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Regulation of the Synthesis of Superoxide Dismutase in Escherichia coli INDUCTION BY METHYL VIOLOGEN*

(Received for publication, May 26, 1977)

H. MOUSTAFA HASSAN AND IRWIN FRIDOVICH

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Paraquat (methyl viologen) caused a rapid and pro- nounced increase in the rate of biosynthesis of the man- ganese-containing superoxide dismutase, in Escherichia coli B growing aerobically, in a trypticase soy-yeast extract (TSY) medium. Removal of the paraquat was followed, after a lag of 30 min, by a marked decrease in cellular content of this enzymic activity. Paraquat also caused a moderate induction of catalase but had little effect on peroxidase or on the iron-containing superoxide dismutase. The concentrations of paraquat, which were effective in inducing the manganese-superoxide dismutase, exerted lit- tle effect on the growth rate and no effect on the net rate of respiration, but did increase that fraction of the total oxygen consumption which was insensitive to 1.0 mu cya- nide. Cells whose content of manganese-superoxide dismu- tase had been augmented by paraquat induction, exhibited increased resistance towards oxygen toxicity and towards the oxygen enhancement of the lethality of streptonigrin.

It seems likely that paraquat subverts a portion of the normal, cyanide-sensitive, electron flow in the cells and transfers that electron flow to oxygen, in a univalent and cyanide-insensitive manner. This results in an increased rate of intracellular production of O,-. 02-, or some product uniquely derived from 02-, then induces or derepresses the biosynthesis of the manganese-superoxide dismutase, which defends the cell against the deleterious reactivities of this radical.

Superoxide dismutases catalytically scavenge O,- and in so doing provide an important defense against oxygen toxicity (l-4). Since, in biological systems, O,- can only be made in the presence of molecular oxygen and since oxygen supply is a variable for many cell types, one might expect that the level of superoxide dismutase would be responsive to the degree of oxygenation. This has been shown to be the case in

* This work was supported by research Grants GM-10287 and HL-17603 from the National Institutes of Health, Bethesda, Md., and by Grant DAHC-0474-G-0194 from the United States Army Research Office, Research Triangle Park, N. C. The costs of publi- cation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertlse- men? in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Streptococcus faecalls (51, Escherichia coli (58), Photobacte- rium l&ognathi (9, lo), Euglena gracilis (111, Saccharomyces cereuisiae (121, rat lung (13-151, and guinea pig leukocytes (16). In E. coli, which contains both manganese- and iron- superoxide dismutases, the manganese enzyme is induced in response to oxygenation, whereas the iron enzyme is not (7, 8). Furthermore, in this organism, induction of superoxide dismutase has been shown to correlate with increased resist- ance towards the toxicity of oxygen and towards the oxygen enhancement of the toxicity of streptonigrin (6, 8). One may then question whether molecular oxygen is itself the inducer (or derepressor) or whether it is 02-, or some product made only from 02-, which serves this control function.

In E. coli it was observed that dependence upon glucose, as

a source of energy, was associated with a low level of super- oxide dismutase; whereas dependence upon the non-carbohy- drate components of a trypticase soy-yeast extract (TSY) medium resulted in enhanced synthesis of this activity (17, 18). It was further demonstrated that this effect of glucose was not due to catabolite repression (18). It appeared likely that the glucose effect was actually due to a diminished rate of production of 02-, in the presence of ample glucose. This supposition was supported by the observation that methyl viologen (paraquat), which is known to increase intracellular production of O,- (19-221, strikingly increased the rate of synthesis of superoxide dismutase (18). The effects of paraquat on the synthesis of superoxide dismutase in E. coli have been further explored. The results of these studies form the body of this report.

