peptides and bacterial growth · peptides and bacterial growth iv. histidine peptides as growth...

PEPTIDES AND BACTERIAL GROWTH

IV. HISTIDINE PEPTIDES AS GROWTH FACTORS FOR LACTOBACILLUS DELBRUECKII 9649*

BY VINCENT J. PETERS, J. M. PRESCOTT, AND ESMOND E. SNELL

(From the Biochemical Institute and the Department of Chemistry, The University of Texas, and the Clayton Foundation for Research, Austin, Texas)

(Received for publication, November 26, 1952)

Addition of partial digests of protein to media containing a complete assortment of free amino acids is essential for growth of some lactic acid bacteria (14) and greatly speeds growth of many others (5-7). In some cases, peptides are unquestionably the active substances involved (1, 4).

In two such cases recently analyzed, two distinctly different mechanisms were found by which peptides exerted growth activity not equaled by their component amino acids. In one instance, assimilation of a free amino acid (L-alanine), but not that of its peptides, was inhibited by an antagonistic amino acid (D-alanine) present in the medium (4). In a second case, a free essential amino acid (tyrosine), but not its peptides, was partially destroyed by the organism before it could be utilized for growth (1).

The identification of additional peptide growth factors and elucidation of their mode of action are of importance to an understanding of the mechanisms involved in growth and protein synthesis. Lactobacillus delbrueckii 730 (ATCC 9649) is an example of an organism whose growth is greatly stimulated by crude natural materials, including casein digests (3). Extensive preliminary work revealed that, peptides were the unidentified growth-promoting compounds involved. The identification of one group of stimulatory peptides for this organism is reported herein.

EXPERIMENTAL

Cultures and Inocula-Stock cultures of L. delbrueckii (ATCC 9649) were maintained in litmus milk containing 0.5 per cent glucose and 0.5 per cent Difco yeast extract.’ They were incubated at 37” for 24 hours or until

* Supported in part by a grant from the Division of R.esearch Grants and Fellow- ships of the United States Public Health Service. For Paper III of this series see Kihara, Klatt, and Snell (1).

* In most cases, 0.5 y of pyridoxamine phosphate and a purified digest of desoxyribonucleic acid sufficient to furnish 1.0 mg. of thymidine per 100 ml. of litmus milk were also added. These supplements are essential for growth of this organism in a synthetic medium (8) but are already present in crude media such as that used above and need not be separately added.

521

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522 PEPTIDES AND BACTERIAL GROWTH. IV

they became acidic to litmus, then stored in the refrigerator. Transfers were made at 7 to 10 day intervals.

The inoculum medium contained, per 5 ml., 2.5 ml. of basal medium (Table I) and 30 mg. of yeast extract. A loop transfer was made from the stock culture to the inoculum medium and incubated at 37” for 18 to 26 hours. The cells were centrifuged, washed once in sterile water, resus-

TABLE I

Composition of Basal Medium

Component

Amino acids Glucose Sodium acetate

KHzPO, 0.10 K*HPOa 0.10 MgSOr.7H20 0.16

MnSOh-4Hs0 FeSOr.7Hs0 NaCl CaCh Adenine sulfate Guanine hydrochloride Uracil Thymidinet

mount per 100 ml. double

strength medium

$-m. *

2.0 2.0

w.

24.0 4.0 4.0 5.5 2.0 2.0 2.0 0.8

--

Oleic acid Tween 40 Ascorbic acid

Calcium pantothenate Nicotinic acid Riboflavin Thiamine

p-Aminobenzoic acid 40 Pyridoxal hydrochloridet 1000 Folk acid 2.0 Biotin 0.4

-

mount per loo ml. double

strength medium

w.

2.0 200

60 Y

80 80 80 40

* The amino acid mixture described by Henderson and Snell (9) was used. t Pyridoxamine phosphate (10 7) permits faster and heavier growth. $ Supplied by a partially purified digest of desoxyribonucleic acid.

pended in sterile water, and diluted to a reading of 90 on the Evelyn calorimeter. Such a suspension shows barely visible turbidity; 1 drop was used to inoculate each 10 ml. of assay medium.

Assay Procedure-Samples for assay were pipetted into 18 X 150 mm. culture tubes and diluted to 5 ml. with distilled water. 5 ml. of the basal medium (Table I) were added. The tubes were capped, then autoclaved at 120’ for 6 to 7 minutes. After cooling, they were inoculated and incubated at 37”. Growth was determined turbidimetrically with the Evelyn calorimeter (660 m/l filter) after the desired incubation period. When materials were limiting, assays were scaled down to a total volume of 6 ml.


