The Catalase Activity of Mycobacteria.
[Received by the Editor, July 14, 1947; issued separately, February, 1949.]
Most bacteria—aerobes and facultative anaerobes—contain catalase but prove sensitive to addition of hydrogen peroxide, a small concentration of which can inhibit growth completely. Since hydrogen peroxide does not accumulate in media which have been used for the cultivation of catalase producers, there has arisen the hypothesis that catalase protects micro-organisms against accumulation of hydrogen peroxide formed during aerobic metabolism. The theory admits that organisms may exhibit different degrees of sensitiveness to hydrogen peroxide.
Anaerobes possess no catalase (Callow, 1923) and are sensitive to such a degree that those species which can produce hydrogen peroxide in the presence of oxygen are incapable of aerobic growth (McLeod and Gordon, 1923, 1925). Other bacteria known to be devoid of catalase (pneumococci, many streptococci and lactobacilli) multiply aerobically, forming hydrogen peroxide which accumulates in the medium. Since these organisms are not more than slightly or moderately sensitive, growth is not inhibited (McLeod and Gordon, loc. cit.). The vagaries of catalase distribution are not simplified by the existence of strains of Shigella dysenteriae and Streptococcus, faecalis, which produce neither catalase nor hydrogen peroxide. These organisms are moderately sensitive to addition of the latter substance.
The mycobacteria are characterized by aerobic metabolism which affords the possibility of hydrogen peroxide formation. Edson (1947). has presented evidence suggesting that hydrogen peroxide may take part in the oxidation of lactic acid by Myco. phlei. In terms of the catalase pretection theory it might be expected that the mycobacteria would develop a high concentration of the enzyme. Only in the case of the tubercle bacillus has catalase activity been measured (Fujita and Kodama, 1931); and the value (QKat = 16) is surprisingly low for a strict aerobe.
Although there have been many studies of the catalase content of bacteria (see Euler, 1934), there have been few systematic comparisons of the activity of species within a single genus. The object of this investigation was to survey the catalase activity of the genus Mycobacterium, and to discover any possible relationships to metabolic behaviour.
The following species of mycobacteria were utilized:—Myco. butyricum (No. 337), Myco. phlei (No. 525), Myco. smegmatis (No. 523), Myco. sp. leprous Kedrowsky (No. 509), Myco. sp. Karlinski (No. 2071), Myco. ranae (No. 2891), Myco stercoris (No. 3820), Myco. tuberculosis hominis (T1 avirulent), Myco. tuberculosis bovis
(B.C.G.), and Myco. avium (T2). Species designated by catalogue numbers only were obtained from the National Collection of Type Cultures, Lister Institute, London, whilst those designated by alphabetical letters were gifts from the Commonwealth Serum Laboratories, Melbourne.
All organisms except Myco. tuberculosis bovis were cultivated on glycerol (5%) — peptone meat-infusion broth at 38°C. Myco. tuberculosis bovis was grown on plain peptone broth at the same temperature. Cultures of the saprophytic mycobacteria and Myco. ranae were used for experiment at the age of 5 to 15 days; whilst cultures of Myco. tuberculosis hominis and bovis and Myco. avium were 3 to 6 weeks old.
Washed Bacterial Suspensions. Organisms were harvested, washed and suspended in distilled water by the procedure of Edson and Hunter (1943). Suitable volumes of suspension were dried to constant weight at 110°C., in order to find the dry weight of bacteria per ml. of suspension.
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Measurement of Catalase Activity. Two methods were employed:—(a) “Katalase fahigkeit” (Kat f) was determined at 0°C. in phosphate buffer, pH 6.98, by the iodometric method of Stern 1932). Kat f = K/g. dry wgt. bacteria, where K is the value of the unimolecular velocity constant extrapolated to zero time. (b) Catalase activity (QKat) was determined manometrically by the method of Fujita and Kodama (1931). Standard Warburg manometers with conical cups and side-bulbs were used. The main compartment contained 2ml. of bacterial suspension and 1ml. of 0·015 M phosphate buffer, pH 7·4. The gaseous phase was air and the inseal contained the usual CO2 absorbent. 0·3 ml. of hydrogen peroxide solution (“Perhydrol,” Merck, made up in 0·005 M phosphate buffer, pH 7·4, to give 0·05 M solution) was added from the side-bulb after 20 minutes' equilibration in the thermostat at 38°C., and pressure changes were observed at 10-minute intervals for a period of 30 minutes.
The density of each bacterial suspension was adjusted so that not more than 50% of the hydrogen peroxide was decomposed in 30 minutes. According to activity, the bacteria used per cup varied from 66μg. to 1·8 mg. dry weight.
The validity of the method was checked by various control experiments. Hydrogen peroxide was found to be stable under the conditions used; and a dilute solution of catalase [beef-liver catalase crystallized by the method of Sumner and Dounce (1937)] gave QKat values of the expected order of magnitude. A control vessel without hydrogen peroxide was set up in each determination of catalase activity for the purpose of measuring respiratory uptake of oxygen. Only in the case of tubercle bacilli (human and bovine) was there a measurable consumption of oxygen amounting to about 3 μl in 30 minutes. The QKat figures were corrected accordingly.
QKat = μl O2 (0°C., 760 mm. Hg) evolved in 30 minutes at 38°C. per mg. dry weight of bacteria.
Typical values of Kat f and QKat are given in Table 1. Although Kat f and QKat determinations were not made on the same cultures, there is fair agreement between the two methods of measuring activity, indicating a consistent formation of catalase by specific organisms, grown on the same medium.
In the genus Mycobacterium catalase production varies over such a wide range that the species may be classified arbitrarily into four groups (Table 2), it being understood that the “high” activity of Myco. phlei relates to members of the genus and not to microorganisms in general.
