the samples, the following adaptation of this counter- activating and emitted beams, respectively. current-distribution procedure was used. All Anthranilic acid standards in buffer at pH 4 were partitioning of solutes between immiscible solvent used to quantitate the fluorescence of the experi- phases was performed in separtory funnels. mental samples. Since anthranilic acid is the Replicate 20-ml samples of culture media were main fluorescent component in the culture acidified to pH 2.5 and subjected to six counter- medium of strain C-83, the fluorescence of the current exchanges between 20-ml volumes of media examined was recorded in terms of a ether saturated with water as the moving phase "fluorescence unit" equivalent to 1 ,umole of and 20 ml of water saturated with ether as the anthranilic acid. This is a convenient means of stationary phase. Anthranilic acid could be expressing the accumulation of fluorescent quantitatively extracted in this manner from products, especially in the exploratory studies culture media and carried in the ether phase which required the routine analysis of many without loss to the lower phases by three volumes samples. The exploratory studies, which involved of ether. Thus, only three upper phases were variations in culture supplements and time of employed in the partition transfers, since addi- growth, focused attention upon certain culture tional extractions would tend to distribute media, and in these the accumulated anthranilic further the components remaining in the aqueous acid was extracted before its fluorimetric deter- phases. For ease of analysis, it was desirable to mination. keep the bulk of these other fluorescent compo- Quantitative determination of anthranilic acid nents in the minimal number of distribution in the culture medium. Anthranilic acid was chambers. identified as the main fluorescent component in The three ether extracts obtained were com- the culture media of strain C-83 by column bined in the seventh chamber, and the ether was chromatography on a Dowex (1 X 4) anion- evaporated off under a stream of nitrogen. Paper exchange resin with 0.1 N HCl as the eluting chromatography of the resulting residue, using solvent. The chromatographic behavior, ultra- several solvent systems, indicated that only one violet spectrum, and observed absorbance to fluorescent compound corresponding to anthranil- fluorescence ratios of the isolated anthranilic ic acid had been extracted. The rest of the acid were indistinguishable from those of an fluorescent material remained in the first four authentic sample. This confirmed the original aqueous phases after the distribution. The observation of Haskins and Mitchell (1949) residue from the ether extracts was dissolved in that anthranilic acid is the main fluorescent 25 ml of acetate buffer (pH 4), and the anthranilic component in N. crassa cultures grown on trypto- acid content was measured fluorimetrically. The phan supplements. fluorescence of the first four aqueous lower The quantitative extraction of anthranilic acid phases was also determined at pH 4. The total from culture media at pH 2.5 could be achieved fluorescence in these fractions was expressed as by three extractions with an equal volume of that equivalent to a known amount of anthranilic VOL. 85, 1963 ANTHRANILATE PRODUCTION IN N. CRASSA 883 acid, since the identity of the fluorescent material TABLE 1. Fluorescent material in the medium of was unknown. 