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Amino acid composition of mycobacteria
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
60, 164-170 (1956)
Amino Acid Composition of Mycobacterial Ben Ginsburg, S,arah L. Lovett and Max S. Dunn From the Chemistry
Laboratory,
University
of California,
Los Angeles,
California
Received July 18, 1955 INTRODUCTION
The amino acid composition of mycobacteria was first determined by Tamura (1) in 1913, and comparable studies were made prior to 1925 by a number of investigators. The data which these workers obtained by classical methods of analysis have been summarized by Camien et al. (2). There has been renewed interest in this problem during the past 10 years with the advent of microbiological, chromatographic, and other methods leading to results which are discussed later in this paper. It was the purpose of the present work to obtain dependable data on the amino acid composition of a number of strains of mycobacteria. EXPERIMENTAL The strains of mycobacteria employed in this study included Mycobacterium tuberculosis var. hominis H37Rv* (human virulent), M. tuberculosis var. hominis H37Ra3 (human avirulent), M. tuberculosis var. hominis 59g4 (human avirulent), M. tuberculosis var. avium 0. V. 5412 (avian virulent), M. tuberculosis var. bovis BCG4 (bovine avirulent), M. smegmatis 0. V. 668312 (saphrophyte), M. phlei 10142 (saphrophyte), M. phlei 336/289B4 (saphrophyte), M. ranae4 (cold-blooded animal virulent), and M. ranae SD4 (streptomycin-dependent variant of M. ranae). The latter two organisms were first isolated by Yegian and Budd (3). 1 Paper No. 106. This work was supported by grants from the Los Angeles Tuberculosis and Health Association, the American Trudeau Society, and the University of California. The authors are indebted to Drs. S. Froman and I. Krasnow, Olive View Sanitarium, Olive View, California, who furnished several strains of mycobacteria and provided facilities for some of the authors’ experiments. 2 Kindly furnished by Dr. S. Froman, Olive View Sanatarium, Olive View, California. 3 Kindly furnished by Mr. William Steenken, Trudeau Laboratory, Trudeau, New York. 4 Kindly furnished by Dr. L. McClaren, U.C.L.A., Medical School. 164
AMINO
ACID
COMPOSITION
OF
MYCOBACTERIA
165
All of the mycobacteria except H37Rv were grown on a modified Long’s medium containing (per 1000 ml.) the indicated quantities of the following components: KH,PO, 3.0 g., NaCl2.0 g., MgSOd.7H20 1.0 g., ferric ammonium citrate 0.05 g., diammonium citrate 5.0 g., Na&OI 12.0 g., glycerol 50 ml., and ammonium oxalate monohydrate 5.4 g. Streptomycin sulfate6 (600 pg./ml.), sterilized by passing the solution through a Seitz filter, was added to the medium on which M. runae SD was grown. Strains H37Rv and M. smegmatis O.V. 66831(S) were grown on Sauton’s medium containing (per 1000 ml.) the indicated quantities of the following components: L-Asparagine monohydrate 4.16 g., citric acid 2.08 g., KtHPO, 0.67 g., MgS04.7HzO 0.07 g., ferric ammonium citrate 0.05 g., and glycerol 63 ml. Each medium was adjusted to pH 7.2, and 2OOml. aliquots were autoclaved at 15 lb. for 15 min. in Roux bottles capped with aluminum foil. Each solution was inoculated by placing a loopful of the culture on the surface, and the inoculated medium was incubated at 37” for a specified time (see Table I) with the bottle placed in a horizontal position. Each organism was transferred on its designated medium at least twice in preparing the cultures used as inocula. When a pellicle formed on the surface of the medium, the growth was harvested and washed on a Biichner funnel three or four times with distilled water. The layer of cells was removed from the funnel and autoclaved in a beaker at 15 lb. for 15 min. Before filtration, M. smegmatis S (40 min.), H37Rv (40 min.), and M. aaium (20 min.) were autoclaved in the Roux bottles at 15 lb. for the stated times. The organisms were defatted by extracting them in a Soxhlet for 24 hr. first with acetone and then with dry ether. The extracted material was dried at 55” for 18-24 hr. giving yields of product varying from 0.24 to 0.93 g. per bottle (see Table I). Moisture was determined by heating 0.5-g. samples to constant weight in a vacuum oven at 55” over P20 5.6Ash was determined by heating the moisture-free material for 4 hr. in a muffle furnace at about 400°.S Nitrogen was determined b) semimicro Kjeldahl.0 The defatted organisms were hydrolyzed by suspending 2-g. quantities in 20 ml. of 3 N HCl and refluxing the mixture for 24 hr. over an oil bath at 130”. The hydrolysis time for M. avium was 42 hr. Each hydrolyzate was filtered, the filtrate was diluted to 100 ml. with distilled water, and aliquots of the solutions were neutralized just prior to analysis for amino acids. Alanine was determined by microbiological assay with Leuconostoc citrovorum [Sauberlich and Baumann (4)], serine with Lactobacillus casei,? methionine with Leuconostoc mesenteroides P-60,’ and all other amino acids with organisms and by methods described previously [Dunn et al. (5)]. The amino acid values reported are the averages of values usually at five levels for which the mean per cent deviation from the mean varied from 1.5 to 4.2 and averaged 2.5. 5 Kindly furnished by Dr. Kark Folkers, Merck and Company, Inc., Rahwap, New Jersey. 6 Analysis by Mrs. Grace Davis. 7 Camien, M. N., and Dunn, M. S., unpublished method.