MATERIALS AND METHODS

Escherichia coli B B,, (provided by D. H. Hall, Georgia Institute of Technology), ATCC No. 29682, was used throughout. Cells were grown in trypticase soy-yeast extract (TSY) medium containing 3% trypticase broth and 0.5% yeast extract. Cultures were grown at 37 on a rotatory water-bath shaker at 200 rpm with a ratio of flask volume to medium volume of 5:l. Growth was estimated, in terms of turbidity, by measuring the absorbance of diluted cell suspensions at 600 nm, using an EU 700 GCA/McPherson spectrophotometer, in cuvettes with l-cm path. Under these conditions 1 mg (dry weight cells)/ml was equal to 3.574 units at 600 nm. Methyl viologen (l,l- dimethyl-4-bipyridinium dichloride) was purchased from Sigma Chemical Co. and was added to the liquid media by dilution from sterile stock solutions.

Cell-free extracts were prepared and assayed for superoxide dis- mutase, catalase, and peroxidase, as previously described (8). Pro-

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7668 Znduction of Superoxide Dismutase by Paraquat

tein was estimated by the method of Lowry et al. (23), using pure bovine serum albumin as a standard.

Assessments of resistance towards hyperbaric oxygen or towards streptonigrin were performed with cells suspended in a glucose minimal medium which contained (per liter): MgS0,.7H,O, 0.2 g; citric acid.H,O, 2.0 g; K,HPO,, 10.0 g; NaNH,HP0.,.4H20, 3.5 g; glucose, 5.0 g; and vitamin B,,, 1.0 mg. Survival was defined as the ability to form colonies on TSY medium, solidified with 2% agar.

Respiration was measured at 37 in the absence and in the presence of 1.0 rnM cyanide, using a Gilson oxygraph. The reaction mixture contained 0.1 ml of cell suspension, whose absorbance at 600 nm was 3.1, 0.1 ml of fresh TSY medium, and 0.05 M potassium phosphate at pH 7.0, to a final volume of 1.8 ml.

RESULTS

Induction of Superoxide Dismutase by Paraquat-Esche- richia coli B, growing in the TSY medium, were very tolerant of paraquat. Growth rates were completely unaffected by concentrations of 0.1 mM or less. At 1.0 mM, paraquat de- creased the specific growth rate by 20% and at 10.0 mM by 50%. Paraquat causes increased production of O,- in both animal and plant systems (19-22) and probably does so in E. coli as well. The remarkable tolerance of E. coli towards this compound therefore suggested some appropriate accommoda- tion to its action; such as an increased synthesis of superoxide dismutase.

As shown in Fig. IA, the addition of 0.5 mM paraquat to an aerobic exponentially growing culture of E. coli resulted, after a brief lag, in a rapid linear increase in superoxide dismutase. Since the units of superoxide dismutase per mg of extractable protein increased sharply, it is clear that paraquat caused a disproportionate increase in the rate of biosynthesis of this enzyme, in comparison to other cell proteins. When the rate of biosynthesis of a particular enzyme increases from one fixed value to a higher fixed value then, as shown by

Marr and Marcus (24) the specific activity of that enzyme should be a linear function of (1 - e-); where K = specific growth rate and t = time. Fig. 1B demonstrates that the expected linear relationship was observed after a brief lag. This lag might have been due to the time needed to establish the effective intracellular level of paraquat and for the para- quat, in turn, to subvert the normal electron flow in the cell.

50 A

FIG. 1. Induction of superoxide dismutase (SOD) by paraquat. FIG. 2. Effect of paraquat on superoxide dismutase isozymes. Escherichia cc& B in late logarithmic growth phase in glucose Escherichia coli B were grown aerobically for 2 h in TSY media, minimal medium were diluted to an initial absorbance of 0.20 at 600 with and without 1.0 mM paraquat. Cell-free extracts were prepared nm into TSY medium containing 0.5 mM paraquat. At intervals and aliquots were applied to 10% polyacrylamide gels. In each case thereafter, aliquots of cells were collected and cell-free extracts 100 pg of total protein was applied per gel. After electrophoresis the were prepared and assayed for protein and superoxide dismutase. A gels were stained for activity and the distribution of isozymes was presents specific activity as a function of time of exposure to the recorded by linear scanning densitometry. Scan A was obtained paraquat. B presents specific activity as a function of 1 - @I from the culture grown in the absence of paraquat, whereas scan B according to model I of Marr and Marcus (24). was from a corresponding culture in its presence.