84-hr cultures of Neurospora crassa strain C-83 grown on increasing levels of tryptophan* RESULTS L-Tryptophan Fluorescencet Fluorescence (umoles per (pmoles per Dry wt of mold per mg Accumulation offluorescent products by wild-type 20 ml) 20 ml) of mold N. crassa. It has been observed (Haskins and Mitchell, 1949) that wild-type and tryptophan- mg requiring mutants of N. crassa produce a blue 1 4.3 3.3 1.3 fluorescence in the medium during the course 2.5 8.5 7.3 1.2 of growth on tryptophan supplements. This 5 11.1 16.1 0.69 fluorescence reaches a maximal intensity with 10 6.4 35.1 0.18 time and then diminishes, except in mutants 20 2.6 40.9 0.06 such as strain C-83 which are unable to utilize 40 1.3 34.3 0.04 anthranilic acid, in which case the fluorescence * Each value is the average of triplicate sam- persists. The accumulation of fluorescent products ples. with time was studied in a wild-type strain of t Expressed as fluorescence units equivalent N. crassa supplemented with increasing levels of to anthranilic acid. tryptophan. Figure 1 shows the accumulation pattern for this strain determined at various less than 2 ,moles of anthranilic acid per culture time intervals after inoculation of the cultures. was observed at the highest tryptophan level The fluorescence was transient, as expected, in a used. strain that could metabolize anthranilic acid. A Accumulation of fluorescent products by strain peak of accumulated fluorescence was obtained C-83. The total fluorescence discharged into the between 48 and 60 hr of incubation, at which medium by a tryptophan-requiring mutant of time an amount of fluorescence equivalent to N. crassa (strain C-83) was determined at various time intervals after inoculation of cultures with levels of trypto- 2.0 supplemented increasing 25 phan. The total fluorescent material accumulated 1~~~~~~~~1 in these cultures increased with the tryptophan supplement, often reaching a maximal value in excess of 10 A.moles (anthranilic acid equivalents) .5~~~~~~~ per culture after 84 hr of incubation on 5 ,umoles of tryptophan (Table 1). At tryptophan concen- trations of 10 ,moles per culture or higher, the () 1.0 total fluorescence accumulated in 84 hr was

*2.5 markedly lower than at the 5-,umole level. Table 1 relates growth response to fluorescence values at the 84-hr incubation interval. Increasing the level of tryptophan reduced the fluorescence released per unit weight of culture by approxi- mately 30-fold over the concentration range used. Optimal growth was attained on the levels of tryptophan at which the accumulation of fluo- CULTURE TIME IN HOURS rescent substances per unit weight of mold was low. FIG. 1. Fluorescent products remaining with time Since the genetic block in strain C-83 favors in the medium of Neurospora crassa strain lA the accumulation of fluorescent substances itn cultured on tryptophan. Each value is the average the culture medium, this organism was used in of quadruplicate samples. The numbers on the curves refer to the amount of tryptophan added (,umoles preference to the normal wild-type strain for per 20 ml of culture). Fluorescence units are ex- further studies on the accumulation of these pressed in terms of ,umoles per 20 ml of culture metabolites. An additional advantage inherent ill (equivalent to anthranilic acid). this choice of organism was the absolute depeni- 884 SOBOREN AND NYC J. BACTERIOL.

TABLE 2. Separation by countercurrent distribution of tryptophan (Table 1). At tryptophan levels of fluorescent substances in 84-hr cultures of which allowed a maximal growth response of Neurospora crassa strain C-83 supplemented strain C-83, the amount of anthranilate accumu- with tryptophan* lated per mg of mold was minimal (Table 2). This suggested that the production of anthranilic L-Trypto- Total Anthra- Fluor- Anthra- phan fluor- nilic acid escence of Dry wt nilic acid acid by cells receiving exogenous tryptophan (umoles escenceinehrauos fmld prg pmrl)20 bafrationt extractt phaset of mold might be an important factor in determining growth. Of the substances known to be in pmoles pmoles Mimoles mng metabolic equilibrium with tryptophan, anthranil- 2.5 7.5 5.1 1.8 7.9 0.65 ic acid and indole were shown to be the most 5 10.2 7.6 3.4 16.8 0 45 potent inhibitors of growth for the wild-type 10 6.3 5.1 2.7 37.8 0.14 strain of N. crassa (Soboren and Nyc, 1961). 