166
GINSBURG,
LOVE’IT
AND
TABLE Moisture,
I
Ash and Nitrogen
Organism
DUNN
Content of hfycobacteria
Growth period
Cells (defatted anddried) Per bottle Moisture Ash Nitrogen4
days
M. smegmatis O.V. 66831 111. smegmatis O.V. 66831 (S) M. phlei 10142~ M. phlei 336/289 B M. ranae M. ranae SD M. tuberculosis var. avium O.V. 541 M. tuberculosis var. bovis BCG M. tuberculosis var. hominia 599 M. tuberculosis var. hominis H37Ra M. tuberculosis var. hominis H37Rv
%
12 7 18 22
12 29 179 79 5
78 47
o!i7
%
% 11.95
0.39
1.93
0.93 0.24 0.73 0.45
3.81 4.16 4.90 6.67
6.43 3.48 7.79 3.16 5.72 3.36 1.86 2.87 3.89
0.61
5.C0
4.23
11.81 9.84
0.67
5.69
6.90
6.97b
0.43 0.50 0.29
5.02 3.95 4.37 6.96
10.80
9.71 10.19 11.83
11.98 9.86 8.04
o Corrected for moisture and ash. b Average of replicate values 6.95, 7.00, 6.92, 7.00. c American Type Culture Collection number. RESULTS
AND
DISCUSSION
As shown by the data in Table II, the strains of mycobact,eria investigated have strikingly similar amino acid composition. This observation is in accord with that of other workers who have found that different strains of yeasts (6, 7) and of bacteria (2) have similar amino acid composition. As is indicated by the data in Table III, the relative order of abundance of amino acids in mycobacteria varies somewhat from that in other microorganisms (8) and in various tissues of vertebrat.es (2). Comparing the authors’ amino acid data with t.hose report.ed in 1951 by Eguchi (9) for a human strain grown on T,ong’s medium (Table IV), it may be noted t.hat the values differ markedly for most of the amino acids. This ma.y be explained at least in part by the presumed lower dependability of the calorimetric and gravimet,ric procedures employed by Eguchi. On the other hand there is reasonable agreement generally (except for aspartic acid) between the authors’ values and those which Boniece (10) found in 1953 by microbiological assay of M. phbi and M. tuberculosis var. avium grown on a chemically defined medium containing ammonium citrate as the source of nitrogen. The values did not vary significantly when peptone or casein hydrolyzate was used as the source of nitrogen. Ion-exchange chromatography has been utilized recently by Stein
a Calculated
to 16% nitrogen
smegmatis O.V. 66831 smegmatis O.V. 66831 (S) phlei 10142 phlei 336/289 B ranae ranae SD tuberculosis v:tr. ~I~UIII O.V. 541 lmvis M. tuberculosis var. BCG X. tuberculosis var. Aominis 599 M. tuberculosis var. howknis H37Ra 111.tuberculosis var. hominis H37Rv
