B12 on acetate oxidation were first observed in the Merck Institute Laboratories,

Transcription

1 THE INFLUENCE OF VITAMIN B12 ON OXIDATION BY A MUTANT STRAIN OF ESCHERICHIA COLI' EVELYN L. OGINSKY, PATRICIA H. SMITH, NICHOLAS E. TONHAZY, AND WAYNE W. UMBREIT Merck Institute for Therapeutic Research, Rahway, New Jersey AND HERMAN C. LICHSTEIN2 AND STANLEY F. CARSON Oak Ridge National Laboratories, Oak Ridge, Tennessee Received for publication February 5, 1951 Recent studies on the functions of vitamin B,2 in bacterial metabolism have shown that it can replace various pyrimidine desoxyribosides for growth of several strains of LactobaciUus lactis, Lactobacillus leichmannii, Lactobacillus acidophilu8, and Lactobacillus delbrueckii (Hoff-J0rgensen, 1949; Kitay, McNutt, and Snell, 1949; Shive, Ravel, and Eakin, 1948; Kitay, McNutt, and Snell, 1950). This vitamin can also replace methionine as a growth requirement for mutant strains of Escherichia coli (Davis and Mingioli, 1950). The data, although suggesting that vitamin Bi2 is involved in the synthesis of these compounds, are not yet sufficiently complete to show the locus of its biochemical function, since in bacteria the sequence of events in these syntheses is not known. It was, therefore, of interest to investigate the possibility that vitamin B12 had still other functions, and to determine whether it would influence other reactions in bacterial cells deficient in the vitamin. For this study we selected a mutant strain of E. coli, isolated by Davis with the ultraviolet-irradiation penicillin method (Davis and Mingioli, 1950). This strain, designated as no , grows rapidly in a synthetic medium containing either DL-methionine at 20 fug per ml or vitamin B,2 at 1 mug per ml, but it will not grow on any of the precursors of methionine or on any other of a variety of substances tested, including desoxyribosides. We considered that, if the organisms were grown in the presence of methionine only, they would presumably be deficient in vitamin B12. Cell suspensions of such organisms were prepared and allowed to oxidize various substrates, in the presence or absence of the vitamin. The results of these studies are described below. This work was done jointly in the two laboratories involved. Although the methods employed were slightly different, the data are sufficiently comparable and confirmatory to be combined into a single publication. The effects of vitamin B12 on acetate oxidation were first observed in the Merck Institute Laboratories, and the possible relations to coenzymes and porphyrins were developed at the Oak Ridge Laboratories. 1 Work at the Oak Ridge National Laboratory was performed under Contract No Eng-26 for the Atomic Energy Commission. 2 Present address, Department of Bacteriology and Immunology, Medical School, University of Minnesota, Minneapolis 14, Minnesota. 581