20 2.1 3.3 1.1 41.1 0.08 Table 3 shows that these compounds also inhibit 25 2.1 2.7 0.7 42.8 0.06 growth when they are added as exogenous sup- 40 0.9 3.4 0.3 38.0 0.09 plements to the tryptophanless mutant strain C-83. * Each value is the average of triplicate sam- Influence of leucine on the accumulation of ples. fluorescent products by strain C-83. It was ob- t Expressed as fluorescence units equivalent that each of the to anthranilic acid. served (Soboren and Nyc, 1961) Fluorimetric determination. amino acids phenylalanine, tyrosine, leucine, and $ methionine could partially negate the growth- inhibitory effect of tryptophan on cultures of dence of strain C-83 on exogenous tryptophan, the wild-type strain of N. crassa. These single which made it possible to control the levels of amino acids also increased the growth response this substance available to the culture during of the tryptophanless mutant strain C-83 to growth. tryptophan supplements (Soboren and Nyc, Accumulation of anthranilic acid by strain C-83. 1961). Since the improved growth response of The quantitation of anthranilic acid in culture strain C-83 to increasing levels of tryptophan is media was accomplished by fluorimetric meas- associated with a decreased production of an- urement of anthranilic acid recovered from these thranilate (Table 2), it was anticipated that the media by countercurrent distribution. Table 2 shows the results for such determinations on TABLE 3. Inhibitory effect of anthranilic acid and cultures of strain C-83 incubated for 84 hr on indole on the growth of Neurospora crassa strain increasing levels of tryptophan. The summation C-83 supplemented with tryptophan of the fluorescence in the ether extracts and in the aqueous phase after countercurrent distribu- Growth of mold (mg) on increasing amounts of inhibitor* tion exceeded the fluorescence of the original Inhibitor media of cultures grown at the higher levels of 0 2.5 5 10 20 40 tryptophan supplementation. Studies in which the ether-extracted material was recombined Anthranilic acid.... 17t 16 18 2 0 0 with the aqueous phase disclosed that these Indole ...... 17 15 9 0 0 media had a quenching effect on fluorescence which would account for the discrepancy of the Anthranilic acid .... 43t 42 28 5 0 0 recovery values. Anthranilic acid was the major Indole .... 35 19 6 1 0 fluorescent component discharged into the culture medium by strain C-83 at all of the levels of * Each value is the average of duplicate sam- tryptophan employed (Table 2). The ratio of ples. The column headings refer to the concentra- tion of either indole or anthranilic acid (,umoles anthranilic acid per unit weight of mold was per 20 ml.). in grown on greatly reduced cultures high t Growth on minimal medium supplemented tryptophan supplements. A similar decrease in with 5 Amoles of tryptophan per 20 ml. the ratio of total fluorescence produced per unit t Growth on minimal medium supplemented weight of mold was observed at increasing levels with 15,moles of tryptophan per 20 ml. VOL. 85, 1963 ANTHRANILATE PRODUCTION IN N. CRASSA 885 improved growth response of this strain to TABLE 5. Separation by countercurrent distribution certain other amino acids might also be asso- of fluorescent substances in 84-hr cultures of ciated with a decreased anthranilate production. Neurospora crassa strain C-83 supplemented with 5 An exploratory study was undertaken to deter- j.moles of tryptophan and the amino mine what effect the growth-stimulatory amino acids that improve growth* acid leucine might have upon the accumulation of Total Anthra- Fluor- Anthra- Supplement fluor- ilic acid escence nilic fluorescent products by strain C-83 supplemented (20 MAmoles escence inete of ofmlDry wt acid per 20 ml) before se- n ether aqueous o mo per mg with a fixed level of 5 ,lmoles of tryptophan per parationt extracttphaset of mold culture. This level of tryptophan, when used as a single supplement, allowed about one-half of the ,umoles pimoles ,umoles mg maximal growth response possible in strain C-83. None ...... 11.4 7.6 2.8 16.7 0.46 This suboptimal growth was associated with the L-Leucine.. 4.1 3.0 0.6 41.8 0.07 accumulation of a large amount of fluorescent L-Phenyla- material in the culture medium (Table 1). lanine.... 