M. M. M. M. M. M. M.
Organism
AW
6.61 7.23 6.33 6.55 6.80 6.51 7.30
6.47
6.98
6.70
6.18
Ala
9.5: 9.7; 9.2( 10.4 8.61 9.1c 10.7
10.3
9.7:
9.51
11.7
= I -
-
6.38
8.57
7.70
7.66
7.54 7.83 7.07 7.21 7.13 7.67 7.76
AG
A*nino
4.46 4 .39 4.49
1.85
9.8 5 2.06 2.02 1.72
11.6
11.3 16.2
4.06
4.35 4.29 4.27 4.32 4.43 4.29 4.50
1.87 2.00 1 .63 1.77 1.87 1.87 2.03
9.8 I 10.4 10.4 13.7 9.6 3 10.0 9.6 8
Is01
II
Hist
-
TABLE Cowlposition
Gh
Acid
-
8.77
9.20
8.85
8.78
8.29 9.05 8.09 8.01 8.26 8.48 9.51
LEUC
Amino
LYS
2.94
3.61
3.96
3.64
4.47 4.37 3.72 3.72 4.56 4.14 3.07
acida
of Mycobacteria
L
-
1.52
1.74
I .62
1 .55
1.49 1.63 1.57 1.57 1.54 1.44 1.59
Meth
-
-
2.80
3.25
3.24
2.75
2.99 3.21 2.93 3.03 2.92 2.95 3.59
Phen
-
-
3.33
3.61
3.43
3.28
3.16 3.60 3.05 3.14 2.87 3.31 3.72
SEX
-
2.16
2.15
2.43
2.01
2.45 2.52 2.06 2.26 2.16 2.00 2.35
TY~
-
6.47
6.70
6.67
7.04
6.59 6.89 5.90 5.92 6.30 6.53 6.82
Val
74.2
72.8
70.9
71.3
69.1 72.8 66.2 71.5 67.1 68.4 72.7
Total
168
QIN8BURG,
LOVETT
AND
DUNN
TABLE III Order of Abundance of Amino Acids in Microorganisms and Tissues of Vertebrates’ Amino acid
Vertebrates
Glutsmic acid Aspartic acid Leucine Lysine Arginine Valine Isoleucine Threonine Phenylalanine Histidine Methionine
Microorgmisms vafgu~ ‘;*a Mycobacteria , , CTbis paper)
(5)
13.9-11.2 10.4-8.1 7.5-6.1 7.9-5.6 6.3-5.8 5.7-4.4 4.7-3.3 4.3-3.7 4.2-3.2 2.1-1.6 2.1-1.7
13.6-7.4 8.34.0 7.9-2.9 6.7-3.2 6.2-3.0 4.8-2.7 4.3-2.4 5.3-2.2 4.8-l .3 2.1-0.8
16.2-9.6 7.3-6.2 9.5-8.0 4.6-3.0 8.6-6.4 6.9-5.9 4.54.1 3.6-2.8 2.1-1.6 1.7-1.4
o Amino acid valuee calculated to 16% nitrogen. TABLE IV Amino Acid Composition of Mycobacteria Organism Amino acid0
Human (9)
Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
12.3 7.36 1.49 2.19 7.38 3.50 2.56 10.6 6.14 11.3 5.85 3.47 4.56 4.88 2.53 1.76 6.41
7.42 4.92 0.25 10.6 1.67 4.55 7.71 3.59 1.47 3.58 4.80 4.96 0.39 2.54 6.95
7.62 4.67 0.21 10.3 1.95 4.48 7.36 3.95 1.37 3.46 4.75 4.98 0.30 2.24 6.83
Nitrogen
lO.lb
11.46
11.6*
(%)
hf. #Mei (10) M. otiwn (10) H37Rv (lZ)c H3’&-~R
a Values calculated to 16% nitrogen. b Not stated if corrected for moisture and rush c Stated to be “relative molar concentration.”
