UTILIZATION OF L- AND D-GLUTAMIC ACIDS AND L-GLUTAMINE BY ASPARTIC ACID-RESISTANT

STRAINS OF LACTOBACILLUS ARABINOSUS*

BY MERRILL N. CAMIEN AND MAX S. DUNN

(From the Chemical Laboratory, University of California, Los Angeles, Califoornia)

(Received for publication, March 28, 1955)

The inhibition of Lactobacillus arabinosus by n-aspartic acid and its re- versal either by L-glutamic acid or, more effectively, by L-glutamine were first reported by Lewis and Olcott (l).l The effectiveness of L-glutamic acid in reversing the inhibition was found to be markedly increased when either the inoculum size or the incubation time was increased, and aspar- agine was shown to be much less inhibitory than aspartic acid (l), thus accounting for the regular, rather than sigmoidal, n-glutamic acid standard curve obtained with the earlier assay medium of Dunn et al. (3) which con- tained asparagine rather than aspartic acid. Subsequently, numerous in- vestigations of L. arabinosus either with L- or nn-aspartic acid as the inhibitor (4-S) or with other somewhat less effective glutamic acid anti- metabolites (9-12) have yielded results substantially the same as those of Lewis and Olcott (1). It has been generally concluded that the observed inhibitions are effected through a blocking of the system involved in glu- tamic acid amidation, and Hat et al. (5) proposed that “glutamine, rather than glutamic acid, is the substance actually utilized by the test organism.” Ayengar et al. (8), however, thought it “doubtful that all of the glutamic acid must be converted to glutamine,” since they showed that combinations of L-glutamic acid and glutamine in an aspartic acid-rich medium gave better growth than equivalent amounts of glutamine alone, and Borek and Waelsch (11) pointed out that some uncertainties existed because of the possibility that inhibitors might block either the metabolism or the pene- tration of a nutrient.

Much less attention has been given to the inhibition of n-glutamic acid than L-glutamic acid utilization of L. arabinosus. Dunn et al. (3) showed that the response of this bacterium to nn-glutamic acid was markedly greater than that to n-glutamic acid in aspartic acid-free media, and this

* Paper 194. This work was aided by grants from Eli Lilly and Company, the Nutrition Foundation, Inc., Swift and Company, the United States Public Health Service, and the University of California. The authors are indebted to Miss Lynn Wyler for technical assistance.

1 Feeney and Strong (2) had previously reported a similar relationship with Lac- tobacillus casei.

125

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126 GLUTAMIC ACID AND L. ARABINOSUS

was subsequently con6rmed by Baumgarten et al. (6). They inferred that “the unnatural antipode of glutamic acid plays some essential r81e in the metabolism of the microorganism.” This conclusion was supported by the subsequent finding of considerable amounts of n-glutamic acid in hy- drolysates of L. arabinosus cells (13, 14), but did not appear to be in accord with the results of a later investigation (15) which showed that n-glutamic acid utilization by L. arabinoms may be completely inhibited by aspartic acid in concentrations so low as to have little effect upon n-glutamic acid utilization.

Recently, a number of aspartic acid-resistant strains of L. arabinosus have been developed and studied in the authors’ laboratory, both for the purpose of elucidating the aspartic acid-glutamic acid interrelationship further and for the possible special applications such strains might have in microbiological assays. The results of these studies are presented in the present paper.

EXPERIMENTAL

Maintenance of the cultures, preparation of inocula, and general experi- mental techniques were as described previously (15, 16). The aspartic acid- and glutamic acid-free basal medium (15), containing 6 mg. per cent of asparagine, was supplemented as required with aspartic acid and with L- and n-glutamic acids. The basal medium supplemented with 4 mg. per cent of n-glutamic acid will be referred to as Medium D, and that supple- mented with 4 mg. per cent of n-glutamic acid as Medium L. The pH (before autoclaving) of the media either with or without supplements was 6.5, unless otherwise indicated.

