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1 STUDIES ON THE METABOLISM OF MYCOBACTERIUM TUBERCULOSIS VI. THE EFFECT OF KREBS' TRICARBOxyLIC ACID CYCLE INTERMEDIATES AND PRECURSORS ON THE GROWTH AND RESPIRATION OF MYCOBACTERIuM TUBERCULOSIS1 NELDA B. HOLMGREN,2 IRVING MILLMAN,3 AND GUY P. YOUMANS Department of Bacteriology, Northwestern University Medical School, Chicago, Illinois Received for publication March 29, 1954 There is no general agreement concerning the oxidation of many of the Krebs' cycle intermediates and precursors by tubercle bacilli. Nakamura (1938) using classical Warburg techniques reported that of 25 substrates tested only glycerol and glutamic acid stimulated respiration. Some of the materials found inactive were glucose, succinic, malic, and citric acids. Cutinelli (1940a,b,c) found that succinic, fumaric, malic, and pyruvic acids caused only small increases in oxygen uptake. Edson and Hunter (1943) confirmed Cutineili's results with pyruvate and succinate using the avirulent Ti strain of Mycobacterium tuberculosis var. hominis and claimed that citrate was not oxidized. Bernheim (1942) reported that glucose and lactic, pyruvic, and succinic acids were not oxidized by strains H37 and B1 while Oginsky, Smith, and Solotorovsky (1950) reported that with the avian Kirchberg strain pyruvic, oxalacetic, and pyruvic plus oxalacetic acids were oxidized slowly. Other members of the citric acid cycle, cis-aconitate, citrate, a-ketoglutarate, succinate, and fumarate, did not permit significant increases in oxygen uptake over a period of 270 minutes. Geronimus and Birkeland (1951) found that avirulent mycobacteria oxidized lactic and low molecular weight fatty acids more rapidly than pathogenic strains. Edson (1951) in a review of the intermediary metabolism of the mycobacteria attempted to integrate most of the scattered information relative to this problem. He came to the conclusion that there were few, if any, clues to the understanding of the intermediary changes. 1 This investigation was aided, in part, by a research grant from Parke, Davis and Company, Detroit 32, Michigan. 2 Medical Research Fellow of the National Tuberculosis Association. ' Predoctorate Fellow of the U. S. Public Health Service. Recently, Holmgren and Youmans (1952) have shown the existence of differences in metabolism between M. tuberculosis var. hominis, strain H37Rv, and certain avirulent strains including the H37Ra strain. The results of these studies indicated a decreased metabolic efficiency of the avirulent mammalian strains tested since these were unable to utilize some as yet undefined substance in bovine serum which markedly stimulated the growth of the H37Rv strain. Differences in the utilization of amino acids (Dubos, 1949; Marshak, 1951), purines and pyrimidines (Marshak, 1951), and of organic acids (Dubos, 1950) also have been reported. A previous report (Youmans and Youmans, 1953a) detailed the effect of certain Krebs' cycle intermediates and precursors on the growth of the virulent H37Rv strain. The present paper is concerned with the effect of these substances upon the growth and respiration of the avirulent variant H37Ra. METHODS Growth 8tudie. The virulent H37Rv and avirulent H37Ra strains of M. tuberculo8i8 var. hominis which had been maintained as surface pellicles on modified Proskauer and Beck medium (P & B) (Youmans and Karlson, 1947) with glycerol as the carbon source were employed. The effect on the growth rate of these strains of 13 substrates as carbon sources to replace glycerol in the chemically defined P & B medium was determined by the small inocula method previously described (Youmans and Youmans, 1949). Each substrate, in the highest concentration employed, was dissolved in the sterile P & B medium. The hydrogen ion concentration was adjusted, when necessary, to a ph of 7.0 with 40 per cent NaOH, and the solution then was sterilized by filtration through UF sintered glass. Dilutions from the highest 405