11.8 9.3 7.3 9.9 7.0 2.4 4.0 7.7 3.7 2.8 2.4 3.2 5.0 5.5 1.7 6.4
12.4 9.4 6.7 11.4 7.9 2.5 3.6 7.9 3.6 2.1 2.5 2.8 4.8 5.7 1.6 6.0
AMINO
ACID
COMPOSITION
OF
MYCOBACTERIA
169
and Moore (11) for the determination of amino acids in H37Rv and H37Ra and by Nishihara (12) in H37Rv and its streptomycin-resistant variant, H37Rv-Sr. Stein and Moore stated that “within the accuracy of the method, no differences in the amino acid composition of t#he two strains are observed.” Since no actual data were reported and no corrections for moisture, ash, or nitrogen were indicated, it appears that more precise analyses are required for substantiation of this conclusion. Nishihara’s replicate “relative molar concentration” values have been averaged by the authors (eliminating values which seem to be in error), and are shown in Table IV. While there is no basis on which to compare Nishihara’s data directly with those in the present paper, it may be noted that in both sets of values the concentrations of certain amino acids (e.g., alanine, valine, glutamic acid, and leucine) are relatively high while those of certain others (histidine, methionine, tyrosine, and phenylalanine) are relatively low. It seems of particular interest that the glutamic acid content of the human virulent strain (H37Rv) exceeded that of the human avirulent strains (H37Ra and M. tuberculosis var. hominis 599) by an average of more than 50 % and exceeded the average of that of the ten representative strains of mycobacteria by nearly 70%. Because of the possible significance of this conclusion, the nitrogen and amino acid values for H37Rv were carefully checked by replicate determinations. Also, the possibility that the medium on which this organism was grown or the method of autoclaving might be responsible for the observed difference in glutamic acid was ruled out by demonstrating that there was no change in the glutamic acid content of M. smegmatis (S) grown and autoclaved under the two sets of conditions defined previously. It may be noted, also, that the glutamic acid content of M. phlei 289B, a strain containing the “cord factor” (13), exceeded by about 35% the average value for the other nine organisms. No appreciable difference in respect to the amino acid pattern was observed between M. ranae and M. ranae SD, its streptomycin-dependent mutant. It has been shown by filter paper chromatography that a-aminobutyric acid, y-aminobutyric acid, (~,e-diaminopimelic acid, and other more common amino acids, not determined in the authors’ studies, occur free in mycobacteria or in hydrolyzates of different st’rains of these organisms (9, 14, 15).
170
GINSBURG, LOVETT AND DUNN SUMMARY
The amino acid composition of ten strains of mycobacteria cultured on chemically defined media has been determined by microbiological assay. It was found that glutamic acid is the most abundant amino acid, and that the relative order of abundance of the other amino acids differs somewhat from that of other microorganisms or vertebrates. While the over-all composition of these organisms is strikingly similar, the human virulent H37Rv is unique in having a glutamic acid content which is much greater than the human avirulent organisms or the average of the other strains assayed. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
TAMURA, S., Z. physiol. Chem. 87, 85 (1913). CAMIEN, M. N., SALLE, A. J., AND DUNN, M. S., Arch. &o&em. 8, 67 (1945). YEGIAN, D., AND BUDD, V., J. Bacterial. 66,459 (1948). SAUBERLICH, H. E., AND BAUMANN, C. A., J. Biol. Chem. 177, 545 (1949). DUNN, M. S., CAMIEN, M. N., MALIN, R. B., MURPHY, E. A., AND REINER, P. J., Univ. Calif. (Berkeley) Publ. Physiol. 8, 293 (1949). BLOCK, R. J., AND BOLLING, D., Arch. Biochem. 7,313 (1945). LINDAN, O., AND WORK, E., Biochem. J. 48,337 (1951). Wu, C., AND HOGG, J. F., J. Biol. Chem. 188, 753 (1952). EGUCHI, S., J. Biochem. (Japan) 38, 85 (1951). BONIECE, W. S., Ph. D. Thesis, Michigan State College, 1950. STEIN, W. H., AND MOORE, S., Cold Spring Harbor Symposia Qua&. Biol. 14, 179 (1940). NISHIHARA, H., J. B&hem. (Japan) 40, 641 (1953). NOLL, H., AND BLOCK, H., J. Biol. Chem. 214,251 (1955). NAGAO, K., Bull. Fat. Fisheries, Hokkaido Univ. (Japan) 2, 128 (1951); Abstr., C. A. 47, 11322 (1953). AUCLAIR, J. C., AND BENOIT, J. C., Rev. Can. BioZ. 11,509 (1953); Abstr., C. A. 47, 6485 (1953). PAULETTA, G., AND DEFRANCESXII, A., Biochim. et Biophys. Acta 9,271 (1952). FREELAND, J. C., AND GALE, E. F., Biochem. J. 41, 135 (1947). STOKES, J. S., AND GUNNESS, M., J. Bacterial. 62, 195 (1946).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
60, 164-170 (1956)
Amino Acid Composition of Mycobacterial Ben Ginsburg, S,arah L. Lovett and Max S. Dunn From the Chemistry
Laboratory,
University
of California,
Los Angeles,
California
Received July 18, 1955 INTRODUCTION