Experiments were first directed toward the isolation of strains capable of utilizing n-glutamic acid in the presence of elevated concentrations of n-aspartic acid. Serial subculture of L. arabinosus 17-5, ATCC 8014, in Medium D, supplemented with gradually increasing concentrations of n-aspartic acid, failed to yield resistant strains, and the use of n-aspartic acid gradient plates, prepared with Medium D containing 2 per cent agar,* was likewise unsuccessful. A strain of considerably increased aspartic acid resistance was readily isolated, however, with the aid of a turbidostatic selector.8 A detailed description of this apparatus and the gradient plate method of isolating resistant strains have been presented previously in de- tail by Bryson and Szybalski (17).

* Special agar-Nobel, supplied by the Difco Laboratories, Inc., was employed in the preparations of solid media.

8 This was of independent design but identical in principle of operation with the instrument developed by Bryson and Szybaleki (17). The authors are indebted to Karl K. Jensen, Ross W. Farmer, Frank Schuster, Andrew Schutz, and Edgar L. Wheeler, of this department, for invaluable aid in the construction of this apparatus.

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hf. N. CAMIEN AND M. 8. DUNN 127

In the present application, Medium D contained in the culture tube of the turbidostatic selector was inoculated with L. arabinosus 17-5, and the resulting growing culture was maintained by the instrument at a constant population density, while L-aspartic acid was automatically introduced into the culture medium in such a manner that its concentration increased proportionately to the progression of growth. After 60 hours of operation the L-aspartic acid concentration within the culture tube had reached 16 mg. per cent and the rate of growth of the culture had dropped to a negli- gible value.

Samples of the final culture from the turbidostatic selector were plated out in Medium D supplemented with 2 per cent agar and 20 mg. per cent of L-aspartic acid to yield isolated colonies4 of aspartic acid-tolerant varie- ties, and the latter were transplanted by stabs into yeast dextrose agar (Difco Laboratories). Since preliminary tests indicated that the cultures isolated in this manner were not significantly different from each other, only one strain, designated L. arabinosus 376-15, was retained for further investigation.

Experiments were next directed toward the isolation of strains capable of utilizing L-glutamic acid in the presence of elevated concentrations of L-aspartic acid. Such strains were found to arise merely upon prolonged incubation of either L. arabinosus 17-5 or L. arabinosus 376-15 in Medium L, supplemented with inhibitory concentrations of L-aspartic acid. They were isolated by plating out in Medium L, which contained 2 per cent agar and was supplemented with 1 per cent r.,-aspartic acid. The strains de- rived in this manner from L. arabinosus 17-5 did not differ significantly among themselves, and only one such strain, designated L. arabinosus 521-17-5, was saved for further study. For the same reason only one strain derived from L. arabinosus 376-15 was retained. It was designated L. arabinmus 521-376-15.

RESULTS AND DISCUSSION

L. arabinmus S76-16-The response of this strain to L-aspartic acid in Medium D is compared in Fig. 1 with that of the wild type strain 17-5. The considerably increased resistance to L-aspartic acid under these con- ditions is apparent. a-Methyl-nn-glutamic acid6 was nearly as effective an inhibitor in Medium D as was L-aspartic acid, and it may be seen (Fig. 2) that the resistance of strain 376-15 to this inhibitor was also considerably greater than that of the parent strain. Likewise, the resistance of strain

4 Agar plate cultures were incubated in an atmosphere of carbon dioxide since this procedure yielded better colony growth than incubation in air.

