JOURNAL OF BACTrOLOGY, May 1973, p. 543-548Copyright 0 1973 American Society for Microbiology

Vol. 114, No. 2Printed in U.SA.

Induction of Superoxide Dismutase byMolecular Oxygen

EUGENE M. GREGORY AND IRWIN FRIDOVICH

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

Received for publication 14 September 1972

Oxygen induces superoxide dismutase in Streptococcus faecalis and inEscherichia coli B. S. faecalis grown under 20 atm of 02 had 16 times more ofthis enzyme than did anaerobically grown cells. In the case of E. coli, changingthe conditions of growth from anaerobic to 5 atm of 0° caused a 25-fold increasein the level of superoxide dismutase. Induction of this enzyme was a response to02 rather than to pressure, since 20 atm of N2 was without effect. Induction ofsuperoxide dismutase was a rapid process, and half of the maximal level was

reached within 90 min after N2-grown cells of S. faecalis were exposed to 20 atmOf 02 at 37 C. S. faecalis did not contain perceptible levels of catalase under anyof the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23),and the concentration of catalase in E. coli was not affected by the presence of02 during growth. S. faecalis, which had been grown under 100% 02 and whichtherefore contained an elevated level of superoxide dismutase, was more

resistant of 46 atm of 0 2 than were cells which had been grown under N2. E. coligrown under N2 contained as much superoxide dismutase as did S. faecalisgrown under 1 atm of 02. The E. coli which had been grown under N2 was as

resistant to the deleterious effects of 50 atm of 02 as was S. faecalis which hadbeen grown under 1 atm of 02. These results are consistent with the proposalthat the peroxide radical is an important agent of the toxicity of oxygen and thatsuperoxide dismutase may be a component of the systems which have beenevolved to deal with this potential toxicity.

Oxygen is toxic (11, 12, 22). The damagingeffects of 02 have been demonstrated in bacte-ria (3, 9, 12, 22), protozoa (26), fungi (25), fish(5), human lymphocytes (21), and whole ani-mals (8). There is, as yet, no adequate explana-tion for the toxicity of oxygen. Superoxidedismutase, which catalyzes the reaction: 02- +03- + 2H+ - H202 + 02 (17) is ubiquitousamong oxygen-metabolizing organisms but isabsent from obligate anaerobes (18). This en-zyme may constitute an important componentof the defenses against oxygen toxicity. Thus,univalent reduction of oxygen, which has beendemonstrated in several enzymatic systems (7,16, 20), generates the superoxide radical whosegreat chemical reactivity may be presumed toconstitute a threat to the integrity of cellularcomponents. If one or more of the reactionswhich generate O2- were not saturated withrespect to oxygen under atmospheric condi-tions, raising the partial pressure of oxygenwould increase the intracellular flux of 02-. Ifthis flux of 02- exceeded the scavenging ability

of the ambient level of superoxide dismutase,cellular damage would result. If this is indeedthe situation, it appeared likely that superox-ide dismutase would be induced in response tooxygen and that cells which had a high level ofthis enzyme would be less sensitive towardsoxygen than those which had a lower level ofthis enzyme. This report describes experimentswith Streptococcus faecalis and with Esche-richia coli which appear to affirm these expec-tation.

MATERIALS AND METHODSTrypticase soy broth was a product of the Bioquest

Division of the Becton and Dickinson Co. Yeastextract and agar were obtained from the Difco Labo-ratories. S. faecalis was obtained from A. Proctor ofthe Department of Microbiology and E. coli B wasprovided by D. Hall of the Division of Genetics ofDuke University Medical Center, Durham, N.C.Cells were exposed to hyperbaric 03 or N2 at 25 C onagar plates or in shallow liquid cultures which weresubjected to continuous magnetic stirring. Pressure

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GREGORY AND FRIDOVICH

was achieved and maintained in stainless steel ves-sels. Superoxide dismutase in the cell-free extractswas assayed as previously described (17) but with themodification (1) that 2 x 10' M cyanide was addedto inhibit cytochrome c peroxidases, which otherwisemay interfere with this assay. Cell-free extracts wereprepared by sonic disruption of cell suspensions in0.05 M potassium phosphate, 10-' M EDTA (pH 7.8)at 0 C for 3 min with a Branson Sonifier, modelW185, operated at a power output of 90 W, followedby centrifugation for 15 min at 27,000 x g. Thesecell-free extracts were concentrated 10-fold by ul-trafiltration over an Amicon PM-10 membrane andwere then restored to their original volume by addi-tion of 0.05 M potassium phosphate, 10-i M EDTAat pH 7.8. This was done to reduce the concentrationsof endogenous low-molecular-weight substances.