The amino acid composition of mycobacteria was first determined by Tamura (1) in 1913, and comparable studies were made prior to 1925 by a number of investigators. The data which these workers obtained by classical methods of analysis have been summarized by Camien et al. (2). There has been renewed interest in this problem during the past 10 years with the advent of microbiological, chromatographic, and other methods leading to results which are discussed later in this paper. It was the purpose of the present work to obtain dependable data on the amino acid composition of a number of strains of mycobacteria. EXPERIMENTAL The strains of mycobacteria employed in this study included Mycobacterium tuberculosis var. hominis H37Rv* (human virulent), M. tuberculosis var. hominis H37Ra3 (human avirulent), M. tuberculosis var. hominis 59g4 (human avirulent), M. tuberculosis var. avium 0. V. 5412 (avian virulent), M. tuberculosis var. bovis BCG4 (bovine avirulent), M. smegmatis 0. V. 668312 (saphrophyte), M. phlei 10142 (saphrophyte), M. phlei 336/289B4 (saphrophyte), M. ranae4 (cold-blooded animal virulent), and M. ranae SD4 (streptomycin-dependent variant of M. ranae). The latter two organisms were first isolated by Yegian and Budd (3). 1 Paper No. 106. This work was supported by grants from the Los Angeles Tuberculosis and Health Association, the American Trudeau Society, and the University of California. The authors are indebted to Drs. S. Froman and I. Krasnow, Olive View Sanitarium, Olive View, California, who furnished several strains of mycobacteria and provided facilities for some of the authors’ experiments. 2 Kindly furnished by Dr. S. Froman, Olive View Sanatarium, Olive View, California. 3 Kindly furnished by Mr. William Steenken, Trudeau Laboratory, Trudeau, New York. 4 Kindly furnished by Dr. L. McClaren, U.C.L.A., Medical School. 164
AMINO
ACID
COMPOSITION
OF
MYCOBACTERIA
165
All of the mycobacteria except H37Rv were grown on a modified Long’s medium containing (per 1000 ml.) the indicated quantities of the following components: KH,PO, 3.0 g., NaCl2.0 g., MgSOd.7H20 1.0 g., ferric ammonium citrate 0.05 g., diammonium citrate 5.0 g., Na&OI 12.0 g., glycerol 50 ml., and ammonium oxalate monohydrate 5.4 g. Streptomycin sulfate6 (600 pg./ml.), sterilized by passing the solution through a Seitz filter, was added to the medium on which M. runae SD was grown. Strains H37Rv and M. smegmatis O.V. 66831(S) were grown on Sauton’s medium containing (per 1000 ml.) the indicated quantities of the following components: L-Asparagine monohydrate 4.16 g., citric acid 2.08 g., KtHPO, 0.67 g., MgS04.7HzO 0.07 g., ferric ammonium citrate 0.05 g., and glycerol 63 ml. Each medium was adjusted to pH 7.2, and 2OOml. aliquots were autoclaved at 15 lb. for 15 min. in Roux bottles capped with aluminum foil. Each solution was inoculated by placing a loopful of the culture on the surface, and the inoculated medium was incubated at 37” for a specified time (see Table I) with the bottle placed in a horizontal position. Each organism was transferred on its designated medium at least twice in preparing the cultures used as inocula. When a pellicle formed on the surface of the medium, the growth was harvested and washed on a Biichner funnel three or four times with distilled water. The layer of cells was removed from the funnel and autoclaved in a beaker at 15 lb. for 15 min. Before filtration, M. smegmatis S (40 min.), H37Rv (40 min.), and M. aaium (20 min.) were autoclaved in the Roux bottles at 15 lb. for the stated times. The organisms were defatted by extracting them in a Soxhlet for 24 hr. first with acetone and then with dry ether. The extracted material was dried at 55” for 18-24 hr. giving yields of product varying from 0.24 to 0.93 g. per bottle (see Table I). Moisture was determined by heating 0.5-g. samples to constant weight in a vacuum oven at 55” over P20 5.6Ash was determined by heating the moisture-free material for 4 hr. in a muffle furnace at about 400°.S Nitrogen was determined b) semimicro Kjeldahl.0 The defatted organisms were hydrolyzed by suspending 2-g. quantities in 20 ml. of 3 N HCl and refluxing the mixture for 24 hr. over an oil bath at 130”. The hydrolysis time for M. avium was 42 hr. Each hydrolyzate was filtered, the filtrate was diluted to 100 ml. with distilled water, and aliquots of the solutions were neutralized just prior to analysis for amino acids. Alanine was determined by microbiological assay with Leuconostoc citrovorum [Sauberlich and Baumann (4)], serine with Lactobacillus casei,? methionine with Leuconostoc mesenteroides P-60,’ and all other amino acids with organisms and by methods described previously [Dunn et al. (5)]. The amino acid values reported are the averages of values usually at five levels for which the mean per cent deviation from the mean varied from 1.5 to 4.2 and averaged 2.5. 5 Kindly furnished by Dr. Kark Folkers, Merck and Company, Inc., Rahwap, New Jersey. 6 Analysis by Mrs. Grace Davis. 7 Camien, M. N., and Dunn, M. S., unpublished method.