2.6 2.0 0.5 43.2 0.05 L-Tyrosine. 2.6 3.1 0.9 41.5 0.08 Leucine was arbitrarily chosen for this explora- L-Methi- tory work since it was one of the amino acids onine .... 6.2 3.7 1.6 46.8 0.08 which gave a large synergistic effect with trypto- phan on the growth of strain C-83 (Soboren and * Each value is the average of triplicate sam- Nyc, 1961). Leucine was added over a concentra- ples. tion range of 2.5 to 40 j,moles per flask to cultures t Expressed as fluorescence units equivalent containing 5 ,umoles of tryptophan, and the to anthranilic acid. fluorescence discharged into the medium was t Fluorimetric determination. measured at the 84-hr incubation interval. The data in Table 4 show that the total fluorescence which greatly improved the growth response of released per unit weight of culture was reduced strain C-83 to tryptophan supplements, were by several fold over the concentration range of tested for their effect on the production of leucine used. Optimal growth was attained on anthranilic acid. The addition of 20 ,umoles of those levels at which the accumulation of fluo- any one of these amino acids at the fixed level of rescent substances was reduced. 5 ,umoles of tryptophan per culture resulted in a Influence of amino acids on the accumulation of maximal growth response and a reduced level of anthranilic acid by strain C-83. The amino acids accumulated anthranilic acid (Table 5). The phenylalanine, tyrosine, leucine, and methionine, ratio of anthranilic acid per unit weight of mold was much lower for the cultures grown with the TABLE 4. Fluorescent material in the medium of supplements than for those grown on 84-hr cultures of Neurospora crassa strain C-83 tryptophan alone. grown on 5 ,umoles of tryptophan and increasing The amino acids , , , and amounts of L-leucine* were chosen at random from the amino acids which have only a small effect on the Leucine Fluorescencet Dry wt of Fluorescence growth response of strain C-83 to tryptophan (jAsmoles per (pumoles per mold per mg of 20 ml) 20 ml) mold supplements (Soboren and Nyc, 1961). These amino acids were individually tested at a level of mg 20 ,umoles per flask for their effect on the produc- 0 7.9 16.8 0.47 tion of anthranilic acid by strain C-83 cultured 2.5 6.8 27.0 0.25 on 5 j,moles of tryptophan. Anthranilic acid was 5 7.0 31.2 0.22 the 10 5.6 33.5 0.17 again major fluorescent component discharged 20 5.0 38.4 0.13 into the medium of all these cultures (Table 6), 40 4.2 40.4 0.10 but these amino acids did not produce any marked decrease in the level of anthranilic acid * Each value is the average of triplicate sam- accumulated. In addition, the values for the ples. ratio of anthranilic acid per unit weight of mold t Expressed as fluorescence units equivalent obtained with these amino acids resembled to anthranilic acid. more closely those of cultures grown on trypto- 886 SOBOREN AND NYC J. BACTERIOL.

TABLE 6. Separation by countercurrent distribution apparent that a good growth response of strain of fluorescent substances in the medium of 84-hr C-83 was attained only when the level of an- cultures of Neurospora crassa strain C-83 thranilate produced was greatly repressed. Thus, supplemented with 5 Amoles of tryptophan and empirical observations relating amino acid the amino acids that do not markedly balance to growth could be related more directly improve growth* as an effect of amino acids on the accumulation Total Ahr-Fluor- Anthra- of a growth inhibitor. escence nilic Supplement fluor- nilic acid Dry wt of (20 MAmoles escence in ether of of mold acid Tryptophan inhibits the accumulation per 20 ml) before se- extract: aqueous per mg parationt e phaset of mold anthranilic acid in tryptophan-requiring mutant strains of N. crassa (Lester, 1961a), Escherichia Mmoles ;tmoles gmoles mg colt (Pardee and Prestidge, 1958), and Aerobacter None . 9.3 6.4 2.4 16.8 0.38 aerogenes (Doy and Pittard, 1960). More specifi- L-Lysine 9.7 6.9 3.3 26.4 0.26 cally, tryptophan has been shown to inhibit L-Arginine. 