S. faecalis was grown in a medium containing 30 gof Trypticase soy broth per liter and 5 g of yeastextract per liter. E. coli was grown in a low phosphatemedium whose composition in grams per liter was:glucose, 40.5; NH4Cl, 5.0; Na2HPO4, 6.0; KH2PO4,3.0; tryptone (Difco), 0.5; MgSO4.7H20, 0.2; NaCl,0.02; FeSO4.7H20, 0.02; ascorbic acid, 0.02. Bacte-rial growth was at 37 C and was monitored tubidi-metrically at 600 nm (14). The protein concentra-tions of cell-free extracts were estimated spectro-photometrically (24). Electrophoretic separationswere performed on polyacrylamide gels, (6) andsuperoxide dismutase activity was localized on thesegels as previously described (1). Gels which had beenstained for enzymatic activity were scanned forabsorbance at 400 nm with a Gilford Gel Scanner,and the amount of activity in a particular band wasestimated from the area under the correspondingabsorbance peak as produced by the Gel Scanner.Catalase was quantitated spectrophotomtrically(2).

RESULTSInduction of superoxide dismutase by ox-

ygen. When S. faecalis, which had been grownanaerobically, was the inoculum, subsequentgrowth was equally rapid under N2, 0.2 atm of02, or 1.0 atm of 02, but was distinctly slowerunder 20 atm of 02. Thus, early stationaryphase was reached in 4 to 4.5 h under theformer conditions, but in 9 h under the lattercondition. Inoculation involved equal dilutionof the cells into fresh media in all of theseexperiments, and the stainless steel pressurevessel was brought to the temperature ofgrowth, 37 C, before the freshly inoculatedsamples were placed in it. It is clear that 20atm of 02 slowed the rate of growth of thesecultures. Cells were grown to late exponentialphase under varying partial pressures of 02 andwere then collected by centrifugation. Cell-freeextracts prepared from these samples werethen assayed for protein content and for super-oxide dismutase activity. The results of these

measurements (Fig. 1) demonstrate thatgrowth under increasing concentrations of 02elicited increased production of superoxide dis-mutase. Control experiments demonstratedthat pressure, per se, was not a factor in theinduction of superoxide dismutase. Cells grownunder 1 atm of N2 gave extracts with the samespecific activity of this enzyme as did cellsgrown under 20 atm of N2.

Extracts from cells grown under N2 or under20 atm of 02 were analyzed by electrophoresison polyacrylamide gels (6), and the gels werethen stained for superoxide dismutase. Only asingle zone of this activity was seen in bothcases and it always exhibited the same mobil-ity. This suggests that the induction by O2 Ofsuperoxide dismutase in S. faecalis involves anincrease in the enzyme which is present at lowlevels even in the anaerobically grown cellsrather than the appearance of a new isoen-zyme.The response of E. coli to changes in the

partial pressure of 02 was examined undercomparable conditions. E. coli, when grownanaerobically, was found to contain more super-oxide dismutase than S. faecalis under thesame conditions. Anaerobically grown S. fae-calis gave extracts whose specific activity was0.8, whereas E. coli gave specific activities of3.8. It was nevertheless true that oxygen in-duced the superoxide dismutase of E. coli. Thisis demonstrated by the results in Table 1. E.coli contains two electrophoretically distinctsuperoxide dismutases (1), and these wereaffected very differently by the partial pressureof 02 under which the cells were grown. Thusisoenzyme I, which was the slower movingspecies, was strikingly induced by 02 whereasisoenzyme II, which showed more rapid ca-thodic migration, was induced to a muchsmaller extent. Polyacrylamide gel electro-pherograms of extracts of E. coli, which hadbeen grown under different partial pressure of02, were stained for superoxide dismutase ac-tivity, and the amount of each isoenzyme wasthen estimated from the area under the cor-responding peaks obtained with a gel scanningdensitometer. The ratios of the amounts ofisoenzyme I to isoenzyme II, so estimated, arepresented in Table 2.Rate of induction of superoxide dismu-

tase. S. faecalis, which had been grown ana-erobically, was transferred to fresh broth andincubated under 20 atm of 02. The doublingtime under these conditions was 1.2 h. Atintervals the turbidity of the culture and thesuperoxide dismutase activity of cell-free ex-tracts, prepared from samples of these cells,