166
GINSBURG,
LOVE’IT
AND
TABLE Moisture,
I
Ash and Nitrogen
Organism
DUNN
Content of hfycobacteria
Growth period
Cells (defatted anddried) Per bottle Moisture Ash Nitrogen4
days
M. smegmatis O.V. 66831 111. smegmatis O.V. 66831 (S) M. phlei 10142~ M. phlei 336/289 B M. ranae M. ranae SD M. tuberculosis var. avium O.V. 541 M. tuberculosis var. bovis BCG M. tuberculosis var. hominia 599 M. tuberculosis var. hominis H37Ra M. tuberculosis var. hominis H37Rv
%
12 7 18 22
12 29 179 79 5
78 47
o!i7
%
% 11.95
0.39
1.93
0.93 0.24 0.73 0.45
3.81 4.16 4.90 6.67
6.43 3.48 7.79 3.16 5.72 3.36 1.86 2.87 3.89
0.61
5.C0
4.23
11.81 9.84
0.67
5.69
6.90
6.97b
0.43 0.50 0.29
5.02 3.95 4.37 6.96
10.80
9.71 10.19 11.83
11.98 9.86 8.04
o Corrected for moisture and ash. b Average of replicate values 6.95, 7.00, 6.92, 7.00. c American Type Culture Collection number. RESULTS
AND
DISCUSSION
As shown by the data in Table II, the strains of mycobact,eria investigated have strikingly similar amino acid composition. This observation is in accord with that of other workers who have found that different strains of yeasts (6, 7) and of bacteria (2) have similar amino acid composition. As is indicated by the data in Table III, the relative order of abundance of amino acids in mycobacteria varies somewhat from that in other microorganisms (8) and in various tissues of vertebrat.es (2). Comparing the authors’ amino acid data with t.hose report.ed in 1951 by Eguchi (9) for a human strain grown on T,ong’s medium (Table IV), it may be noted t.hat the values differ markedly for most of the amino acids. This ma.y be explained at least in part by the presumed lower dependability of the calorimetric and gravimet,ric procedures employed by Eguchi. On the other hand there is reasonable agreement generally (except for aspartic acid) between the authors’ values and those which Boniece (10) found in 1953 by microbiological assay of M. phbi and M. tuberculosis var. avium grown on a chemically defined medium containing ammonium citrate as the source of nitrogen. The values did not vary significantly when peptone or casein hydrolyzate was used as the source of nitrogen. Ion-exchange chromatography has been utilized recently by Stein
a Calculated
to 16% nitrogen
smegmatis O.V. 66831 smegmatis O.V. 66831 (S) phlei 10142 phlei 336/289 B ranae ranae SD tuberculosis v:tr. ~I~UIII O.V. 541 lmvis M. tuberculosis var. BCG X. tuberculosis var. Aominis 599 M. tuberculosis var. howknis H37Ra 111.tuberculosis var. hominis H37Rv