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V. J. PETERS, J. M. PRESCOTT, AND E. E. SNELL

Results

523

E$ect of Supplementary Amino Acids on Growth of L. delbrueckii-In the instances previously examined (1, 4), high concentrations of individual amino acids duplicated the growth-promoting effects shown by their peptides at much lower concentrations. As a means of implicating peptides possibly involved in the nutrition of L. delbrueckii, the effects on growth of high levels of supplemental amino acids were determined. Those amino acids showing a definite effect are listed in Table II. Histidine greatly stimulates growth; phenylalanine, tyrosine, tryptophan, and lysine show much less pronounced effects. The medium in which these effects are

TABLE II

E$ects of Supplementary Amino Acids on Growth of L. delbrueckii

Addition to basal medium

None ......................... n-Histidine.HCl .............. n-LysineeHCl.. ............... nn-Phenylalanine ............. nn-Tryptophan ............... n-Tyrosine ....................

T

Am60ui%pe’

:I

5.2 4.8 4.8 4.8 4.8

T

Per cent of incident light transmitted*

Incubation time ~__

15 hrs. 20 hrs. 28 hrs.

100 92 82 89 77 65 98 86 76 99 89 77 99 88 77 97 87 73

* Uninoculated medium = 100.

noted contains each of these amino acids in what by ordinary standards is considered an excess (9). It must therefore be concluded that some im- pediment to utilization of these free amino acids exists in this test organism.

Comparative Activities of Histidine and Histidine Peptides2-The stimu- lating effects of histidine are most noticeable between 15 and 28 hours of incubation (Table II). Histidine was omitted from the basal medium and the activities of histidine and several of its peptides were determined during this time interval (Fig. 1). Histidine peptides proved far more active than histidine in promoting growth. The amount of the most active histidine peptides required for growth supplies an amount of histidine very similar to that required by organisms such as Streptococcus faecalis, which utilize the free amino acid efficiently. The most active of the peptides tested are

1 We are indebted to Dr. V. du Vigneaud for samples of each of the histidine peptides tested.


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carnosine (B-alanyl-L-histidine), n-alanyl-L-hi&dine, and cz-amino-n-butyryl-L-histidine, which contain amino acid residues not normally found in proteins. However, peptides of histidine with the common L-amino acids also show enhanced activity whether these are tested in a histidine-free medium or in the histidine-containing basal medium. The comparative activities of histidine and several peptides under the latter conditions are shown in Fig. 2. This high activity of histidine peptides indicates that they are primarily responsible for the growth-promoting effects of partial protein

MICROMOLES OF HISTIDINE OR PEPTIDE PER 6 ML.

FIG. 1. The comparative activities of histidine and its peptides in supporting growth of L. delbrueckii. Histidine was omitted from the basal medium used. Curves A, B, C, and D show the responses to carnosine, n-alanyl-L-hi&dine, o(- amino-n-butyryl-L-histidine, and L-histidine, respectively. Incubation time, 17 hours.

hydrolysates for this organism. This supposition is confirmed by trials summarized elsewhere (10).

E$ect of Histidine on Comparative Activities of Tyrosine and Tyrosine Peptides-As indicated in Table II, certain other amino acids show growth effects in the basal medium used here. These effects are much less pronounced than those of histidine, and separate tests have shown that they are not observed when the medium contains sufficient histidine to permit maximal growth. Tyrosine was chosen as representative of these amino acids, and its activity was compared with that of several of its peptides. The peptides proved many times more active than free tyrosine (Fig. 3). When an excess of histidine was added and tyrosine was omitted, however,


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V. J. PETERS, J. M. PRESCOTT, AND E. E. SNELL 525

MICROGRAMS PER 6 ML

FIG. 2. The comparative activities of histidine peptides in supporting growth of L. delbrueckii. The basal medium contains 1 mg. of L-histidine per 10 ml. Incuba- tion time, 18 hours. Curve A, L-or-amino-n-butyryl-n-histidine; Curve B, n-alanyl- n-histidine; Curve C, glycyl-n-histidine; Curve D, r,-alanyl-L-histidine; Curve E, P-n-aspartyl-n-histidine; Curve F, L-histidine.