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02 INDUCTION OF SUPEROXIDE DISMUTASE

c,0 4 8 12 16 20P02 (Atmospheres)

FIG. 1. Effect of oxygen upon the level of superox-ide dismutase in S. faecalis. S. faecalis, grown underN2, was used to inoculate broth cultures which werethen grown to late exponential phase at 37 C underthe 0, tensions indicated on the abscissa. These cellswere harvested, and cell-free extracts prepared fromthem were assayed for superoxide dismutase and forprotein. The specific activity of these extracts is heregraphed as a function of the 0° tension duringgrowth.

TABLE 1. E. coli superoxide dismutase and catalaselevelsa

Superoxide CatalaseGrowth conditions dismutase units

units (mg-1) (mg- 1)

Nitrogen 3.8 11.3Air 13.5 12.51 atmO, 21.2 _b5 atm °2 (45 min) 42.5 _b5 atm °2 (19 h) 92.8 12.6

a Cell-free extracts from E. coli grown under theindicated condition were assayed for superoxide dis-mutase and catalase activity as described in Materi-als and Methods. Catalase was measured on freshcell-free extracts which had never been frozen.

b Catalase was not measured under these condi-tions.

were measured. The results of these manipula-tions (Fig. 2) demonstrate that exposure of S.faecalis to 02 caused a marked increase in thecontent of superoxide dismutase. It is alsoapparent that this enzyme induction was morerapid than was the proliferation of cells underthe same conditions. Thus, the amount ofsuperoxide dismutase activity increased nearly10-fold within 2.5 h whereas the cells could haveincreased in number by only fourfold in thesame interval had they been growing exponen-tially. There was, in fact, some lag in the cellgrowth, so that the 10-fold increase in enzymeactivity, seen in the first 2.5 h under 20 atm of

02, was actually accompanied by a twofoldincrease in turbidity. During the exponentialphase of cell proliferation, under the conditionsshown in Fig. 2, the amount of superoxidedismutase per milliliter of culture was a linearfunction of the turbidity of the culture. Theseresults indicate that the increase in superoxidedismutase activity, observed upon transferringS. faecalis from anaerobic conditions to 20 atmof 02, was due to enzyme induction rather thanto selection of an enzyme-rich strain. It wasalso shown that S. faecalis, grown to stationaryphase under N2, exhibited a fourfold increasein the specific activity of superoxide dismutaseduring a 30-min exposure to 20 atm of 02- E.coli, under the same conditions, showed atenfold gain in their content of this enzymeduring 45 min of exposure to 02.Catalase. Early attempts to explain the

inability of obligate anaerobes to grow in airproposed that H202 was the toxic product ofoxygen metabolism and that the presence ofcatalase distinguished 02-tolerant from 02-in-

TABLE 2. Relative amounts of E. coli isoenzymesa

Condition of growth Isoenzyme U/isoenzyme II

Nitrogen.... 0.4Air.... 1.25 atmO°2......... 7.6

a Cell-free extracts of E. coli grown under theindicated condition were subjected to electrophoresison 10% acrylamide gels and stained for activity asdescribed in Materials and Methods. The gels werescanned and the relative areas for each peak werenormalized for isoenzyme II as 1 area unit.

1.6

IE 1.2c'0

o

0.0.

0.41

- . .............SODActivity

- . ................./Turbidityx

1

x

I /

4l16

211

_ 8a-.