Acetone treatment (see Edson and Hunter, 1947) of Myco. phlei was followed by great reduction of activity (Table 1). Bacterial suspensions which had been heated in a water-bath at 100°C. for 5 minutes showed no catalase activity.
The accuracy of the methods used is about ± 5%. Duplicate determinations on a single culture agreed within these limits; but the variance of activity in different cultures of the same species was occasionally as great as 50%.
|Myco. sp. leprous Kedrowsky||0.65||305|
|Myco. sp. Karlinski||0.30||47|
|Myco. tuberculosis hominis||0.08||16|
|Myco. tuberculosis bovis||0.15||6|
|Myco. phlei, acetone-dried powder||0.35||—|
|(1) Very low||Myco. tuberculosis bovis|
|Myco. tuberculosis hominis|
|(2) Low||Myco. sp. Karlinski|
|(3) Moderate||Myco. sp. leprous Kedrowsky|
|(4) High||Myco. phlei|
Without exception representative species of the genus Mycobacterium have been found to contain catalase, but the QKat values vary from 6 (Myco. tuberculosis bovis) to 2,080 (Myco. phlei). Nevertheless, the catalase activity of Myco. phlei is considerably lower than that recorded for species of other aerobic genera examined by Fujita and Kodama (1931), who found the QKat values of Pseudomonas
aeruginosa, Neisseria gonorrhoeae, and Corynebacterium, diphtheriae to be 15,000, 9,200, and 9,000 respectively.
Since the metabolism of the mycobacteria is predominantly or exclusively aerobic, it would follow as a prediction from the “H2O2 protection” theory that the catalase activity of a species should be related to over-all metabolism and to the rate of growth on the surface of nutrient broth. The prediction is valid only if the mycobacteria possess no alternative means of destroying hydrogen peroxide. Since no alternative reaction is established, the assumption may be retained.
Some degree of parallelism between endogenous respiration of washed suspensions and rate of growth on broth was observed by Edson and Hunter (1943), who examined most of the species investigated in the present work. Saprophytic species have a distinctly higher respiration than the tubercle bacillus; in general, the growth rate and the catalase activity of these species are also of a higher order. These facts are consistent with the hypothesis that there is a relationship between catalase activity and the over-all metabolic rate.
Certain facts, however, are not consistent with the hypothesis:— (1) The endogenous respiration is of a similar order in all saprophytic species (Edson and Hunter, 1943), whereas the catalase activity varies from “low” to “high” (Table 2); and (2) Myco. avium, which respires and grows at approximately the same rate as Myco. tuberculosis haminis and bovis, possesses catalase activity of a totally different order. On this account it is necessary to question —and probably reject—the hypothesis that catalase activity is directly related to over-all metabolism.
A more satisfactory explanation is likely to be found in a relationship between catalase activity and the velocity of oxidations promoted by particular enzyme systems. It is well known that hydrogen peroxide is formed in catalytic cycles which involve autoxidizable flavoproteins. Edson (1947) has presented evidence for the existence in Myco. phlei of a powerful flavoprotein system (lactic acid oxidase) containing flavine-adenine-dinucleotide. In this connection the relatively high catalase activity of Myco. phlei becomes significant.
It is possible that catalase may protect Myco. phlei from the toxic effect of hydrogen peroxide arising in the oxidation of lactic acid. Alternatively, another automatic safeguard may exist. Pyruvic acid, which is an intermediate in the breakdown of lactic acid, interacts with hydrogen peroxide rapidly and spontaneously according to the equation (Holleman, 1904):—
CH3COCOOH + H2O2 = CH3COOH + CO2 + H2O.
Sevag (1933), indeed, has suggested that Holleman's reaction may function protectively in organisms like pneumococci which possess no catalase.
No doubt there are several specific reactions of acid-fast metabolism—other than lactate oxidation—in which hydrogen peroxide may be formed and in which there is no alternative safeguard. If
so, catalase may serve an important protective function in the mycobacteria, and the catalase activity of a given species may develop in relation to its particular enzymic equipment. This question could be studied by measuring the catalase and other enzymatic activities of a species grown on synthetic media containing single sources of carbon.
On the other hand, the role of catalase may be somewhat different. Keilin and Hartree (1936, a, b) have brought forward evidence to show that catalase may be concerned not merely in destroying hydrogen peroxide, but in promoting coupled oxidations. Attempts to investigate this aspect of catalase function in mycobacteria would be premature until there is a greater accumulation of knowledge regarding the metabolism and enzymic constitution of the genus.
1. The catalase activity of representative species of the genus Mycobacterium has been measured by two different methods and found to vary widely from species to species.
2. The significance of catalase in bacterial metabolism is discussed.
Callow, A. B., 1923. J. Path. Bact., 26, 320.
Edson, N. L., 1947. Biochem. J., 41, 145.
Edson, N. L., and Hunter, G. J. E., 1943. Ibid., 37, 563.
—— 1947. Ibid., 41, 139.
von Euler, H., 1934. Chemie der Enzyme, Zweiter Teil, Abschnitt 3, Bergmann, Munich, p. 17.
Fujita, A., and Kodama, T., 1931. Biochem. Z., 232, 20.
Holleman, A. F., 1904. Rec. trav. chim., 23, 169.
Kellin, D., and Hartree, E. F., 1936a. Proc. Roy. Soc., B., 119, 114.
—— 1936b. Ibid., 119, 141.
MoLeod, J. W., and Gordon, J., 1923. J. Path. Bact., 26, 332.
—— 1925. Ibid., 28, 147.
Sevag, M. G., 1933. Biochem. Z., 267, 211.
Stern, K. G., 1932. Hoppe-Seyl. Z., 204, 259.
Sumner, J. B., and Dounce, A. L., 1937. J. Biol. Chem., 121, 417.