9.3 8.7 2.4 24.0 0.36 the conversion of shikimate-5-phosphate to L-Proline... 11.9 8.3 3.4 19.5 0.42 anthranilate in cell-free extracts of E. coli L-Aspartic (Moyed, 1960). Significantly, this reaction is the acid ...... 11.8 8.9 3.7 21.9 0.41 earliest one specific to tryptophan biosynthesis * Each value is the average of triplicate sam- (Doy, 1960). The inhibitory effect of tryptophan ples. on the formation of anthranilic acid has been t Expressed as fluorescence units equivalent described as a feedback control mechanism to anthranilic acid. involved in the regulation of tryptophan bio- I Fluorimetric determination. synthesis (Doy and Pittard, 1960; Moyed, 1960). The observed decrease in the level of anthranilic phan alone, in contrast to the values obtained acid produced by strain C-83 in the presence of with the amino acids in Table 5. high levels of tryptophan (Table 2) is probably the result of feedback effects by tryptophan on DISCUSSION one or more in the pathway of its bio- Certain correlations have been noted between synthesis. However, to date, there are no defini- the level of tryptophan added as supplement, the tive studies at the enzyme level in N. crassa amount of anthranilic acid accumulated in the which identify the involved in the feed- medium, and the growth achieved by a trypto- back control by tryptophan, nor is it known phan-requiring mutant of N. crassa strain C-83. whether this control is by repression or by At tryptophan levels which allowed a nearly feedback inhibition. maximal growth response of this strain, the The aromatic metabolites phenylalanine, amount of anthranilic acid accumulated per tyrosine, tryptophan, p-hydroxybenzoic acid, unit weight of mold was always a minimal value. and p-aminobenzoic acid are metabolically This suggested that the production of anthranilic interrelated through certain common precursors acid by cells receiving exogenous tryptophan in N. crassa (Tatum et al., 1954) and in E. coli might be an important factor in determining (Davis, 1951). Davis (1951, 1952) noted that growth, since anthranilic acid is a potent growth phenylalanine and tyrosine inhibit the accumu- inhibitor of N. crassa. The accumulation of an- lation of some of these early precursors in mutant thranilic acid was also related to the amino acids strains of E. coli with aberrations in the synthesis which improve the growth response of strain of the aromatic compounds. A more specific C-83 to tryptophan. The addition of any one of investigation carried out on an aromatic-requiring the amino acids phenylalanine, tyrosine, leucine, mutant strain of E. coli (Fradejas, Ravel, and or methionine resulted in a near maximal growth Shive, 1961) disclosed that either phenylalanine response and a reduced level of accumulated or tyrosine can partially inhibit the formation anthranilic acid. Lysine, proline, arginine, and of cyclic intermediates related to shikimic acid. aspartic acid, which do not markedly improve the In combination, these amino acids exert a syner- growth response of strain C-83 to tryptophan, gistic effect on the level of these intermediates in had little effect on the level of anthranilic acid the cell. Significantly, the early precursors whose accumulated. From these studies, it became levels are affected in these studies are inter- VOL. 85, 1963 ANTHRANILATE PRODUCTION IN N. CRASSA 887 mediates in the biosynthesis of several aromatic of strains of Salmonella (Rydon, 1948), E. coli metabolites. It appears that the biosynthesis of (Doy, 1960), and N. crassa (Lester and Yanofsky, the aromatic compounds belongs to a complex 1961). The endogenous production of anthranilic metabolic situation in which one product exerts acid in strain C-83 and its accumulation as a control over the production of intermediates result of the metabolic block in this strain common to several other metabolites. However, probably accounts for the anthranilate found in relationships of this kind have not been exten- cultures of this organism. The resultant "self- sively studied as systems in which feedback