Paraquat did not cause any induction of superoxide dismutase when added to cultures growing in the rigorous exclusion of oxygen.

Znduction of Specific Superoxide Dismutase Zsozymes by Paraquat - Electrophoresis of crude extracts of aerobically grown E. coli, followed by staining for superoxide dismutase activity (251, reveals three bands (8). The slowest migrating of these is due to the manganese-superoxide dismutase (261, the fastest to the iron-superoxide dismutase (27) and the middle band to a newly recognized superoxide dismutase, which appears to be a hybrid composed of one subunit from each of the other two.

As shown in Fig. 2 growth of E. coli in the presence of 1.0 mM paraquat occasioned a striking increase in the man- ganese-superoxide dismutase, a small but perceptible increase in the hybrid superoxide dismutase and a barely detectable decrease in the iron-superoxide dismutase. We have previ- ously seen that the manganese enzyme, but not the iron enzyme, was induced by oxygenation (7, 8). In this earlier work the rate of O,- production in the cells was presumably increased by raising the p02, whereas in the present work oxygenation was constant but the degree of conversion of molecular oxygen to O,-, which has been called the per cent univalent reduction (29), was increased by paraquat. The modest increase in the hybrid enzyme and the decrease in the iron enzyme is exactly what would be expected. Thus in the presence of an increased supply of manganese enzyme more of the hybrid would be generated and some of the subunits of the iron enzyme would be diverted into the hybrid form.

Effects of Varying Paraquat Concentrations-E. coli B was grown for 2 h in TSY media, containing graded amounts of paraquat. Cell-free extracts were prepared and the level of each superoxide dismutase isozyme in these extracts was measured by applying 3 units of total activity to polyacryl-

H. Dougherty, submitted for publication.

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Induction of Superoxide Dismutase by Paraquat 7669

amide gels. After electrophoresis, staining for enzymatic ac- tivity and linear scanning densitometry, the areas above the activity troughs were measured. These areas are demonstra- bly proportional to the activities of each isozyme. Fig. 3 presents the specific activity of each isozyme, as well as of total superoxide dismutase, as a function of the level of paraquat in the growth medium, plotted on linear and loga- rithmic coordinates. It is apparent that large increases in total superoxide dismutase can be achieved at fixed PO,, by adding paraquat to the medium and further, that these increases are almost entirely due to increases in the man- ganese enzyme.

Deinduction- The very rapid increase in superoxide dis- mutase, following addition of paraquat to the growth medium strongly suggested that induction in all of the cells, rather than selection of a small subpopulation of hyperproducers, was being observed. One way to gain confidence in this conclusion is to abruptly remove paraquat and observe subse- quent changes in superoxide dismutase content. In the case of induction, removal of the source of the inducer should lead to a decline in activity. In contrast, once selected, a strain of hyperproducers would continue to generate the high level of

s A

t 70

[Paraquot] , mh4

I.6

1.6

[Poraquat] log,, fit4

FIG. 3. Effects of varying the concentration of paraquat. Esche- richza coli B in late logarithmic growth phase, in a glucose minimal medium, were diluted to an initial absorbance of 0.20 at 600 nm, into TSY media containing various concentrations of paraquat. After 2 h (four generations) of growth on a shaker at 200 rpm, cell- free extracts were prepared and assayed for total superoxide dismu- tase (SOD) activity. Aliquots of these extracts were also placed onto 10% polyacrylamide gels which were developed, stained for activity, and scanned as in Fig. 2. In this case, amounts of the extracts bearing 3.0 units of total activity were loaded onto each gel. The data allowed calculation of the amount of each isozyme present in each extract. A presents specific activity as a function of paraquat concentration in the growth medium, while B presents the same data on logarithmic coordinates.

activity, even when the selection stress was removed. E. coli B were grown for 90 min in a TSY medium contain-

ing 0.5 mM paraquat. The cells were then collected by centrif- ugation, washed twice in cold sterile TSY medium and then resuspended in prewarmed (37) TSY medium. At intervals thereafter cells were collected, cell-free extracts were prepared and assayed for superoxide dismutase content. Fig. 4 demon- strates that the high rate of production of superoxide dismu- tase, caused by paraquat, continued for 30 min following its removal and then declined sharply. The temporary continued high rate of synthesis, following removal of paraquat, could have been due to the completion of partially synthesized molecules and to the persistence of the superoxide dismutase messenger RNA. The sharp decline in activity seen after 30 min is certainly consistent with the induction model and inconsistent with the selection model.