2 582 OG5INSKY ET AL. [VOL. 61 METHODS The medium used was that described by Davis and Mingioli; it contained 20,ug DL-methionine per ml. The organisms were grown either in large flasks (2 to 4 liters) containing medium at approximately four-fifths of the flask volume ("deep grown") or in 500-ml flasks containing 100 ml of medium aerated continuously by means of a reciprocal shaker ("air grown"). The cells were generally harvested by centrifugation after 24 hours' incubation at 32 C, although occasionally the incubation period was extended to 48 hours if the earlier growth appeared scanty. The packed cells were suspended in distilled water, recentrifuged, and resuspended in distilled water to give a final concentration of 0.5 or 2.0 mg nitrogen per ml. The volume of the cell suspensions added to each Warburg flask was adjusted to give a final concentration of 0.2 or 0.25 mg nitrogen per ml. Conventional manometric methods were applied throughout. Phosphate buffer, M/300 at ph 7.0, was used, except in the instances noted. Substrates were adjusted to the appropriate ph with KOH. Cobione (vitamin B12 Merck) was employed at a final concentration of 70 m,g per ml and was usually added 30 to 60 minutes before the substrates were tipped in. RESULTS Cell suspensions prepared from cultures "deep grown" in the presence of methionine were capable of oxidizing a variety of substrates, such as pyruvate, succinate, and some other members of the tricarboxylic acid cycle, in the absence of vitamin B12. The addition of the vitamin to flasks containing freshly harvested cells did not alter the oxidation rate to any great degree. However, when the suspensions were "aged" by being held in the refrigerator, their oxidation pattern became susceptible to the action of the vitamin. The endogenous respiration was in no case stimulated by vitamin Bn; in contrast, in most experiments, vitamin B12 lowered the oxidation rate slightly, but only after 2 to 3 hours had elapsed. Acetate was the first substrate whose oxidation by "deep grown" cells was stimulated by vitamin B12. Figure 1 shows the typical effect on suspensions aged for 10 days or more. The rate of oxidation of acetate was markedly increased, although the total oxygen uptake remained essentially the same. This effect of vitamin B12 was apparent with some suspensions within 2 days after harvest, with others only after 5 to 6 days. Within this period, there was usually little or no effect on the oxidation of succinate or other substrates. It should be noted, however, that the oxidation of acetate by these suspensions did not begin immediately on addition of substrate, but only after a lag period. In some experiments the lag period was considerably shorter in the presence of vitamin B12 (see figure 1). In others, there was no effect on the lag period, although there was still a marked effect on the oxidation rate. This phenomenon of "adaptation" to oxidation of acetate will be discussed later. If the suspensions were stored for longer than 5 days, the oxidation of other substrates was also markedly affected. Data obtained with several substrates in the presence or absence of vitamin B12 are presented in table 1. The rate of

3 1951] INFLUENCE OF VITAMIN B12 ON OXIDATION 583 JI 0 MINUTES Figure 1. The effect of vitamin B1, on the oxidation of acetate by E. coli strain Ten AM acetate, cell suspension equivalent to 0.75 mg bacterial nitrogen, 0.1 ml 0.1 M phosphate buffer, ph 7.0, 210 mjug vitamin B12, distilled water to volume of 3.0 ml. Cells aged 17 days. Acetate added 60 minutes after vitamin B12, and manometer readings begun (zero time on graph). All oxygen uptake data in this and subsequent figures and tables were corrected for endogenous respiration. TABLE 1 Effect of vitamin B12 on the oxidation of various substrates by E. coli, 8train OXYGEN UTAX TOTAL Os SUBSTRATE Without Bs With Bu px Os per ox substrate,m P i % theor Qo0(N) Qos(N) Found Theory Acetate Pyruvate Oxalacetate Pyruvate oxalacetate 20 Succinate Glutamate Fumarate Malate Acetoacetate Stearate

4 584 OGINSKY ET AL. [VOL. 61 oxidation was much more rapid with vitamin B12. At the time of the rate break with vitamin B12 (comparable to 180 minutes in figure 1), the oxygen uptake of the cells without vitamin B12 was about one-half to two-thirds that of the former. The rate in the absence of vitamin B12 remained constant until the total oxygen uptake reached that of the cells with vitamin B12, when the rate decreased sharply. Thus the presence of the vitamin influenced the rate of oxidation but not the total oxygen uptake. Except for acetate and stearate, all the substrates named in table 1 were oxidized rapidly, with no lag period. E. coli oxidizes stearate only after a considerable lag, as reported previously (Oginsky, Smith, and Solotorovsky, 1950). This lag period was shortened by adding vitamin Bn1 to the cell suspensions used in this study. The total oxygen uptake with any of the substrates was below that required for theoretical oxidation to completion. We have considered the possibility of the accumulation of formate, which might account for the differences between the actual and the theoretical oxygen uptake. Although these cell suspensions neither oxidize formate nor possess the hydrogenlyase system, we have not been able to demonstrate any accumulation of the compound, by the method of Grant (1947). It is impossible to say at this time whether the incomplete oxidation is due to the specific oxidation sequence in these cells, to nonspecific factors involved in hydrogen or oxygen transport, or to oxidative assimilation. At any rate, whatever its cause, it is not discernibly influenced by vitamin B12. The mechanism of the aging process is not known. A study of the effect of aging on the acetate oxidation rate of one "deep grown" cell suspension showed that, in the absence of vitamin B12, the rate dropped rather sharply during the first 8 days after harvest, and thereafter dropped more slowly. However, if the acetate was oxidized in the presence of vitamin B12, the rate decreased much more gradually. The net effect of the addition of the vitamin was to restore, at least partially, the acetate oxidation rate to that of freshly harvested cells. Since the curves for acetate oxidation appeared to indicate adaptation to the substrate, several attempts were made to determine whether vitamin B12 also influenced the rate of acetate oxidation of acetate-adapted cells. If this organism is grown with aeration ("air grown" cells), the acetate oxidation proceeds immediately on the addition of substrate. The addition of the vitamin to a 1-dayold suspension of "air grown" cells did not affect the oxidation rate, but 5 days later the acetate oxidation rate of the same suspension was increased by the vitamin, in the absence of a lag period. Again, as with the other substrates, a vitamin B12 effect was noted in the absence of adaptation. "Air grown" cell suspensions differ from those prepared with "deep grown" cells in that the vitamin B12 effect does not appear first with acetate oxidation but concomitantly with all substrates tested. Several other methods for determining whether the vitamin affects the adaptation to acetate were also used. First, the usual "deep grown" cells were allowed to oxidize 10 Mm of acetate in the presence or absence of vitamin B12. After the oxygen uptake due to added substrate was complete in all flasks, the manometers were opened and the flasks allowed to remain in the 37 C water bath over-