6 The authors are indebted to Dr. Karl Pfister of Merck and Company, Inc., for a generous supply of this product.

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128 GLUTAMIC ACID AND L. ARABINOSUS

376-15 in Medium D to L-asparagine, a relatively feeble inhibitor, was greater than that of strain 17-5 (Fig. 3). The same relationship held also with nn-methionine sulfoxide, previously described (18) (data not shown), which under the present conditions was an even less effective inhibitor than L-asparagine.6 Much higher concentrations of n-aspartic acid were re-

16 14

a 7

25 50

FIG. 1 FIQ. 2 FIG. 1. Response of strain 17-5 (Curves A, A’, A”) and strain 376-15 (Curves B,

B’, B”) to L-aspartic acid (inhibitor) in Medium D after 2 days (Curves A, B), 4 days (Curves A’, B’), and 6 days (Curves A”, B”) incubation. The values on the hori- zontal scale are the concentrations of inhibitor as mg. per cent. Those on the verti- cal scale are the titration values (ml. of 0.01 N sodium hydroxide per ml. of culture).

FIG. 2. Same as Fig. 1, except that the inhibitor is cu-methyl-DL-glutamic acid.

14 14

7 7

600 1200 200 400 Fra. 3 FIQ. 4

FIG. 3. Same as Fig. 1, except that the inhibitor is natural asparagine. FIG. 4. Same as Fig. 1, except that the response to n-aspartic acid was determined

in Medium L. 6 day responses, corresponding to Curves A” and B” in Fig. 1, were not determined.

quired to effect the inhibition of these strains in Medium L than in Medium D, and it may be seen (Fig. 4) that the resistance of strain 376-15 to L-

aspartic acid was only slightly greater than that of strain 17-5 in Medium L. With prolonged incubation under these conditions, however, good growth of either strain resulted (Curves A’, B’, Fig. 4), presumably through

6 The relatively high inoculum concentration and prolonged incubation time largely account for the impotence of methionine sulfoxide in the present experiments as contrasted with those of Borek el al. (9).

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M. N. CAMIEN AND M. S. DUNN 129

the spontaneous appearance of new strains of which representative types (strains 521-17-5 and 521-376-15) are described in a later section.

The increased resistance of strain 376-15 to L-aspartic acid (Fig. 1) and other glutamic acid antimetabolites (Figs. 2 and 3) appears to be perma- nent, since no change in these properties has been observed over a 13 year period during which strain 376-15 has been serially subcultured at 3 week intervals in yeast dextrose agar. In all respects other than that of aspartic acid resistance, L. arabinosus strain 376-15 has appeared to be identical with strain 17-5.

Strains 621-17-6 and &?l-.!J76-l&-These strains responded to L-aspartic acid in Medium D in a similar manner to the strains (17-5 and 376-15, respectively) from which they were derived (Fig. 5), but, unlike the parent strains, they appeared to be entirely resistant to L-aspartic acid in Me- dium L (Fig. 6). Aside from the differences in common with their respec- tive parent strains in Medium D (Fig. 5), the two new strains differed in the following important respects. (a) Strain 521-376-15 produced a floc- culent growth in liquid media, whereas strain 521-17-5 produced a uniform turbidity indistinguishable from that produced by either strain 17-5 or 376-15. All four strains possessed identical morphology of individual cells, but the cells of strain 521-376-15 were linked into long intertwining chains, whereas those of the other three strains occurred predominantly as either isolated cells or very short chains. (5) Strain 521-376-15 was stable to ten serial subcultures in yeast dextrose agar, whereas strain 521-17-5 lost its resistance to aspartic acid after a corresponding series of subcultures. (c) The response curve of strain 521-17-5 with n-aspartic acid in Medium L tended to be U-shaped in repeated experiments (one set of data shown in Fig. 6), whereas the corresponding curve with strain 521-376-15 was essen- tially a horizontal line (Fig. 6).