2 406 N. B. HOLMGREN, I. MILLMAN, AND G. P. YOUMANS [VOL. 68 TABLE 1 A stattstical comparison of the generation time obtained by estimating and by calculating the slope of the line to fit the experimental data STRUD 0l MYCOBACTRIM TUBERCULOS H37Rv Slope constant H37Ra Slope constant Esti- Calcu- Esti- Calcumated lated mated Iated Number of experiments Mean generation time in hours Standard deviation.... A =1.41 t1.81 Standard error = per cent confidence limits concetration were made aseptically into sterile basal medium. Five ml of each dilution were delivered aseptically into twenty 18 by 150 mm pyrex tubes. These tubes had been rinsed previously with water redistilled from glass, capped with aluminum caps, and sterilized by autoclaving for 30 minutes at 20 lb. Two tubes of each medium then were inoculated with each small inocuum (10-2, 10 $, 10-, 10-, and 10-6 mg) of the virulent H37Rv strain, and two tubes, with each small inoculum of the avirulent H37Ra strain of M. tuberculosu var. hominis. After inoculation, the tubes were incubated at 37 C and examined daily for evidence of growth as described earlier (Holmgren and Youmanm, 1952; Youmans and Youmans, 1949). By recording the first day when growth appeared with each inoculum, the generation time for the tubercle bacilli with each substrate could be calculated. In a former paper (Holmgren and Youmans, 1952), the best straight line to fit the experimental data was evaluated by the regression equation Y - a + bx. The slope, b, derived in this manner was used to calculate the generation time. In the present paper, the generation times were calculated from the data by this method and also by estimating the best straight line to fit the data visually and calculating the generation time from the slope (Youmans and Youman, 1949). A minimum of two determinations was performed using each substrate as a carbon source in an otherwise complete medium. Manometric 8tudies. H37Ra cells were grown on the surface of the modified P & B medium in one liter Erlenmeyer flasks containing 400 ml of the medium. Pellicles from 30 day old cultures were removed by filtration through coarse sintered glms and washed with cold distilled water. The cell mass was ground in a sterile mortar adding 0.01 m phosphate buffer, ph 7.0, slowly until a relatively stable suspension was obtained. This suspension was standardized in a Hopkins vaccine centrifuge tube and diluted to contain 40 mg of cells per ml. One ml of this suspension was pipetted into each of 3 Warburg vessels. The substrates were dissolved in 1.0 ml of 0.01 M phosphate buffer solution, ph 7.0, and neutralized with NaOH when necesary. The final concentrations pipetted into the side arms were such as to result in concentrations of 0.5 m, 0.05 m, and M when mixed with the bacterial suspension in the Warburg vessel. For endogenous controls, 1.0 ml of buffer solution was added to the side arm of a fourth flask containing the same amount of cell susion. After 30 minutes of equilibration the contents of each side arm was tipped into the main compartment, and readings were recorded. Three separate determinations were performed with each substrate. RESULTS In table 1 the results obtained by the two methods of calculating the mean generation time of 20 determinations of the growth rate of H37Rv and H37Ra in the basal medium with glycerol as a carbon source are compared statistically. The results show that very little difference exists between the generation times obtained by the two methods and reflects the linear relationship between the time of first appearance of growth and the numbers of organisms used as inocula. Since the generation time calculated by either method is essentially the same, only the results obtained by the method of visually estimating the slope were subsequently employed. In table 2 the generation times of the avirulent variant H37Ra are given for those concentrations of the utilizable substrates which supported growth of all 5 inocula used in the test. These substrates were glucose, glycerol, lactic acid, pyruvic acid, acetic acid, a-ketoglutaric acid, and L-malic acid. No growth was obtained

19541 METABOLISM OF MYCOBACTERIUM TUBERCULOSIS 407 TABLE 2 The effect of Krebs' tricarboxylic acid cycle intermediates and precursors on the growth of Mycobacterium tuberculosis var.

3 19541 METABOLISM OF MYCOBACTERIUM TUBERCULOSIS 407 TABLE 2 The effect of Krebs' tricarboxylic acid cycle intermediates and precursors on the growth of Mycobacterium tuberculosis var. hominis, strain HS7Ra SUB"MSS GROWTH ESUREXENNTS Concentration of substrate in per cent Glucose... * * * * Glycerol Lacticacid.0 * * Pyruvicacid Acetic acid ṫ 0 0 * Citric acid a-ketoglutaric acid * Succinic acid " 10-l ' 10 $' Fumaric acid ' 10-"' L-Malic acid * * * * DL-Alanine " 10-" 10-2' L-Glutamic acid Aspartic acid...o None Calculated as the sodium salt. t All values are generation times in hours, except: 0 no growth; * growth of inocula not constant (see text); ' smallest inoculum showing growth. in the controls which contained asparagine and citrate but no glycerol or other carbon source. The results obtained with the virulent H37Rv strain have not been given since they differed from those reported by Youmans and Youmans (1953a,b) only in that growth was obtained with all 5 inocula in the same concentrations of L-malic acid that supported growth of H37Ra. In the presence of glycerol, pyruvic acid, acetic acid, and ca-ketoglutaric acid, the generation time was independent of the concentration of the substrate. However, the highest concentration of these substrates permitting growth of all 5 inocula differed. Growth was supported by higher concentrations of glycerol than of pyruvic or a-ketoglutaric acid and by higher concentrations of the latter two than of acetic acid. With glucose, lactic acid, and a-malic acid as substrates, the range of concentrations giving a linear relationship between the size of inoculum and the first appearance of growth was narrow. In concentrations above and below this range the growth of the inocula was not constant. For example, with glucose TABLE 3 Respiration of Mycobacterium tuberculosis var. hominis, strain HS7Ra, in the presence of various substrates SUBSTRATES (QO,) Molar concentration of substrate Glucose Glycerol.0.33 Lactic acid Pyruvic acid Acetic acid Citric acid.; cis-aconitic acid... 0 Oxalsuccinic acid a-ketoglutaric acid Succinic acid O Fumaric acid L-Malic acid Oxalacetic acid * Per mg wet wt/hr; corrected for endogenous respiration. 0 - No stimulation.