poisoning" by endogenous anthranilate could mechanisms may function. An inhibitory effect explain the observation (Haskins and Mitchell, of tyrosine and phenylalanine on the availability 1949) that mutant strains of N. crassa which of intermediates required for anthranilate syn- are blocked in the formation of tryptophan from thesis could explain the decreased anthranilate anthranilic acid grow poorly as compared with production by strain C-83 when this organism other mutants of the tryptophan series, even has an exogenous source of these aromatic when they are supplemented with adequate amino acids (Table 5). At the supplemental amounts of tryptophan. Strain C-83, which falls levels of tyrosine and phenylalanine which were into this class of tryptophan mutants, gives employed in these studies, the decreased accumu- growth approaching that of a normal strain when lation of anthranilic acid was associated with an anthranilic acid production is repressed by increase in culture growth. Apparently, the pro- proper supplementation. duction of aromatic precursors was not depressed Wainwright and Bonner (1959) and Lester to growth-limiting levels by feedback effects in (1961b) showed that anthranilic acid may these experiments. affect the activity of several enzymes in the cell. Leucine and methionine were also effective in Gibson and Yanofsky (1960) showed that an- reducing the amount of anthranilic acid accumu- thranilate inhibits the activity of a partially lated by strain C-83. There is no direct evidence purified preparation of indole-3-glycerol phos- to relate these amino acids to aromatic biosyn- phate synthetase obtained from a tryptophan thesis, but indirect evidence indicates that some auxotroph of E. coli. The inhibitory effect of metabolic relationships may exist among these anthranilic acid on this enzyme has been cor- compounds. Barratt and Ogata (1954) described related with the observed inhibitory effect of a mutant of N. crassa with an aberration at a anthranilate on the growth of a wild-type strain single genetic locus that can utilize for growth of E. coli (Lester and Yanofsky, 1961). At low phenylalanine, tyrosine, tryptophan, or leucine levels of anthranilate, the growth-inhibitory when any one of these amino acids is employed effect on this strain of E. coli could be completely as a single supplement. The relationship of these reversed by tryptophan, but at high levels of supplements is apparently not due to an inter- anthranilate this effect could not be reversed by conversion among them (Barratt, Fuller, and tryptophan. It was suggested that, at low levels, Tanenbaum, 1956). These workers suggested anthranilic acid limits tryptophan synthesis by that perhaps the primary genetic lesion in this an inhibitory effect on indole-3-glycerol phos- strain caused the accumulation of some unidenti- phate synthetase, but at higher concentrations fied growth inhibitor whose effect can be relieved a second unknown inhibitory effect is produced by any one of the effective, yet apparently un- which cannot be reversed by tryptophan. The acid Also of interest related, amino supplements. latter type of inhibition be responsible for is the fact that a clustering of several single loci could concerning methionine, phenylalanine, tyrosine, the growth retardation produced in strain C-83 tryptophan, and leucine synthesis occurs in group by endogenously formed anthranilic acid when IV of the linkage map of N. crassa (Barratt and this organism is cultured on tryptophan supple- Strickland, 1961). Whether there is any relation- ments adequate to fulfill its minimal growth ship between the observed clustering of these requirements. The production of anthranilate genes and the ability of these amino acids to and its effect on growth involves regulating repress anthranilate production is not presently mechanisms which will need extensive probing known. at the enzyme level before they can be properly Exogenous anthranilic acid inhibits the growth evaluated. 888 SOBOREN AND NYC J. BACTERIOL.