M. M. M. M. M. M. M.
Organism
AW
6.61 7.23 6.33 6.55 6.80 6.51 7.30
6.47
6.98
6.70
6.18
Ala
9.5: 9.7; 9.2( 10.4 8.61 9.1c 10.7
10.3
9.7:
9.51
11.7
= I -
-
6.38
8.57
7.70
7.66
7.54 7.83 7.07 7.21 7.13 7.67 7.76
AG
A*nino
4.46 4 .39 4.49
1.85
9.8 5 2.06 2.02 1.72
11.6
11.3 16.2
4.06
4.35 4.29 4.27 4.32 4.43 4.29 4.50
1.87 2.00 1 .63 1.77 1.87 1.87 2.03
9.8 I 10.4 10.4 13.7 9.6 3 10.0 9.6 8
Is01
II
Hist
-
TABLE Cowlposition
Gh
Acid
-
8.77
9.20
8.85
8.78
8.29 9.05 8.09 8.01 8.26 8.48 9.51
LEUC
Amino
LYS
2.94
3.61
3.96
3.64
4.47 4.37 3.72 3.72 4.56 4.14 3.07
acida
of Mycobacteria
L
-
1.52
1.74
I .62
1 .55
1.49 1.63 1.57 1.57 1.54 1.44 1.59
Meth
-
-
2.80
3.25
3.24
2.75
2.99 3.21 2.93 3.03 2.92 2.95 3.59
Phen
-
-
3.33
3.61
3.43
3.28
3.16 3.60 3.05 3.14 2.87 3.31 3.72
SEX
-
2.16
2.15
2.43
2.01
2.45 2.52 2.06 2.26 2.16 2.00 2.35
TY~
-
6.47
6.70
6.67
7.04
6.59 6.89 5.90 5.92 6.30 6.53 6.82
Val
74.2
72.8
70.9
71.3
69.1 72.8 66.2 71.5 67.1 68.4 72.7
Total
168
QIN8BURG,
LOVETT
AND
DUNN
TABLE III Order of Abundance of Amino Acids in Microorganisms and Tissues of Vertebrates’ Amino acid
Vertebrates
Glutsmic acid Aspartic acid Leucine Lysine Arginine Valine Isoleucine Threonine Phenylalanine Histidine Methionine
Microorgmisms vafgu~ ‘;*a Mycobacteria , , CTbis paper)
(5)
13.9-11.2 10.4-8.1 7.5-6.1 7.9-5.6 6.3-5.8 5.7-4.4 4.7-3.3 4.3-3.7 4.2-3.2 2.1-1.6 2.1-1.7
13.6-7.4 8.34.0 7.9-2.9 6.7-3.2 6.2-3.0 4.8-2.7 4.3-2.4 5.3-2.2 4.8-l .3 2.1-0.8
16.2-9.6 7.3-6.2 9.5-8.0 4.6-3.0 8.6-6.4 6.9-5.9 4.54.1 3.6-2.8 2.1-1.6 1.7-1.4
o Amino acid valuee calculated to 16% nitrogen. TABLE IV Amino Acid Composition of Mycobacteria Organism Amino acid0
Human (9)
Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
12.3 7.36 1.49 2.19 7.38 3.50 2.56 10.6 6.14 11.3 5.85 3.47 4.56 4.88 2.53 1.76 6.41
7.42 4.92 0.25 10.6 1.67 4.55 7.71 3.59 1.47 3.58 4.80 4.96 0.39 2.54 6.95
7.62 4.67 0.21 10.3 1.95 4.48 7.36 3.95 1.37 3.46 4.75 4.98 0.30 2.24 6.83
Nitrogen
lO.lb
11.46
11.6*
(%)
hf. #Mei (10) M. otiwn (10) H37Rv (lZ)c H3’&-~R
a Values calculated to 16% nitrogen. b Not stated if corrected for moisture and rush c Stated to be “relative molar concentration.”