The somewhat U-shaped response of strain 521-17-5 to L-aspartic acid in Medium L (Curve a, Fig. 6) is probably correlated with the instability of this strain and is probably a result of the presence of non-resistant, to- gether with resistant, cells in the inoculum. Analogous U-shaped responses of a histidineless Eschwichia coli mutant to histidine have been explained in detail by Ryan and Schneider (19)’ on the basis of mutual interference between the histidineless strain and its histidine-independent derivatives. Experiments were not carried out to confirm the suspected relationship between strain 521-17-5 and its non-resistant counterpart, but an analo- gous investigation of strain 376-15 showed that its development in Medium D supplemented with 20 mg. per cent of L-aspartic acid is markedly im-

7 It is of interest that U-shaped histidine response curves observed earlier with Lactobacillus fetmenti (20) were essentially the same as those described by Ryan and Schneider. Analogous threonine responses with L. fermenti were also observed (20).

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130 GLUTAMIC ACID AND L. ARABINOSUS

paired by the presence of cells of its non-resistant parent, strain 17-5 (Fig. 7), and it was established that this effect resulted from acidity developed (to pH values below 4.9) in the medium through glycolytic activity of the

IO 6

5 3

20 40

FIQ. 5 Fxo. 6 FIN. 5. Response of strains 17-5 (Curve A), 521-17-5 (Curve a), 376-15 (Curve B),

and 521-376-15 (Curve b) to L-aspartic acid in Medium D with 40 hours incubation. The coordinates are as in Fig. 1.

FIG. 6. Same as Fig. 5, except with Medium L and 20 hours incubation. The broken line denotes a change in scale at 100 mg. per cent of L-aspartic acid.

8

4 5

6 7 8 5 IO

Fxa. 7 FIG. 8

FIQ. 7. 3 day acid production (values on the vertical scale) of strain 376-15 (Curve A) in Medium D supplemented with 20 mg. per cent of L-aspartic acid and with varying numbers of cells of strain 17-5 per ml. (Logs of these numbers are shown on the horizontal scale.) The initial numbers of strain 376-15 cells were 106 per ml. The acidity produced by the glycolytic action of the non-growing strain 17-5 cells alone is shown in Curve B. This acidity was subtracted from the total acidity of the mixed cultures to obtain the values plotted in Curve A.

FIQ. 8. 3 day response of strain 17-5 to L-aspartic acid in Medium D, adjusted before autoclaving to the pH values indicated on the curves. The titration values have been corrected by subtracting the corresponding values for the uninoculated media (pH 4.5,8.26; pH 5.0, 5.27; pH 5.5,2.&I; pH 6.0, 1.37; pH 6.5,0.68). Otherwise the coordinates are the Bame as in Fig. 1.

non-proliferating cells of strain 17-5. The effects of reduced pH on sensi- tivity of strains 17-5 and 376-15 to L-aspartic acid in Medium D are pre- sented in Figs. 8 and 9, respectively. The results at pH values between 6.5 and 8.0 were excluded from Figs. 8 and 9 to avoid crowding, but it was

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M. N. CAMIEN AND M. S. DUNN 131

observed that L-aspartic acid sensitivity of either strain increased with in- creasing pH above 6.5. With strain 17-5 the L-aspartic acid response curves at pH 7.0,7.5, and 8.0 fell between those at pH 5.5 and 5.0 in Fig. 8. The corresponding curves at pH 7.0 and 7.5 for strain 376-15 fell slightly below those at pH 6.5 and 6.0, respectively, in Fig. 9, while the curve at pH 8.0 was nearly superimposable on that at pH 5.0 (Fig. 9). These pH effects were comparatively small in the range from pH 6.0 to 7.0, and ap- pear, therefore, to be distinct from those concerned in the inhibition of L-glutamic acid utilization. Ayengar et al. (8), for example, employing a medium containing 100 mg. per cent of aspartic acid, observed that shifting the pH from 6.0 to 7.0 resulted in a marked, frequently complete depression