4 408 N. B. HOLMGREN, I. MILLMAN, AND G. P. YOUMANS [VOL. 68 < 180- LL d 160/ ~~~A 140 / 120 l i- oo O~~~~~O floe TIME IN HOURS DL-alanine were unique in that growth appeared only in the higher concentrations. Finally, citric, aspartic, and glutamic acids were unable to serve as energy or carbon sources under the conditions of these experiments. Manometric studies. The quotients (Qo.) obtained from manometric studies of 13 substrates are shown in table 3. Lactic, pyruvic, and acetic acids have a far greater stimulatory effect on respiration than do glucose, glycerol, and the Krebs' cycle intermediates. Figure 1 shows graphically these differences at optimal concentrations. In optimal concentrations, the respiration of H37Ra was stimulated approximately twice as much by lactic acid as by pyruvic acid and again twice as much by pyruvic acid as by acetic acid. It is interesting that citric and -x-h succinic acids in the highest concentrations 4 used (0.5M) were inhibitoryandyetshowed no stimulation in lower concentrations. Oxalacetic Lctic acid, acid produced as much stimulation in 0.05 M Figure 1. The effect of (A) 0.5 M la (B) 0.5 M pyruvic acid, (C) M a( cetic acid, concentrations as did 10 times this concentration (D) 0.1 m fumaric acid, (E) 0.05 M a-ke toglutaric of glucose and glycerol. Of the Krebs' cycle acid, (F) 0.05 M oxalacetic acid, 0.5 M gl[ucose and intermediates tested, only oxalacetic, fumaric, glycerol, (G) M L-malic acid, (1El) 0.05 M L-malic, and a-ketoglutaric acids produced citric, oxalsuccinic, succinic acids, anid M slight stimulatory action on the respiration cis-aconitic acid, on the respiration of Af. tubercu- of the H37Ra strain. lowis var. hominis, strain H37Ra. E7ach flask contained 40 mg washed cells suspended in one ml DlSCUSSION of 0.01 M phosphate buffer, ph 7.0, arad one ml of substrate dissolved in 0.01 M phosphlate buffer It has been reported that the avirulent mutant neutralized to ph 7.0 with 10 per cent NiaOH. H37Ra differs from the parent H37Rv strain Qo2 values corrected for endogenous reespiration. in its inability to multiply in the host tissues (Suter, 1952; Pierce et al., 1953). This difference the best linear relationship was obitained in between the two strains may reflect a loss of a the 0.5 to 2.0 per cent concentraltions and metabolic ability in H37Ra, rendering it unable with L-malic acid in the 0.1 and 0.25; per cent to use a substrate found in vivo which the virulent concentrations. In the presence of lswctic acid, H37Rv can metabolize, thus enabling the the 0.05 and 0.1 per cent concentrat ions gave virulent strain to grow and compete with the the best results whereas the relationship with cell for the substrate. A more complete knowledge 0.01, 0.025, and 0.25 per cent coneentrations of the enzymatic equipment for both growth departed slightly from linearity anid in the and respiration may aid in disclosing a metabolic 0.5 and 1.0 per cent concentrations became difference correlated with virulence. nonlinear. Within the limitations of the methods used in Growth was either absent or slig] ht in the these studies, no evidence that the two strains presence of the various concentrations ( of succinic differ essentially in the utilization for energy and or fumaric acid and the 3 amino acids, D)i-alanine, growth of intermediates and precursors of the L-glutamic, and aspartic acids. Growtth of only Krebs' tricarboxylic acid cycle has been found. the large inocula was obtained witth certain With the substrates which supported growth of concentrations of succinic and fumaric3 acid and both H37Rv and H37Ra, not only were similar with DL-alanine. Higher concentrations of succinic generation times obtained but their effective and fumaric acid were inhibitory. GlLucose and concentrations were in a similar range.