11.8 9.3 7.3 9.9 7.0 2.4 4.0 7.7 3.7 2.8 2.4 3.2 5.0 5.5 1.7 6.4
12.4 9.4 6.7 11.4 7.9 2.5 3.6 7.9 3.6 2.1 2.5 2.8 4.8 5.7 1.6 6.0
AMINO
ACID
COMPOSITION
OF
MYCOBACTERIA
169
and Moore (11) for the determination of amino acids in H37Rv and H37Ra and by Nishihara (12) in H37Rv and its streptomycin-resistant variant, H37Rv-Sr. Stein and Moore stated that “within the accuracy of the method, no differences in the amino acid composition of t#he two strains are observed.” Since no actual data were reported and no corrections for moisture, ash, or nitrogen were indicated, it appears that more precise analyses are required for substantiation of this conclusion. Nishihara’s replicate “relative molar concentration” values have been averaged by the authors (eliminating values which seem to be in error), and are shown in Table IV. While there is no basis on which to compare Nishihara’s data directly with those in the present paper, it may be noted that in both sets of values the concentrations of certain amino acids (e.g., alanine, valine, glutamic acid, and leucine) are relatively high while those of certain others (histidine, methionine, tyrosine, and phenylalanine) are relatively low. It seems of particular interest that the glutamic acid content of the human virulent strain (H37Rv) exceeded that of the human avirulent strains (H37Ra and M. tuberculosis var. hominis 599) by an average of more than 50 % and exceeded the average of that of the ten representative strains of mycobacteria by nearly 70%. Because of the possible significance of this conclusion, the nitrogen and amino acid values for H37Rv were carefully checked by replicate determinations. Also, the possibility that the medium on which this organism was grown or the method of autoclaving might be responsible for the observed difference in glutamic acid was ruled out by demonstrating that there was no change in the glutamic acid content of M. smegmatis (S) grown and autoclaved under the two sets of conditions defined previously. It may be noted, also, that the glutamic acid content of M. phlei 289B, a strain containing the “cord factor” (13), exceeded by about 35% the average value for the other nine organisms. No appreciable difference in respect to the amino acid pattern was observed between M. ranae and M. ranae SD, its streptomycin-dependent mutant. It has been shown by filter paper chromatography that a-aminobutyric acid, y-aminobutyric acid, (~,e-diaminopimelic acid, and other more common amino acids, not determined in the authors’ studies, occur free in mycobacteria or in hydrolyzates of different st’rains of these organisms (9, 14, 15).
170
GINSBURG, LOVETT AND DUNN SUMMARY
The amino acid composition of ten strains of mycobacteria cultured on chemically defined media has been determined by microbiological assay. It was found that glutamic acid is the most abundant amino acid, and that the relative order of abundance of the other amino acids differs somewhat from that of other microorganisms or vertebrates. While the over-all composition of these organisms is strikingly similar, the human virulent H37Rv is unique in having a glutamic acid content which is much greater than the human avirulent organisms or the average of the other strains assayed. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
TAMURA, S., Z. physiol. Chem. 87, 85 (1913). CAMIEN, M. N., SALLE, A. J., AND DUNN, M. S., Arch. &o&em. 8, 67 (1945). YEGIAN, D., AND BUDD, V., J. Bacterial. 66,459 (1948). SAUBERLICH, H. E., AND BAUMANN, C. A., J. Biol. Chem. 177, 545 (1949). DUNN, M. S., CAMIEN, M. N., MALIN, R. B., MURPHY, E. A., AND REINER, P. J., Univ. Calif. (Berkeley) Publ. Physiol. 8, 293 (1949). BLOCK, R. J., AND BOLLING, D., Arch. Biochem. 7,313 (1945). LINDAN, O., AND WORK, E., Biochem. J. 48,337 (1951). Wu, C., AND HOGG, J. F., J. Biol. Chem. 188, 753 (1952). EGUCHI, S., J. Biochem. (Japan) 38, 85 (1951). BONIECE, W. S., Ph. D. Thesis, Michigan State College, 1950. STEIN, W. H., AND MOORE, S., Cold Spring Harbor Symposia Qua&. Biol. 14, 179 (1940). NISHIHARA, H., J. B&hem. (Japan) 40, 641 (1953). NOLL, H., AND BLOCK, H., J. Biol. Chem. 214,251 (1955). NAGAO, K., Bull. Fat. Fisheries, Hokkaido Univ. (Japan) 2, 128 (1951); Abstr., C. A. 47, 11322 (1953). AUCLAIR, J. C., AND BENOIT, J. C., Rev. Can. BioZ. 11,509 (1953); Abstr., C. A. 47, 6485 (1953). PAULETTA, G., AND DEFRANCESXII, A., Biochim. et Biophys. Acta 9,271 (1952). FREELAND, J. C., AND GALE, E. F., Biochem. J. 41, 135 (1947). STOKES, J. S., AND GUNNESS, M., J. Bacterial. 62, 195 (1946).