ACKNOWLEDGMENTS HASKINS, F. A., AND H. K. MITCHELL. 1949. Evidence for a tryptophan cycle in Neuro- This study was supported in part by grant spora. Proc. Natl. Acad. Sci. U.S. 35:500-506. RG-5794 from the National Institutes of Health, LESTER, G. 1961a. Some aspects of tryptophan U.S. Public Health Service, and by the Cancer synthetase formation in Neurospora crassa. Research Fund of the University of California. J. Bacteriol. 81:964-973. LITERATURE CITED LESTER, G. 1961b. Repression and inhibition of indole-synthesizing activity in Neurospora BARRATT, R. W., R. C. FULLER, AND S. W. TANEN- crassa. J. Bacteriol. 82:215-223. BAUM. 1956. Amino acid interrelationships in LESTER, G., AND C. YANOFSKY. 1961. Influence certain leucine- and aromatic-requiring of 3-methylanthranilic and anthranilic acids strains of Neurospora crassa. J. Bacteriol. on the formation of tryptophan synthetase 71:108-114. in Escherichia coli. J. Bacteriol. 81:81-90. BARRATT, R. W., AND W. OGATA. 1954. A strain of MITCHELL, H. K., AND J. LEIN. 1948. A Neuro- Neurospora with an alternative requirement spora mutant deficient in the enzymic syn- for leucine or aromatic amino acids. Am. J. thesis of tryptophan. J. Biol. Chem. 175:481- Botany 41:763-771. 482. BARRATT, R. W., AND W. N. STRICKLAND. 1961. MOYED, H. S. 1960. False feedback inhibition: Neurospora information conference, p. 75-94. inhibition of tryptophan biosynthesis by Natl. Acad. Sci. Natl. Res. Council. 5-methyltryptophan. J. Biol. Chem. 235:1098- BEADLE, G. W., AND E. L. TATUM. 1945. Methods 1102. of producing and detecting con- PARDEE, A. B., AND L. S. PRESTIDGE. 1958. Effects cerned with nutritional requirements. Am. J. Botany 32:678-686. of azatryptophan on bacterial enzymes and CRAIG, L. C. 1944. Identification of small amounts bacteriophage. Biochim. Biophys. Acta 27: of organic compounds by distribution studies. 330-344. Separation by counter-current distribution. PARTRIDGE, C. W. H., D. M. BONNER, AND C. J. Biol. Chem. 155:519-534. YANOFSKY. 1952. A quantitative study of DAVIS, B. D. 1951. Aromatic biosynthesis. The the relationship between tryptophan and role of shikimic acid. J. Biol. Chem. 191:315- niacin in Neurospora. J. Biol. Chem. 194: 325. 269-278. DAVIS, B. D. 1952. Aromatic biosynthesis. V. RYDON, H. N. 1948. Anthranilic acid as an inter- Antagonism between shikimic acid and its mediate in the biosynthesis of tryptophan precursor, 5-dehydroshikimic acid. J. Bac- by Bacterium typhosum. Brit. J. Exptl. Pathol. teriol. 64:749-763. 29:48-57. Doy, C. H. 1960. Tryptophan biosynthesis in SOBOREN, J., AND J. F. Nyc. 1961. Amino acid microorganisms. Rev. Pure Appl. Chem. interactions in Neurospora crassa. J. Bac- 10:185-205. teriol. 82:20-25. DoY, C. H., AND A. J. PITTARD. 1960. Feedback TATUM, E. L., S. R. GROSS, G. EHRENSVARD, AND control of tryptophan biosynthesis. Nature L. 185:941-942. GARNJOBST. 1954. Synthesis of aromatic FRADEJAS, R. G., J. M. RAVEL, AND W. SHIVE. compounds by Neurospora. Proc. Natl. 1961. The control of shikimic acid synthesis Acad. Sci. U.S. 40:271-276. by tyrosine and phenylalanine. Biochem. WAINWRIGHT, S. D., AND D. M. BONNER. 1959. Biophys. Res. Commun. 5:320-323. On the induced synthesis of an enzyme re- GIBSON, F., AND C. YANOFSKY. 1960. The partial quired for the biosynthesis of an essential purification and properties of indole-3-gly- metabolite: induced synthesis in cerol phosphate synthetase from Escherichia Neurospora crassa. Can. J. Biochem. Physiol. coli. Biochim. Biophys. Acta 43:489-500. 37:741-750.