Effects of Paraquat on Respiration-Oxygen consumption by cells can be divided into two categories. One, which is due to the action of cytochrome c oxidase, involves the reduction of 0, to H,O without production of detectable intermediates and is inhibited by cyanide and the second, which is due to

all other oxygen consuming reactions, which may involve O,- and H,O, as intermediates and which may be insensitive to cyanide. This is an arbitrary division, but useful nevertheless.

E. coli B, grown in TSY medium for 2 h were transferred to fresh diluted TSY medium containing varying amounts of paraquat and their rates of respiration were then immediately measured, both in the absence and in the presence of 1.0 mM cyanide. Paraquat had no effect on total oxygen consumption but it did markedly increase the cyanide-resistant respiration. These effects are illustrated by Fig. 5. It is apparent that the levels of paraquat which increased cyanide-resistant respira- tion were the same as those previously seen to be effective in inducing the biosynthesis of superoxide dismutase. The corre- lation between the level of superoxide dismutase induced by a given concentration of paraquat and the amount of cyanide- resistant respiration seen in the presence of that concentration of paraquat, is shown in Fig. 6. These results are in accord

801

Om 0 Minutes

FIG. 4. Deinduction by removal of paraquat. Escherichia coli B were induced with respect to superoxide dismutase (SOD) by aerobic growth for 90 min in TSY medium containing 0.5 rnM paraquat. The cells were chilled, collected by centrifngation, and twice washed in cold, sterile TSY medium devoid of paraquat. The washed cells were resuspended in prewarmed (37) TSY medium and allowed to resume aerobic growth. At intervals samples were removed and the cells were washed and cell-free extracts were prepared and assayed. This figure presents total activity as a function of time after resumption of growth in the TSY medium devoid of paraquat.

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7670 Induction of Super-oxide Dismutase by Paraquat

I OO

I 1 1 I 2 3

[Paroquot] , mM

FIG. 5. Effects of paraquat on cyanide-sensitive and cyanide-in- sensitive respiration. Cells were grown aerobically for 2 h in TSY medium to an absorbance of 3.1 at 600 nm. One-tenth-milliliter aliquots of this cell suspension, plus 0.10 ml of fresh TSY medium, were diluted with 0.05 M potassium phosphate, pH 7.0, to a final volume of 1.8 ml, which contained the indicated final concentrations of paraquat, with and without 1.0 rnM cyanide. Oxygen consumption was followed at 37 and is presented in the ordinate as nanoatems of 0, per min per mg dry weight of cells.

I(11 I I I I I Ill1

OO 20 40 60 80 loo N-atom 02/min/mg cells

(m presence of ImM CN-1

FIG. 6. Correlation between the effects of paraquat on cyanide- resistant and on induction of superoxide dismutase (SOD). Data were taken from Figs. 3 and 5 and correlated.

with the view that cyanide-resistant respiration is a measure of O,- production and that superoxide dismutase induction was a response to increased O,- production, caused by para- quat. Paraquat has been reported to increase the antimycin A and the amytal-resistant respiration of rat liver mitochon- drial fragments (30).

Effects of Paraquat on Catalase and Peroxidase - Cyanide- insensitive respiration should result in the production of H,O, as well as of O,-. In that case paraquat might also cause an induction of one or more of the enzymes which function to scavenge HzOz. This supposition was explored by measuring levels of catalase and of dianisidine-peroxidase in cells grown in the presence of graded amounts of paraquat. As shown in Fig. 7, paraquat did cause induction of catalase, but not nearly to the degree that it induced superoxide dismutase. The level of peroxidase was not notably affected.