19541 METABOLISM OF MYCOBACTERIUM TUBERCULOSIS Ang Several types of response in the growth of H37Ra to the presence of the substrates tested are noted.

5 19541 METABOLISM OF MYCOBACTERIUM TUBERCULOSIS Ang Several types of response in the growth of H37Ra to the presence of the substrates tested are noted. The cell wall of H37Ra is apparently permeable to glucose, glycerol, lactic acid, pyruvic acid, acetic acid, a-ketoglutaric acid, and L-malic acid and to a limited extent, also, to succinic acid, fumaric acid, and DL-alanine. The generation time in the presence of glycerol, pyruvic acid, acetic acid, and ax-ketoglutaric acid is independent of concentration of substrate over a definite, relatively wide but characteristic range of concentrations for each substrate. For example, with pyruvic acid as substrate, a concentration of 0.01 per cent gives essentially the same generation time as 0.5 per cent. If the 0.01 per cent concentration is sufficient to saturate the enzyme surface and to permit a maximal growth rate under the conditions of these experiments, then some mechanism perhaps is operative to remove the excess substrate over this amount up to a certain limit since the 1.0 per cent concentration inhibited growth. It is possible that any excess of these substrates over that utilized in respiration is removed by participating in synthesis of compounds essential to the organism. Glucose, lactic acid, and L-malic acid supported growth in a narrow range of concentrations. Above and below this range, the growth of the different inocula was not constant, resulting in a nonlinear relationship between the size of inoculum and the first appearance of growth. Therefore, a generation time could not be calculated from the results with these concentrations. It is of interest that growth occurred only in the higher concentrations of glucose, a finding which suggests slow penetration. Penetration of the cell by glucose may be by diffusion, the internal concentration depending on the extracellular concentration, or the affinity of the enzyme for glucose, possibly hexokinase, may be of such a low order as to require greater concentrations of substrate molecules in order to increase the probability of contact and subsequent reaction. From similar results Schaefer (1948) suggested the same possibilities. Finally, the lack of growth in citric, aspartic, and glutamic acids may indicate that the cell wall of H37Ra is impermeable to these substrates or that the implicated enzymes are already saturated with intermediates furnished by the high endogenous respiration. The Warburg studies show that the substrates, lactate, pyruvate, acetate, oxalacetate, fumarate, malate, a-ketoglutarate, glucose, and glycerol, were utilized to varying degrees. An explanation for the lack of stimulation with citric, cis-aconitic, oxalsuccinic, and succinic acids might be that these substrates either do not penetrate the cell membrane or that the particular enzymes concerned are saturated with oxidizable products furnished by the endogenous respiration. Moreover, certain discrepancies are seen which might indicate that cell permeability is not the sole factor involved. Limited availability of enzyme sites due to saturation by endogenous products may explain both the inhibition obtained with the higher concentrations of citrate and succinate and the lack of stimulation or inhibition of respiration by lower concentrations. If we accept this lack of stimulation in the lower concentrations as being due to impermeability of the cell membrane to these relatively large molecules, then the inhibitory effect of higher concentrations remains to be explained. With the exception of succinate, a correlation is apparent between growth and respiration since substrates permitting growth also stimulate oxygen uptake. Succinic acid, on the other hand, inhibited respiration in high concentration and showed no stimulation over the endogenous respiration in lower concentrations. The results cannot be compared quantitatively, however, because of the difference in methodology: minimal inoculum of growing cells in one case and massive amounts of resting suspensions in the other. The presence of a tricarboxylic acid cycle in H37Ra is not established by the results obtained from these growth and respiratory studies; however, additional evidence for the operation of such a cycle will be presented in a subsequent publication (Millman and Youmans, 1954). SUMMARY The effect on the growth of Mycobacterium tuberculosis var. homini8, strain H37Ra, of 13 substrates as carbon sources was determined by the small inocula method. Glucose, glycerol, lactate, pyruvate, acetate, a-ketoglutarate, and L-malate supported growth. Only limited growth was obtained with succinate, fumarate, and DL-alanine. Citric acid, L-glutamate, and aspartate did not support growth. The utilization

410 N. B. HOLMGREN, I. MILLMAN, AND G. P. YOUMANS [vol. 68 of these substrates for growth by the virulent and avirulent strains, H37Rv and H37Ra, appeared to be similar.