25 50 100 200

FIG. 9 FIQ. 10

Fro. 9. Same as Fig. 8, except with strain 376-15. FIG. 10. 42 hour response of L. arabinosus 17-5 to L-aspartic acid in the basal medium

containing the following supplements: Curve A, 2 mg. per cent of L-glutamine, Curve A’, 4 mg. per cent of L-glutamine, Curve B, 4 mg. per cent of L-glutamic acid, Curve B’, 2 mg. per cent of L-glutamic acid plus 2 mg. per cent of L-glutamine, Curve C, 4 mg. per cent of n-glutamic acid, Curve C’, 2 mg. per cent of n-glutamic acid plus 2 mg. per cent of L-glutamine. The coordinates are the same as in Fig. 1.

of the response of L. arabinosus to L-glutamic acid, and Borek and Waelsch (11) showed that the marked inhibitions of L-glutamic acid utilization, in aspartic acid-containing media, by either oxalacetate or its spontaneous decomposition product, bicarbonate, were due to the mild alkalizing prop- erties of the latter substance.

A further important distinction between the aspartic acid-inhibited system involved in D-glutamic acid utilization and that involved in L-glu- tamic acid utilization is revealed by the relationships shown in Fig. 10. It may be seen that, whereas the utilization of L-glutamic acid in an equal mixture with L-glutamine (2 mg. per cent each) was not inhibited by L-

aspartic acid (Curve B’, Fig. lo), the utilization of L-glutamic acid alone was completely inhibited (Curve B, Fig. 10). The utilization, however, of n-glutamic acid in an equal mixture with L-glutamine (2 mg. per cent of each) was apparently completely inhibited by L-aspartic acid, since with

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132 GLUTAMIC ACID AND L. ARABINOSUS

excess inhibitor the response of L. arabinosus to the mixture (Curve C’, Fig. 10) was the same as that to 2 mg. per cent of L-glutamine alone (Curve A, Fig. 10). Moreover, L-glutamic acid in moderate concentrations (2 to 8 mg. per cent) markedly stimulated the response of L. arabinosus to L-

glutamine in the basal medium supplemented with a large excess (1 per cent) of L-aspartic acid (Fig. ll), whereas D-ghlbnk acid, under compa- rable conditions, was without effect. * These results suggest, as has been proposed previously, that the amidation of L-glutamic acid is blocked by excess inhibitor, and further that the requirement of L. arabinosus 17-5 for L-glutamine is quantitatively much smaller than for L-glutamic acid, so that, when even very small amounts of L-glutamine are supplied in the

FIQ. 11. 20 hour response of L. arabinosus 17-5 to L-glutamine in the basal me- dium, supplemented with 1 per cent L-aspartic acid and the indicated (0,2,4,6, and 8) mg. per cent of L-glutamic acid. The values on the horizontal scale are the micro- grams of L-glutamine per ml. of medium. Otherwise, the coordinates are the same as in Fig. 1.

medium (Fig. ll), the inhibited reaction is no longer essential for the utili- zation of L-glutamic acid.

n-Glutamic acid utilization, on the other hand, may apparently be com- pletely inhibited by L-aspartic acid even in the presence of either L-gluta- mine (Fig. 10) or L-glutamic acid (15), suggesting that the inhibition in this case affects either the penetration of D-glutamic acid into the cell or its conversion to L-glutamic acid. The alternative appears unlikely, how- ever, from the following evidence. First, the glutamic racemase activity of L. arabinosus cell preparations (21, 22), through which L-glutamic acid may be formed from n-glutamic acid, is not inhibited by aspartic acid (22). Second, L. arabinosus 17-5 cells harvested from Medium L with and without a supplement of 20 mg. per cent of L-aspartic acid, hydrolyzed, and assayed

8 A stimulation of L-glutamine response by n-glutamic acid in medium containing only 100 mg. per cent of aspartic acid has been reported by Ayengar et al. (8), but the n-glutamic acid concentrations employed by these authors were necessarily much higher than the L-glutamic acid concentrations used in the present experiments (Fig. 11).