5 1951] INFLUENCE OF VITAMIN B12 ON OXIDATION 585 night. The next morning, fresh acetate was tipped in, and the oxygen uptake was measured. The results of this experiment are shown in table 2. In the first oxidation of acetate, vitamin B12 increased the rate, not only in the early stage, but also in the stage of rapid oxidation. However, the data obtained on the second day indicate a stimulatory effect during the first hour only. These results are somewhat at variance with those obtained with "air grown" cells. The possibility exists that vitamin B12 is involved in increasing the rate of synthesis of a compound required in the oxidative process. Under the conditions of the experiment, enough of this compound might have been produced in the absence of vitamin B12 to raise the oxidation rate. Second, in the course of studies on the mode of action of streptomycin, we had observed that this antibiotic, in common with several others such as chloramphenicol and aureomycin, inhibited adaptive enzyme formation, as had been shown previously for streptomycin and benzoic acid oxidation by mycobacteria (Fitz- TABLE 2 Acetate oxidation by E. coli strain, Qos(N) Hour H our 1st 2nd 3rd 4th 5th 6th Total _Uptake First day No B1, With B Second day No B * _ 291 With B, * Not determined. gerald and Bernheim, 1948; Fitzgerald, Bernheim, and Fitzgerald, 1948). Since the acetate oxidation by the "deep grown" cells appeared to be adaptive, it was of interest to determine whether streptomycin would inhibit the reaction. It was found that the addition of 36,ug of streptomycin per ml, added with the acetate, inhibited the oxidation completely in the presence or absence of vitamin B12, whereas no effect of streptomycin was evident if it was added 60 minutes after the oxidation had begun. Third, if the vitamin was involved only in increasing the rate of adaptation, its addition after oxidation had begun, rather than before, should not influence the rate of oxidation. To test this hypothesis, 70 m,ug per ml of vitamin B12 were tipped into the reaction vessels at various times before or after the addition of acetate. The results of three such experiments are given in table 3. The acetate oxidation in the absence of the vitamin began at approximately 60 minutes after tipping; if vitamin B12 had been added previous to this, the oxidation began,