90 ’ ’ I I I I I I I I I 0 .05 0.1 0.2 0.3 0.4 0.5 1.0 2.0 4.0 6.0 6.0 10.0

MICROMOLES PER 6 ML.

FIG. 3. Stimulation of L. delbrueckii by L-histidine, n-tyrosine, and their peptides on the complete basal medium. Incubation time, 17 hours. Curve A, carnosine; Curve B, L-leucyl-n-tyrosine; Curve C, L-prolyl-n-tyrosine; Curve D, L-

histidine; Curve E, n-tyrosine.


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526 PEPTIDES AND BACTERIAL QROWTH. IV

maximal growth effects were obtained on addition of small amounts of tyrosine, and tyrosine and its peptides showed approximately equimolar activities (Fig. 4).

These observations defy detailed interpretation, but may indicate a recip- rocal relationship in which an excess of either tyrosine or histidine promotes more efficient utilization of the other amino acid. Under conditions that prevent efficient utilization of free amino acids, utilization of their peptides proceeds unhindered. Under these latter conditions, therefore, the pep-

MICROMOLES PER 6 ML.

FIG. 4. The comparative activities of tyrosine and tyrosine peptides for L. delbrueckii in a tyrosine-free medium supplemented with excess (5 mg. per 6 ml.) histidine. Incubation time, 17 hours. Curve A, L-leucyl-L-tyrosine; Curve B, L-tyrosine; Curve C, L-prolyl-L-tyrosine.

tides show greatly enhanced activities as compared with the free amino acids.

Hydrolysis of Histidine Peptides by L. del6rueckii-In previous instances examined, cells have proved capable of hydrolyzing growth-promoting peptides, even though these surpassed their component amino acids in growth- promoting activity (1, 4). By exactly similar techniques, disappearance of n-alanyl-L-h&dine by incubation with resting cells of L. delbrueckii with simultaneous appearance of alanine was readily demonstrated. Hy- drolysis of carnosine could not be demonstrated by the same technique; this, however, does not preclude such hydrolysis by growing cultures of the test organism.

Development of Histidine-Sensitive Mutant of L. clelbrueckii-During a 16 to 24 hour assay period only those tubes which contained 300 to 800 y or more of free histidine showed detectable growth (Fig. 1). If incubation is


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v. J. PETERS, J. M. PRESCOTT, AND E. E. SNELL 527

continued, the sequence of events illustrated by the growth curves of Fig. 5 occurs. The culture does not grow without added histidine, but does utilize lower levels of histidine with increasing efficiency. Higher levels of histidine (above about 300 y per 10 ml.) prevent development of this ability, as evidenced by the dip in the dose-response curve at these concentrations.

Cells that have grown in this way on small amounts of histidine behave quite unlike the parent culture. Upon subculture, they grow as rapidly

MICROGRAMS HISTIDINE HYDROCHLORIDE PER 6 ML.

FIG. 5. Effect of time on the response of L. delbrueckii to histidine. Histidine was omitted from the basal medium. Incubation at 37” for the time indicated.

with low levels of histidine as with peptides of histidine (Fig. 6). Indeed, on the molar basis, histidine is slightly more active than carnosine. This ability to utilize small amounts of histidine is retained through repeated transfers in litmus milk.

This change of L. delbrueckii in its behavior toward histidine appears to be a true mutation rather than an adaptation. Emergence of the mutant as the predominant element in the population occurs when histidine peptides or large amounts of free histidine are not available. Separate trials have shown that other nutritional requirements of the mutant culture of L. delbrueckii are exactly the same as those of the parent culture.

Attempts to Explain In.w@ciency of Small Amounts of Histidine for Parent Culture of L. delbrueckii-The possibility that one or more of the constit-


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uents normally present in the synthetic medium might be antagonistic to histidine and thus prevent the effective assimilation of low levels of this amino acid was investigated. Each substance employed in the medium was omitted individually; then the effect of adding increasingly large amounts on the response to a constant level of histidine was observed. No antagonistic effects were noted.

100 ’ ’ I I I Al I I I I 0 .05 0.1 02 0.3 0.4 jl.0 2.0 4.0 6.0 8.0 I

MICROMOLES OF HISTIOINE OR CARNOSINE PER 6 ML. 0

FIG. 6. The comparative growth responses of parent and mutant cultures of L. delbrueckii to histidine and carnosine. Histidine was omitted from the basal medium. Incubation time, 18 hours. Curve A, response of mutant culture to L-histidine; Curve B, response of mutant (0) and parent (A) culture to carnosine; Curve C, response of parent culture to L-histidine.