Correlation between Level of Superoxide Dismutase and Resistance to Oxygen Toxicity-The effects of paraquat, de- scribed above, provide an excellent opportunity for further exploring the thesis that superoxide dismutase constitutes a

FIG. 7. Effects of paraquat on catalase and peroxidase. Condi- tions of growth and of preparation of cell free extracts were as described in the legend of Fig. 3 but the extracts were assayed for catalase and for dianisidine peroxidase.

defense against oxygen toxicity. Thus paraquat, at concentra- tions which exhibited minimal toxicity, caused marked in- creases in the cellular content of manganese-superoxide dis- mutase with much smaller increases in catalase and virtually no change in iron-superoxide dismutase, hybrid-superoxide dismutase or peroxidase. Furthermore this was achieved at

fixed ~0, and without significant effects on the specific growth rate or rate of total respiration.

E. coli, taken from the logarithmic phase of growth in glucose-minimal medium, were transferred to TSY media containing 0.00, 0.10, and 1.00 mM paraquat. After 2 h of growth in these media, at 37 and at 200 rpm, the cells were collected by centrifugation in the cold and were twice washed

with cold TSY medium. They were then incubated for 90 min in ice-cold minimal medium to allow complete removal of residual paraquat from the cells, by diffusion into the cold medium (this step was essential). The washed cells were then collected by centrifugation, suspended at lO/ml in glucose minimal medium containing 0.5 mglml of chloramphenicol, to prevent subsequent enzyme induction and were then ex- posed, for 4l/z h to 20 atm of 0, or N,. Survival was assessed

by plating appropriate dilutions on TSY plates and counting colonies after 24 to 48 h of incubation at 37. The results, shown in Table I, demonstrate that induction of manganese- superoxide dismutase, by exposure to paraquat, is correlated with the cells being resistant to oxygen toxicity.

Correlation between Level of Superoxide Dismutase and Resistance to Streptonigrin - Streptonigrin is a p-quinone antibiotic whose lethality is enhanced by oxygen probably be- cause it can, by oxidation-reduction cycling, divert electrons from pathways which do not involve the production of O,-, to those that do (6, 8, 31-33). Increased cellular content of superoxide dismutase, caused by growth under increased oxygenation (6, 81, has been shown to correlate with increased resistance towards streptonigrin. The profound induction of the manganese enzyme by paraquat offered an opportunity to further test the superoxide theory of the oxygen enhancement of the lethality of streptonigrin.

E. coli were exposed to 0.00, 0.10, and 1.00 mM paraquat in TSY medium and were then thoroughly washed free of para- quat as described in the preceding section. The washed cells were diluted to 1 x 107/ml in a glucose-minimal medium containing 2 Fg/ml of streptonigrin and were incubated at 37, on a shaker at 200 rpm. At intervals samples were removed, appropriately diluted and plated on TSY agar for

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Induction of Superoxide Dismutase by Paraquat 7671

TABLE I

Effects of hyperbaric oxygen or nitrogen on survival ofparaquat

grown cells

Growth conditions and cell properties were the same as in Fig. 8. -_____.

Viable counts Paraquat

At zero time After 4'12 h at 20 After 4'12 h at 20 atm of N, atm of 0, mM

0 1.12 X 10 1.2 x 10 (107%) 2.2 x lo6 (19.6%) 0.1 8.2 x 10fi 8.6 x 106 (105%) 6.4 x lo6 (78%) 1.0 1.05 x 10 1.17 x 10 (111%) 8.5 x 106 (81%)