-4 .2

C,0

-o 4 8 12 16 20 24Hours

FIG. 2. Rate of induction of superoxide dismutasein S. faecalis under 20 atm of 02. S. faecalis, grownunder N2, was used to inoculate (102 cells per ml)broth cultures which were then incubated at 37 Cunder 20 atm of 02. Growth was followed by measur-ing optical density (0) while superoxide dismutase(x) was assayed on cell-free extracts prepared at theindicated intervals.

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GREGORY AND FRIDOVICH

tolerant cells (4, 19). Subsequent discoveries ofacatalasic aerobes (10, 13) weakened the im-pact of this hypothesis. It was neverthelessconsidered important to assay cell-free extractsfor catalase. S. faecalis, whether grown underN2 or under 20 atm of 02, did not containdetectable catalase. They may contain peroxi-dases, which serve to scavenge H202, but thesewere not investigated. E. coli did containcatalase, but the specific activity of this en-zyme was not significantly changed in cellsgrown under 5 atm of 02 as compared to cellsgrown under N2. Thus, extracts of oxygen-grown E. coli contained 12.6 units of catalaseper mg of protein, whereas the correspondingfigure for N2-grown cells was 11.3. It is thusapparent that catalase, unlike superoxide dis-mutase, was not induced by 02.Deleterious effects of hyperbaric 02. S.

lOG

80 -

.0

0

40\

20

I I I I I00 2 4 6 8

HoursFIG. 3. Deleterious effects of hyperbaric 02 on S.

faecalis. S. faecalis, grown under N2 and containing0.8 units of superoxide dismutase per mg of solubleprotein (0) or grown under 02 and containing 4.8times more of this enzyme (X), were diluted intofresh broth (107 cells per ml) containing 0.5 mg ofpuromycin per ml and were incubated at 37 C under46 atm of 02- At intervals portions were removed,diluted with fresh broth which was free ofpuromycin,and plated onto agar plates. After incubation over-night at 37 C under 20 atm of 02, colonies werecounted and the percentage of survivors of theoriginal inoculum was calculated. In a control experi-ment the effects of 46 atm of N2 upon the survival ofN2-grown cells was determined (0). These measure-ments are of cells which produce colonies after a 12-hincubation on agar plates under 20 atm of oxygen.

faecalis, grown anaerobically or alternatelygrown under 1 atm of 02, was suspended infresh medium containing 0.5 mg of puromycinper ml. This inhibitor of protein synthesis wasused to prevent the induction of superoxidedismutase during the subsequent exposure ofthese cells to hyperbaric oxygen. It was ob-served that, in the presence of puromycin, thelevel of superoxide dismutase in these cellsslowly decreased during incubation under oxy-gen, and the half-time of this decrease wasapproximately 4 h. These suspensions of cellswere exposed to 46 atm of 02 at 25 C, and atintervals samples were removed from the pres-sure vessel and, after suitable dilution, wereplated onto agar. To control for the possibledamaging effects of pressure, per se, a suspen-sion of N2-grown cells was exposed to 46 atm ofN2 and then plated under otherwise identicalconditions. Exposure to hyperbaric 02 wasseen to cause a progressive increase in fre-quency of aberrant colonies of S. faecalison the agar plates. These colonies were ab-errant in the sense that they were smaller,after 12 h of growth at 37 C, than were thenormal colonies. Aberrant colonies wereproduced under hyperbaric 02 but not un-der hyperbaric N2 and, furthermore, the rateof production of aberrant colonies was muchfaster in the case of N2-grown, low-superoxidedismutase cells than in the case of 02-grown,high-superoxide dismutase cells. To obtainquantitative data, it was necessary to count theproportion of aberrant colonies, but this wasinconvenient because such counting requiredthe decision, with respect to each colony, ofwhether it was normal or aberrant. The situa-tion was simplified by the discovery that 20atm of 02 suppressed the growth of the aber-rant colonies but not the normal colonies. Theprecedure, then, was to expose cells to 46 atmof 02 and, at intervals, to spread dilutedsamples onto sterile agar plates which wereincubated for 12 h at 37 C under 20 atm of 02and then counted. Figure 3 presents the resultsof the experiment. It is apparent that cellswhich had been induced, with respect to super-oxide dismutase, by previous growth under100% 02 were more resistant towards the dele-terious effects of 46 atm of 02 than were cellswhich had been grown under N2 and whichtherefore contained less superoxide dismutase.It is also clear that 46 atm of N2 was withouteffect of these cells. The appearance of normalcolonies of S. faecalis may be compared withthat of the aberrant colonies in Fig. 4.The defect which was progressively intro-