Certain bacteria possess a histidine decarboxylase which attacks histidine to form histamine (11, 12). The possibility that free histidine, but not its peptides, was being destroyed in this or some other way was tested. Di- rect manomet,ric tests with resting cells showed no histidine decarboxylase to be present. Similarly, no histamine could be detected on paper chro- matograms of a histidine solution incubated with cells and buffers in the range pH 3 to 7. Finally, heavy suspensions of cells were incubated for 48 hours in the complete basal medium containing histidine. Growth oc- curred under these conditions. Following incubation, the fate of the histidine was determined by microbiological assay. The results (Table IIT) failed to show any destruction of histidine by the test organism.

Possible degradation products and derivatives of histidine were tested


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for antagonistic effects against histidine. None of the compounds tested (urocanic acid, imidazolecarboxylic acid, imidazolelactic acid, imidazole aldehyde) had such effects.

From these experiments, it appears that neither is free histidine destroyed nor its assimilation prevented by antagonistic amino acids in the

TABLE III

Recovery of Histidine from Growing Cultures of L. delbrueckii*

Y

n-Histidine added. . . . . . . . . n-Histidine foundt

480

Incells...................................... 34 “ medium...................................... 442

* Culture incubated 48 hours at 37” (see the text). t Histidine was determined by assay with 8. faecdis. The cells were hydrolyzed

before assay by autoclaving at 120” with 3 N KC1 for 15 hours.

TABLE IV

Comparative Uptake of Histidine or Carnosine by Parent and Mutant Strains of L. delbrueckii*

L-Histidine added.. . . . . . . n-Histidine found after incubation

In supernatantliquid........... “ washings....................

Uptake of n-histidine.. . Carnosine added. Carnosine found after incubation

In supernatant liquid. “ washings.

Uptake of carnosine. . .

7 Y

196 105

103 69 24

56 27 22

190

90 85 54 54 46 51

Parent strain

i2

22 15 15

Mutant strain

Y 7 Y

196 105 52

75 68 53

40 12 28 14 37 26

190

* See the text for conditions.

medium.3 Thus, neither of the explanations previously found (1, 4) for the heightened activity of peptides as opposed to their component amino acids is applicable in this case.

3 Since most of the amino acids of the basal medium are essential for growth of the test organism, they cannot be eliminated entirely from the medium without preventing growth. It is possible that certain of them reach maximal concentrations within the cell at very low external concentrations. If such an amino acid were antagonistic to histidine, increases in its concentration in the medium would not necessarily depress the response to a limiting amount of histidine. For this reason, the possibility that the results described here result from action of an antagonistic amino acid cannot be wholly eliminated.


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Comparative Uptakes of His&line and Carnosine by Cells of Parent and Mutant Strains--The availability of the mutant culture, differing from the parent strain only in its ability to utilize small amounts of histidine, made possible an experiment to determine whether cells of the parent strain were freely permeable to histidine. Cells of both cultures were obtained by growing them separately in the histidine-free basal medium supplemented with a slightly suboptimal (5 y per ml.) amount of carnosine. They were centrifuged, washed several times with water, and finally packed by prolonged centrifuging. To 0.5 ml. of packed cells (about 150 mg., dry weight) of the appropriate culture was added 0.5 ml. of a solution containing either L-histidine or carnosine. The cells were suspended uniformly and incubated at 27” for 45 minutes with occasional gentle shaking. They were then recentrifuged, the supernatant liquid removed, and the cells washed once with water. Supernatant fluid and washings were separately diluted, then assayed for L-histidine with S. faecalis, or for carnosine with the parent strain of L. delbrueckii. The results (Table IV) demonstrated that the parent strain of L. debbrueckii took up distinctly less histidine than did the mutant strain. Equal amounts of carnosine were taken up by the two cultures. The experiment was repeated several times with similar results.