6 58-06 OGINSKY ET AL. [VOL. 61 generally, about 10 minutes earlier. When the vitamin was added just as oxidation began, no increase in rate was evident until 60 minutes had elapsed. At that time the oxidation rate rose until it equaled that noted in experiments in which vitamin B12 had been present previous to the commencement of oxygen uptake. If, however, the addition of the vitamin was made when the oxidation was well advanced, no such rate increase occurred. We have therefore concluded that the action of vitamin B12 was primarily upon the rate of oxidation of acetate and that its effect on the adaptation to acetate oxidation was minor. It was of interest to determine how much vitamin B, was required to produce a maximum effect on acetate oxidation. A series of vitamin B12 concentrations from m,ug to 1,000 mug were used with a suspension equivalent to 0.25 mg bacterial nitrogen in each flask. No stimulation occurred until the concentration reached 0.07 mug; maxium activity was attained at a concentration of about 2.0 miug. Althougb the majority of the experiments had been run in the presence of 70 m,ug vitamin Bu per ml (or 210 mug), it appeared that this concentration TABLE 3 Effect of time of addition of vitamin B12 on acetate oxidation rate TIM B, ADDED Max. Qos (N) ON ACETATE 1st expt. 2nd expt. 3rd expt 60 min. before acetate min. before acetate With acetate min. after acetate min. after acetate min. after acetate Without B Without B was far in excess of that required for maximum stimulation. Calculations based on the dry weight of the cell suspension and on the assumption of uniform weight (0.3 X g per cell) and sensitivity of each bacterial cell indicate that at the minimum effective concentration the ratio is two molecules of vitamin B12 per cell. At the maximum effective concentration, the ratio is 82 molecules of vitamin per cell. A ratio of approximately 170 vitamin B12 molecules per red blood cell has been calculated by Ockrent (1950). The effect of ph on the acetate oxidation was investigated by allowing the reaction to proceed in the presence or absence of 70 m,ug of vitamin B,2 per ml in M/75 phosphate buffer at ph 5.3, 6.2, and 7.7. The results shown in table 4 indicate that at more alkaline ph levels, acetate oxidation in the absence of vitamin B12 is increased in both rate and extent to a greater degree than in the presence of the vitamin. The data presented thus far demonstrate the following phenomena. "Deep grown" cells differ from "air grown" cells in that the latter possess an acetateoxidizing system, whereas the former make it "adaptively" in the resting cell

7 1951] INFLUENCE OF VITAMIN B2 ON OXIDATION 587 state. Except for this difference the following points summarize the observations on both types of cells: (a) When freshly harvested, the cells were not stimulated by vitamin B12. (b) After storage for 2 to 5 days, acetate oxidation was stimulated by vitamin B12. The oxidation of other substrates by "air grown" cells was, in some cases, affected during this period. (c) After storage for longer than 5 days, the oxidation of several substrates by "deep grown" cells (cf. table 1) was stimulated by vitamin B12. (d) The stimulation produced by the vitamin was generally considerably greater with "deep grown" than with "air grown" cells. (e) The amount of vitamin B12 required was exceedingly small (approximately 2 molecules per cell), and a brief but discernible time was required before its effect could be observed. (f) With respect to the oxidation of all substrates tested, vitamin B12 infuenced the rate of oxidation, not the amount of oxygen uptake. TABLE 4 Effect of ph on acetate oxidation ph No B12 Max. Qo2 (N) Total uptake With B12 Max. Qo2 (N) Total uptake It is evident from these facts that the action of vitamin B12 in this system is complex. The pathway of acetate oxidation in this organism is not known. No positive evidence has been obtained that the oxidation proceeds either by way of condensation with oxalacetate (Stern and Ochoa, 1949) or by way of "self-condensation" to succinate (Foster et al., 1949; Ajl, 1950; Swim and Krampitz, 1950). However, from the fact that the amount of oxygen utilized is independent of vitamin B12, it would appear that whatever the metabolic pathway of the carbon moiety, the vitamin influences only the rate at which oxygen is taken up. This would suggest that the vitamin is concerned in the oxidative pathways, rather than with the carbon compound transformations. The amount of the vitamin required and the time relationship suggest that vitamin B12 may be concerned with the synthesis of a hydrogen- or electron-transporting system. One might presume that, with excess methionine, either the same or a supplementary pathway to oxygen is available and that this system is of relatively more importance to acetate oxidation by "deep grown" cells than to that of the other substrates. This system appears to be broken down during