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02 INDUCTION OF SUPEROXIDE DISMUTASE

FiG. 4. Appearance of normal and aberrant colonies of S. faecalis. N.-grown cells were exposed tohyperbaric 0, as described in the legend of Fig. 3 but after dilution and plating were grown out under airrather than under 20 atm of 02. The difference in colony size is apparent.

duced into anaerobically grown S. faecalis,during exposure to 46 atm of 02 in the presenceof puromycin, was a persistent one. Thus, cellstaken from an aberrant colony and spread ontosterile agar, gave rise to aberrant colonies whengrown in air. Whatever the nature of thisdefect, it diminished the growth rate in air andvirtually halted growth under 20 atm of 02.When E. coli were exposed to 46 atm of 02,

under conditions which had caused the appear-ance of abberant colonies of S. faecalis, nochanges in colony morphology were observed.The resistance of E. coli to hyperbaric 02 wasalso demonstrated by the fact that 80 to 85% ofthe cells gave rise to colonies after 8 h ofexposure to 46 atm of 02 at 25 C.

DISCUSSIONSuperoxide dismutase was induced by 02 in

both S. faecalis and E. coli, whereas catalasewas not detectable in S. faecalis and wasunaffected by 02 in E. coli. S. faecalis, whichhad been grown under N2 and which had a lowlevel of superoxide dismutase, suffered somedamage when exposed to 46 atm of 02 at 25 C.This damage was progressive with time ofexposure to the hyperbaric O2. Damaged cellsformed colonies which were smaller than nor-mal if incubated in air for 12 h at 37 C and didnot form colonies if incubated under 20 atm of02 for 12 h at 37 C. S. faecalis, in which super-oxide dismutase had been induced by growth

in 100% 02, was resistant towards this effect ofhyperbaric oxygen. These results are consistentwith the proposal that superoxide radical is anagent of oxygen toxicity and that superoxidedismutase is an important factor in the defenseagainst the toxicity of 02. It is, of course, pos-sible that growth in the presence of 100% 02induced enzymes other than superoxide dismu-tase and that these were actually responsiblefor the effects observed. We cannot, at present,exclude this possibility, but we can state thatcatalase was not induced by 02 in these orga-nisms and, therefore, was not involved in theprotection afforded by prior growth under 100%02. Growth under hyperbaric 02 has beenreported (15) to raise the level of catalase in E.coli, but this was not the case under theconditions reported above. It is pertinent thatE. coli, which contained as much superoxidedismutase when grown under N2 as did S.faecalis when grown under 1 atm of 02, wasvery resistant to the deleterious effects of 46atm of 02.

ACKNOWLEDGMENTSThis work was supported in part by Public Health Service

grant GM-10287 from the National Institute of GeneralMedical Sciences.

E.M.G. was a postdoctoral fellow of the National HeartInstitute, Public Health Service grant no. HE-05736.

LITERATURE CITED1. Beauchamp, C. O., and I. Fridovich. 1971. Superoxide

dismutase: improved assay and an assay applicable to

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acrylamide gels. Anal. Biochem. 44:276-287.2. Beers, R. F., and I. W. Sizer. 1952. A spectrophotometric

method for measuring the breakdown of hydrogenperoxide by catalase J. Biol. Chem. 159:133-140.

3. Bornside, G. H. 1969. Bactericidal effects of hyperbaricoxygen determined by direct exposure. Proc. Soc. Exp.Biol. Med. 130:1165-1167.

4. Callow, A. B. 1953. Further observations on the produc-tion of hydrogen peroxide by anaerobic bacteria. J.Pathol. Bacteriol. 66:527-538.

5. D'Aoust, B. G. 1969. Hyperbaric oxygen toxicity to fishat pressures present in their swimbladders. Science163:576-578.

6. Davis, B. J. 1964. Disc electrophoresis. II. Methods andapplication to human serum proteins. Ann. N.Y.Acad. Sci. 121:404-436.