DISCUSSION

These results indicate that the high activity for L. delbrueckii of histidine peptides as compared with that of free histidine, cannot be explained in terms of either of the mechanisms found to apply in other instances of this phenomenon (1, 4).3 Rather, it appears due to the fact that the parent strain of this organism cannot remove histidine from dilute solutions with the same facility as it does histidine peptides. Whether this inability to remove histidine from solutjon results from a lowered permeability of the cell wall 60 this amino acid, or from a lack or inactivity of receptor mechanisms for histidine inside the cell, cannot be determined from these results. Whatever the metabolic defect may be, it is not shared by the mutant strain, which utilizes free histidine efficiently. In contrast to their behavior toward free histidine, both strains absorb carnosine equally well, as would be expected from the fact that this peptide is equally active in supporting growth of the two organisms.

The most active of the histidine peptides contain amino acids (e.g. p- alanine, a-amino-n-butyric acid) that do not occur in proteins. This indicates rather conclusively that the peptides cannot be incorporated directly into proteins of the test organism. As appears from this and preceding papers of this series (1, 4), postulation of such direct utilization is not necessary to explain the heightened growth-promoting properties of peptides as opposed to the corresponding free amino acids.


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Hydrolysis of at least some of the active peptides (e.g. D-alanyl-L-histidine) can be effected by the test organism, but whether such hydrolysis is a prerequisite to utilization of the amino acid, or whether transfer of the histidine to other peptidic combinations can occur without hydrolysis, cannot be decided from present data.

SUMMARY

The growth-promoting action of partial hydrolysates of casein for Lac- tobacillus delbrueclcii in synthetic mediums is duplicated by very high amounts of histidine. Phenylalanine, tyrosine, tryptophan, or lysine is considerably less active. Histidine peptides are far more active than is free histidine. Similarly, tyrosine peptides are more active than is tyrosine, although much less active than histidine peptides in supporting growth. The stimulatory action of partial protein hydrolysates for this organism is ascribed to the combined action of peptides of these amino acids. The most active single peptides found were n-alanyl-L-histidine, cw-amino-n-butyryl-L-histidine, and carnosine. These peptides contain amino acids not normally found in proteins; the peptides are not, therefore, utilized by direct incorporation into the protein molecule.

No amino acids or other nutrients antagonistic to histidine were found in the basal medium. Neither resting nor growing cultures of L. delbrueclcii destroyed histidine. It is probable, therefore, that neither of the previously discovered (1, 4) mechanisms by which peptides exert growth effects not shared by their component amino acids can explain the high activity of histidine peptides for this organism.

On prolonged incubation of L. dclbrueclcii with small amounts of histidine, a mutant develops which utilizes free histidine equally or more effectively than histidine peptides. The latter promote growth of the mutant and parent culture with equal effectiveness. Heavy suspensions of mutant cells removed free histidine from solution more effectively than did cells of the parent culture. Both cultures were equally effective in removing histidine peptides from solution. This inability of the parent culture to con- centrate free histidine rapidly from dilute solution, an ability which it retains for histidine peptides, may explain the requirement for peptides of histidine for rapid growth.

BIBLIOGRAPHY

1. Kihara, H., Klatt, 0. A., and Snell, E. E., J. Biol. Chem., 197,801 (1952). 2. Wright, L. D., and Skeggs, H. R., J. Bact., 48, 117 (1944). 3. Kitay, E., and Snell, E. E., J. Bact., 60, 49 (1950). 4. Kihara, H., and Snell, E. E., J. Biol. Chem., 197, 791 (1952). 5. Kihara, H., McCullough, W. G., and Snell, E. E., J. Biol. Chem., 197,783 (1952). 6. Snell, E. E., Ann. Rev. Microbial., 3, 97 (1949).


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7. Woolley, D. W., J. Biol. Chem., 166, 783 (1946). 8. McNutt, W. S., and Snell, E. E., J. Biol. Chem., 182, 557 (1950). 9. Henderson, L. M., and Snell, E. E., J. Biol. Chem., 1’72, 15 (1948).

10. Prescott, J. M., Peters, V. J., and Snell, E. E., J. Biol. Chem., 202, 533 (1953). 11. Gale, E. F., Advances in Enzymol., 6, 1 (1946). 12. Laderborg, V. A., and Clapper, W. E., J. Bact., 63, 393 (1951).


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E. SnellVincent J. Peters, J. M. Prescott and Esmond

DELBRUECKII 9649FACTORS FOR LACTOBACILLUS

IV. HISTIDINE PEPTIDES AS GROWTH PEPTIDES AND BACTERIAL GROWTH:

1953, 202:521-532.J. Biol. Chem.

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peptides and bacterial growth · peptides and bacterial growth iv. histidine peptides as growth...

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