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M. N. CAMIEN AND M. S. DUNN 133

microbiologically (14) for L- and n-glutamic acids, were found in the present experiments to contain identical proportions (60 per cent L- and 40 per cent D-) of the two enantiomorphs. It may be concluded from the latter exper- iment that the racemization of L-glutamic acid to yield n-glutamic acid in growing cells of L. arabinosus 17-5 is not significantly inhibited by an amount of L-aspartic acid (20 mg. per cent) sufficient to block completely the utilization of n-glutamic acid by this microorganism (Fig. 1). It is concluded, therefore, that L-aspartic acid inhibits n-glutamic acid utiliza- tion in L. arabinosus by blocking the penetration of this nutrient into the bacterial cell, whereas L-glutamic acid utilization is evidently inhibited through a blocking of the amidation of this acid. (The inhibition by as- partic acid of L-glutamic acid uptake in Staphylococcus aureus (23) appears to be a related phenomenon.)

Other differences between the two aspartic acid-inhibited systems are also in evidence. The gene governing the system involved in n-glutamic acid utilization is apparently very stable, since strains (comparable to L. arabinosus 376-15) evidencing a mutation in this gene developed from strain 17-5 only under carefully controlled selective conditions. On the other hand, the gene correspondingly involved in L-glutamic acid utilization is apparently highly mutable, since strains showing alteration in this gene invariably appeared when either L. arabinosus 17-5 or L. arabinosus 376-15 was incubated in Medium L supplemented with inhibitory concentrations of L-aspartic acid. It is of interest that strain 376-15, which is mutated in respect to n-glutamic acid utilization, also shows some increase in re- sistance to inhibition of L-glutamic acid utilization (Fig. 4), suggesting that this strain might actually be a double, rather than a single, mutant. Continuous growth in the turbidostatic selector under the conditions which led to the isolation of strain 376-15 could easily have favored the selection of a double mutant, and t’he second mutated gene, though apparently of minor consequence in strain 376-15, might account for the interesting characteristics observed in strain 521-376-15, a derivative of strain 376-15, but not in strain 521-17-5, a derivative of strain 17-5.s

The high degree of cross resistance of aspartic acid-resistant strain 376-15 to cr-methyl-nn-glutamic acid (Fig. 2), L-asparagine (Fig. 3), and nn-methi- onine sulfoxide appears to be worthy of comment. It has been suggestedlO that in L. arabinosus 376-15 the reactive sites of n-glutamic acid penetration may, through mutation, have become more specific for this nutrient, and hence less reactive not only with L-aspartic acid but also with other glu-

@The characteristics referred to are the stability of strain 521-376-15, but not strain 521-17-5, to serial subculturing in yeast dextrose agar and the flocculent growth of strain 521-376-15 in liquid media contrasted to the homogeneous growth of the other strains under the same conditions.

10 Private communication from Dr. D. E. Atkinson of this department.

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134 GLUTAMIC ACID AND L. ARABINOSUS

tamic acid antimetabolites. Tests for cross resistance in strains 521-17-5 and 521-376-15 were not carried out.

The present experimental results have led to a proposed scheme, outlined in Scheme 1, for the utilization of L- a,nd n-glutamic acids and L-glutamine”

Ertrncelluln~ !

(a) /

Intracellular

n-Glutamic acid --+---+ n-glutamic acid ___f n-glutamic acid (b) Jr racemization I residues

L-Glutamic acid -j- L-glutamic acid -- + I

Cell wall + hy-

(rl) drol- (c) ami-

ysis I( dation

L-glutamic acid residues

L-Glutamine -l----3 L-glutamine >

Protein or other essential com- plex protoplas- mic constitu- ent

SCHEME 1. Scheme of L-glutamine and L- and n-glutamic aaid utilization by L. arabinosus.