8 588 OGINSKY ET AL. [VOL. 61 storage of the cells. We have therefore made some studies on hydrogen-transporting systems. The over-all respiratory system is sensitive to cyanide, and thus presumably involves heavy metal catalysis. No difference in cyanide sensitivity was observed between cells respiring in the presence or absence of vitamin B12. It was found that the addition of 1 to 2 mg of coenzyme I per ml resulted in only a small, temporary effect on the rate of oxidation. Nor could the components of coenzyme Downloaded from MINUTES Figure S. A comparison of cytochrome c and vitamin B12 effects on acetate oxidation. Ten pm acetate, cell suspension equivalent to 0.75 mg bacterial nitrogen, 0.2 ml 0.2 M phosphate buffer, ph 6.2, 210 mpg vitamin B12, 500 pg cytochrome c, distilled water to volume of 3.0 ml. Cells aged 29 days. Acetate added 45 minutes after vitamin B12 or cytochrome c. on July 26, 2018 by guest I (nicotinic acid, ribose, and adenine) replace the vitamin B12. The addition of 167,ug of cytochrome c per ml, instead of the vitamin, resulted in a marked rise in the rate of oxidation, and in an increase in the total oxygen uptake as well. Figure 2 compares the action of vitamin B12 and the cytochrome c preparation on acetate oxidation. This stimulatory effect of cytochrome c was somewhat surprising, since some strains of E. coli have been shown to lack both cytochrome c and the typical cytochrome oxidase (Keilin and Harpley, 1941). Part of the effect, at least, could be accounted for by apparent contamination of the cytochrome c preparation with vitamin B12 (as measured by growth activity for L.

9 1951] INFLUENCE OF VITAMIN B12 ON OXIDATION 589 lactis Dorner no. 8000). One preparation was found to contain 3.64 mpg vitamin B12 per mg (or 0.61 m,;g per 167,ug), and another cytochrome c preparation (crystalline) contained 5.82 m,ug per mg, which might account for the rate stimulation but not for the increase in total oxygen uptake.3 One should consider, as well, the possibility that the apparent vitamin B12 content of the cytochrome c preparations was due to activity of the cytochrome c itself in replacing the vitamin B12 requirement. It was then investigated whether organisms grown with vitamin B12 contained more or different cytochrome from that of deficient cells, which should have been the case if the vitamin was involved in the synthesis of porphyrins. To test this theory two types of "air grown" cells were used. The medium for growth of both types contained 20,g of DL-methionine per liter, and in addition one set contained 10 mpg vitamin B12 per liter. Cell suspensions of each type were prepared to yield a concentration of 2 mg dry weight per ml. Absorption spectra of these suspensions were determined over the ranges from 395 to 420 m,u, and from 500 to 700 m,u, on a Beckman DU spectrophotometer. Small peaks were observed with both cell suspensions at the following wave lengths: 402 to 410, 515, 555 to 560, 605, and 635 m,u. Slightly increased absorption at 402 to 410, 515 to 520, 555 to 560, and 605 m,u but no peaks differing in wave length were observed when the ceils grown in the presence of vitamin B12 were compared to those grown in its absence. It is possible that these increments were due to experimental error, although they were rather consistent. The location of these absorption peaks does not agree, except for those at 555 to 560 and 635 m,u, with those previously reported for the cytochromes of E. coli, i.e., 532 and 562 m,u (Frei, Riedmiller, and Almasy, 1934); 528 to 532, 558 to 567, 587 to 595, and 625 to 635 m,u (Fujita and Kodama, 1934); 560, 590, and 628. myt (Keilin and Harpley, 1941). The reasons for the peaks observed in our experiments at 402 to 410, 515, and 605 m,u are not known, but they may be due to experimental factors unaccounted for, such as strain differences and methods of cultivation. The peak at 402 to 410 m,u would presumably be due to total porphyrin content, and the increased adsorption of vitamin B12-treated cells might result, therefore, from increased porphyrin synthesis. The present data provide some indication that vitamin B12 may be concerned in the synthesis of porphyrins. However, the data are not conclusive, and further study is required. The effect of cytochrome c in stimulating respiration cannot be employed as evidence of such porphyrin synthesis for two reasons. First, the cytochrome c preparations contained sufficient vitamin B12, as determined by standard microbiological assay, to show independent activity. Second, there are several points of evidence which show that vitamin B12 and cytochrome c influence oxygen uptake in different manners. For example, the "deep grown" cells responded to vitamin B12 earlier in the aging process than they did to the cytochrome c preparation. After prolonged aging, when the response to both was marked, addition of the cytochrome c preparation resulted in an increase in total oxygen uptake, whereas the addition of vitamin B12 did not. "Air grown" cells 3 We are greatly indebted to Mrs. Jeanne Wall of the Research Laboratories of Merck and Company, Inc., for careful and repeated analyses of these preparations.