Q6-

0.4-

0.21 0 5 IO 15 20

Mmutes

-I

FIG. 8. Correlation between content of superoxide, dismutase and resistance towards streptonigrin. Escherichia coli B were grown for 2 h in TSY media containing 0.00, 0.10, and 1.00 mu paraquat in order to modify their content of superoxide dismutase. Cells were chilled, collected by centrifugation, and twice washed in cold, sterile TSY devoid of paraquat. The washed cells were then suspended for 90 min in ice-cold minimal medium to allow diffusion of residual paraquat from the cells. The cells were again collected by centrifu- gation and were suspended to lO?/ml in glucose minimal medium containing 0.5 mg/ml of chloramphenicol and 2 pglml of streptoni- grin and were then incubated at 37 at 200 rpm. At intervals samples were taken, diluted, and plated onto TSY agar for assess- ment of survival. Lzne 1, cells grown in the absence of paraquat and containing 10.1 units/mg of superoxide dismutase, 16.2 units/mg of catalase, and 0.24 units/mg of peroxidase. Line 2, cells grown in the presence of 0.10 rnM paraquat and containing 36 unitsimg of super- oxide dismutase, 22.8 unitsimg of catalase, and 0.21 units/mg of peroxidase. Line 3, cells grown in the presence of 1.0 rnM paraquat and containing 75 units/mg of superoxide dismutase, 35 units/mg of catalase, and 0.36 unitsimg of peroxidase.

estimation of survival in terms of colony forming units. The results, illustrated in Fig. 8 clearly demonstrated that prior exposure to paraquat, with its concomitant induction of man- ganese-superoxide dismutase, provided protection against streptonigrin.

7. Gregory, E. M., Yost, F. J., Jr., and Fridovich, I. (1973) J. Bacterial. 115, 987-991

8. Hassan, H. M., and Fridovich, I. (1977) J. Bacterial. 129, 1574- 1583

9. Puget, K., and Michelson, A. M. (1974) Biochem. Biophys. Res. Commun. 58, 830-838

DISCUSSION

10. Puget, K., and Michelson, A. M. (1974) Biochimie 56, 1255-1267 11. Asada, K., Kanematsu, S., Takahashi, M., and Kona, Y. (1976)

Adv. Exp. Med. Biol. 74, 551-564 E. coli, grown in the absence of oxygen, are devoid of the 12. Gregory, E. M., Goscin, S. A., and Fridovich, I. (1974) J.

manganese-superoxide dismutase and exposure to oxygen induces rapid synthesis of this enzyme (5-8). Nitrate, which can serve as an electron sink in place of oxygen and which causes the anaerobic induction of the electron transport chain, does not induce this enzyme (17). Oxygen is thus clearly required for the biosynthesis of the manganese enzyme in E. coli and we could suppose that: (a) molecular oxygen directly acts as the inducer or derepressor; (b) molecular oxygen is reduced to O,m which is then the inducer or derepressor; (c) O,- uniquely reacts with some precursor to generate a more stable product, which then acts as inducer or derepressor. The profound induction of manganese-superoxide dismutase by paraquat, at a constant p0, and a constant rate of respira- tion, clearly eliminates proposal a. Since paraquat is known to favor the production of O,- (20, 34) and markedly increases the cyanide-resistant respiration, we are left with proposals b and c and cannot, at present, distinguish between them.

The profound induction of the manganese-superoxide dis- mutase in E. coli by paraquat and the great tolerance of this organism for this toxin are probably related. Thus the in- creased production of 02-, caused by paraquat, is an important factor in its toxicity and the increased synthesis of superoxide dismutase is a defense against this aspect of its toxicity. There are numerous compounds which exhibit toxicity or

carcinogenicity, in which O,- may play a role (35). We may suppose that superoxide dismutase provides a defense also against these agents and predict that paraquat and related compounds would be more toxic towards E. coli under condi- tions that interfered with the induction of the manganese- superoxide dismutase. Preliminary results indicate that this is the case. Certainly streptonigrin is more toxic to E. coli which have low cellular levels of superoxide dismutase, than to cells which have higher levels of this activity.

The iron-superoxide dismutase unlike the manganese en- zyme, was not responsive to induction by oxygen (8) or by paraquat. The control of its biosynthesis remains unknown. It may be constitutive, as previously suggested (81, or under the control of factors we have yet to explore. In a facultative microorganism it is clearly advantageous to retain some level of superoxide dismutase (i.e. iron enzyme), even during an- aerobic growth, as a standby defense against sudden exposure to oxygen. The present studies strongly support the view that O,- is an important agent of oxygen toxicity and that the manganese-superoxide dismutase is an essential defense against this toxicity.

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