by L. arabinosus. The first step in the utilization of these substances is pictured as penetration of the cell wall, since n-glutamic acid utilization may apparently be inhibited at this point (Scheme 1, a). All three com- pounds are considered to be essential intracellular metabolites, even though L-glutamine has proved to be an adequate nutrient even in the absence (extracellularly) of L- and D-glutamic acids under all conditions thus far tested. D-Glutamic acid is considered to be an essential metabolite for the following reasons. (a) Ample proportions of n-glutamic acid are appar- ently invariably present in hydrolysates of L. arabinosus cells, as shown both in the present experiments and in those of earlier investigations (13, 14). (b) Combinations of n-glutamic acid with either L-glutamic acid (3, 6) or L-glutamine (8) (Curve C’, Fig. 10) promote better growth of L. arabi- ~OSUS in inhibitor-free media than the same total amounts of either L-glu- tamic acid or L-glutamine alone. L-Glutamic acid is considered to be an essential metabolite because in its absence in the basal medium, supple- mented with 1 per cent L-aspartic acid, the amount of L-glutamine required to produce half maximal response is increased approximately 30-fold (Fig. ll), and the extra supply of L-glutamine is apparently needed, under these conditions, to form the essential L-glutamic acid. The intracellular inter-

11 n-Glutamine is excluded because it apparently is not utilizable by L. arabinosus (unpublished data by Mr. Albert Lepp of this department with the present authors).

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M. N. CAMIEN AND M. S. DUNN 135

conversions of the three essential metabolites become essential when one or two of the three are lacking from the external environment. These in- terconversions are represented in the proposed scheme as racemization (Scheme 1, b) of either L- or n-glutamic acid to yield the opposite enantio- morph, amidation (c) of L-glutamic acid to yield L-glutamine, and hydroly- sis (d) of L-glutamine to yield L-glutamic acid. Of these three pot,entially inhibitable reactions, only that represented at Scheme 1, c, has been inhib- ited sufficiently to produce an effect on L. arabinosus growth. It is of considerable interest, however, that the racemase activity of acetone-dried cells of L. arabinosus is markedly inhibited by cY-ketoglutaric acid (22), that the hydrolysis by S. aureus of L-glutamine to yield L-glutamic acid is inhibited by various r-alkylamides of glutamic acid (24), and that y-gluta- mylhydrazine inhibits the hydrolysis of L-glutamine by streptococcal ex- tracts (25).

In Scheme 1, all three of the essential metabolites are pictured, for sim- plicity, as leading to the corresponding amino acid residues in a single essential complex protoplasmic constituent. It seems probable, however, that at least several such constituents are involved and that the different complexes contain residues of either all three or only one or two of the des- ignated amino acids. An interesting example of one type of complex which might be derived in the suggested manner from n-glut’amic acid (Scheme 1) is the uridine 5’-pyrophosphate derivative which was isolated from S. aureus and shown to contain a peptide moiety composed of one n-glutamic acid, one L-lysine, and three alanine residues (26).

SUMMARY

The utilization of L- and n-glutamic acids and L-glutamine by Lactobacil- lus arabinosus 17-5 and by aspartic acid-resistant strains derived from this microorganism has been investigated. The aspartic acid-inhibited system involved in L-glutamic acid utilization by L. arabinosus was shown to be genetically and metabolically distinct from that involved in n-glutamic acid utilization. Reasons were given for considering both L- and D-gh-

tamic acids as well as L-glutamine essential metabolites for L. arabinosus, and a scheme of the probable pathways of these metabolites was proposed. The requirement of L. arabinosus for L-glutamine was shown to be quanti- tatively much smaller than the simultaneous (metabolic) requirements of this microorganism for L- and n-glut’amic acids.

BIBLIOGRAPHY

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Merrill N. Camien and Max S. DunnOF LACTOBACILLUS ARABINOSUS

ASPARTIC ACID-RESISTANT STRAINSACIDS AND l-GLUTAMINE BY

UTILIZATION OF l- AND d-GLUTAMIC

1955, 217:125-136.J. Biol. Chem. 

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