10 590 OGINSKY ET AL. [vol. 61 on aging have been shown to develop a marked response to vitamin B12, while at the same time their response to cytochrome c was very slight. In addition, organisms were grown in the basal medium plus 100 m,ug of the vitamin per ml, so that they were presumably saturated with vitamin B12 and with the compounds in whose synthesis it may be involved. These cells responded to the cytochrome c preparation in a fashion similar to the vitamin-b12-deficient cells, but they did not respond to vitamin B12. At the moment, therefore, we have been unable to identify more closely the reaction or reactions in which vitamin B1n is involved. SUMMARY Vitamin B12 has been shown to increase markedly the rate of oxidation of a variety of substrates by resting cell suspensions of a mutant strain of Escherichia coli that requires methionine or vitamin B12 for growth. REFERENCES AJL, S. J Acetic acid oxidation by Escherichia coli and Aerobacter aerogenes. J. Bact., 59, DAVIS, B. D., AND MINGIOLI, E. S Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bact., 60, FITZGERALD, R. J., AND BERNHEIM, F The effect of streptomycin on the formation of adaptive enzymes. J. Bact., 55, FITZGERALD, R. J., BERNHEIM, F., AND FITZGERALD, D. B The inhibition by streptomycin of adaptive enzyme formation in mycobacteria. J. Biol. Chem., 175, FOSTER, J. W., CARSON, S. F., ANTHONY, D. S., DAVIS, J. B., JEFFERSON, W. E., AND LONG, M. V Aerobic formation of fumaric acid in the mold Rhizopus nigricans: synthesis by direct C2 condensation. Proc. Nat. Acad. Sci. U. S., 35, FREI, W., RIEDMtTLLER, L., AND ALMASY, F ttber Cytochrom und das Atmungsystem der Baktieren. Biochem. Z., 274, FUJITA, A., AND KODAMA, T Untersuchungen ilber Atmung und GArung pathogener Baktieren III. Biochem. Z., 273, GRANT, W. M Colorimetric micromethod for determination of formic acid. Ind. Eng. Chem., Anal. Ed., 19, HOFF-J0RGENSEN, E Difference in growth promoting effect of desoxyribosides and vitamin B1, on three strains of lactic acid bacteria. J. Biol. Chem., 178, KEILIN, D., AND HARPLEY, C. H Cytochrome system in Bacterium coli commune. Biochem. J., 35, KITAY, E., McNuTT, W. S., AND SNELL, E. E The nonspecificity of thymidine as a growth factor for lactic acid bacteria. J. Biol. Chem., 177, KITAY, E., McNuTT, W. S., AND SNELL, E. E Desoxyribosides and vitamin B1, as growth factors for lactic acid bacteria. J. Bact., 59, OCKRENT, C Relation between vitamin B12 and the red blood cells. Nature, 165, OGINSKY, E. L., SMITH, P. H., AND SOLOTOROVSKY, M The action of streptomycin. IV. Fatty acid oxidation by Mycobacterium tuberculosis, avian type. J. Bact., 59, SaT1VE, W., RAVEL, J. M., AND EAKIN, R. E An interrelationship of thymidine and vitamin B12. J. Am. Chem. Soc., 70, STERN, J. R., AND OCHOA, S Enzymatic synthesis of citric acid by condensation of acetate and oxalacetate. J. Biol. Chem., 179, SWIM, H. E., AND KRAMPITZ, L Evidence for the condensation of acetic acid to succinic acid in Escherichia coli. Bact. Proc., 1950,