6 410 N. B. HOLMGREN, I. MILLMAN, AND G. P. YOUMANS [vol. 68 of these substrates for growth by the virulent and avirulent strains, H37Rv and H37Ra, appeared to be similar. Respiratory studies using whole cells of H37Ra indicated that the substrates lactate, pyruvate, acetate, oxalacetate, funmarate, L- malate, a-ketoglutarate, glucose, and glycerol were utilized. Lactate, pyruvate, and acetate stimulated oxygen uptake to a greater extent than did glucose, glycerol, or the Krebs cycle intermediates. Of the Krebs' cycle intermediates only oxalacetate fumarte, L-malate, and a-ketoglutarate showed slight stimulations of respiratory activity. Citrate and succinate inhibited respiration in high concentrations but showed no stimulation in lower concentrations. With the exception of succinate, substrates which permitted growth also stimulated oxygen uptake. REFERENCES BEENHEM, F The oxidation of benzoic acid and related substances by certain mycobacteria. J. Biol. Chem., 1U, CUINELL, C. 1940a La ossidazione degli acidi grassi da parte del bacillo di Koch. Boll. ist. sieroterap. milan., 19, CUMNrEu, C. 1940b Sul meccanismo di ossidazione degli grassi da parte del bacillo di Koch. Boll. ist. sieroterap. milan., 19, CUmNJsIJ, C. 1940c Ricerche sul metabolismo glucidico del bacillo di Koch. Boll. ist. sieroterap. milan., 19, DuBos, R. J Toxic effects of dl-serine on virulent human tubercle bacilli. Am. Rev. Tuberc., 60, 385. DuBos, R. J The effect of organic acids on mammalian tubercle bacilli. J. Exptl. Med., 92, EDSON, N. L The intermediary metabolism of the mycobacteria. Bacteriol. Revs., 15, EDSON, N. L., AND HUNTER, G. J. E Respiration and nutritional requirements of certain members of the genus Mycobacterium. Biochem. J. (London), 37, GERONIMsUS, L. H., AND BIRKELAND, J. M The relationship between the virulence of tubercle bacilli and the origin of their oxidative attack upon certain substrates. Am. Rev. Tuberc., 64, HOLMGREN, N. B., AND YOUMANs, G. P Studies on the metabolism of virulent and avirulent mycobacteria. Am. Rev. Tuberc., 86, MARSHAK, A Differences in response of a virulent strain of the tubercle bacillus and its avirulent variant to metabolites and their genetic significance. J. Bacteriol., 61, MITLTAN, I., AND YomANs, G. P Studies on the metabolism of Mycobacterium tuberculosis. VII. Terminal respiratory activity of an avirulent strain of Mycobacterium tuberculosis. J. Bacteriol., 68, NAKAMURA, T ttber die Atmung die Tuberkelbazillen. T6hoku J. Exptl. Med., 34, OGINSKY, E. L., SMiTH, P. H., AND SOLOTOROV5KY, M The action of streptomycin. IV. Fatty acid oxidation by Mycobacterium tuberculosis, avian type. J. Bacteriol., 59, PIERCE, C. H., Du3os, R. J., AND SCAEFER, W. B Multiplication and survival of tubercle bacilli in the organs of mice. J. Exptl. Med., 97, SCHAEFER, W Recherches sur la croissance du Mycobacterium tuberculosis en culture homogene. Ann. inst. Pasteur, 74, SUTER, E The multiplication of tubercle bacilli within normal phagocytes in tissue culture. J. Exptl. Med., 96, YOUMANS, A. S., AND YoumANs, G. P. 1953a Studies on the metabolism of Mycobacterium tuberculosis. II. The effect of compounds related to the Krebs tricarboxylic acid cycle on the growth of Mycobacterium tuberculosis var. hominis. J. Bacteriol., 65, YOUMANS, A. S., AND YOUMANS, G. P. 1953b Studies on the metabolism of Mycobacterium tuberculosis. III. The growth of Mycobacterium tuberculosis var. hominis in the presence of various intermediates of the dissimilation of glucose to pyruvic acid. J. Bacteriol., 65, YOUMANS, G. P., AND KARL5ON, A. G Streptomycin sensitivity of tubercle bacilli; studies on recently isolated tubercle bacilli and the development of resistance to streptomycin in vivo. Am. Rev. Tuberc., 55, YOUMANS, G. P., AND YOUMANS, A. S A method for the determination of the rate of growth of tubercle bacilli by the use of small inocula. J. Bacteriol., 58,

Received for publication February 8, 1960

Received for publication February 8, 1960 EFFECT OF HOMOGENATES OF ORGANS FROM IMMUNIZED GUINEA PIGS ON THE RESPIRATION OF MYCOBACTERIUM TUBERCULOSIS' ANNE S. YOUMANS, GUY P. YOUMANS, AND ANDREW HEGRE, JR. Department of Microbiology, Northwestern