7. Fridovich, I. 1970. Quantitative aspects of the produc-tion of superoxide anion radical by milk xanthineoxidase. J. Biol. Chem. 245:4053-4057.

8. Gershman, R., L. Gilbert, and D. Caccamise. 1958.Effect of various substances on survival times of miceexposed to different high oxygen tensions. Amer. J.Physiol. 192:563-571.

9. Gifford, G. D. 1968. Mutation of an auxotrophic strain ofE. coli by high pressure oxygen. Biochem. Biophys.Res. Commun. 33:294-298.

10. Gledhill, W. R. and L. E. Casida, Jr. 1969. Predominantcatalase-negative soil bacteria. III. Agromyces, gen N.,microorganisms intermediary to Actinomyces and No-cardia. Appl. Microbiol. 18:340-349.

11. Gottleib, S. F. 1971. Effects of hyperbaric oxygen onmicro organisms. Annu. Rev. Microbiol. 25:111-152.

12. Haugaard, N. 1968. Cellular mechanisms of oxygentoxicity. Physiol. Revs. 48:311-373.

13. Jones, D., J. Watkins, and D. J. Meyer. 1970. Cyto-chrome composition and effect of catalase on growth ofAgromyces ramnosus. Nature (London) 226:1249-1250.

14. Koch, A. Z. 1970. Turbidity measurements of bacterialcultures in some available commercial instruments.Anal. Biochem. 38:252-259.

15. Luck, H. 1954. Einfluss von Komprimierten Saurstoff

,NIED FRIDOVICH J. BACTERIOL

auf einige Stoftwechselvorgange in Bakterien. SchweizZ. Allg. Pathol. Bakteriol. 17:106-117.

16. McCord, J. M., and I. Fridovich. 1968. The reduction ofcytochrome c by milk xanthine oxidase. J. Biol. Chem.243:5753-5760.

17. McCord, J. M., and I. Fridovich. 1969. Superoxidedismutase: an enzymatic function for erythrocuprein.J. Biol. Chem., 244:6049-6055.

18. McCord, J. M., B. B., Keele, Jr., and I. Fridovich. 1971.An enzyme based theory of obligate anaerobiosis: thephysiological function of superoxide dismutase. Proc.Nat. Acad. Sci. U.S.A. 68:1024-1027.

19. McLeod, J. W., and J. Gordon. 1923. The problem ofintolerance of oxygen by anaerobic bacteria. J. Pathol.Bacteriol. 26:332-343.

20. Misra, H. P., and I. Fridovich. 1971. The generation ofsuperoxide radicals during the autoxidation of fer-redoxins. J. Biol. Chem. 246:6886-6890.

21. Mizrahi, A., G. U. Vosseller, Y. Yagi, and G. E. Moore.1972. The effect of dissolved oxygen partial pressure ongrowth, metabolism, and immunoglobulin productionin a permanent human lymphocyte cell line culture(36092). Proc. Soc. Exp. Biol. Med. 139:118-112.

22. Moore, B., and R. S. Williams. 1911. The growth ofvarious species of bacteria and other micro organismsin atmospheres enriched with oxygen. Biochem. J.5: 181-187.

23. Stanier, R. Y., M. Doudoroff and E. A. Adelberg. 1970.The Microbial World, 3rd. ed. p. 663. Prentiss Hall,Inc., E4glewood, N.J.

24. Warburg, O., and W. Christian. 1941. Isolierung undKristallisation des Garungsferments Enolase. Bio-chem. Z. 310:384-421.

25. Wiseman, G. M., F. C. Violago, E. Roberts, and I. Penn.1966. The effects of hyperbaric oxygen upon aerobicbacteria. I. In vitro studies. Can. J. Microbiol.12:521-529.

26. Whittner, M. 1957. Effects of temperature and pressureon oxygen poisoning of Paramecium. J. Protozool.4:20-23.

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ERRATA

Induction of Superoxide Dismutase byMolecular Oxygen

EUGENE M. GREGORY AND IRWIN FRIDOVICHThe Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Volume 114, no. 2, p. 543, abstract, line 19: Change "peroxide" to read "superoxide."

Thymineless Death in Escherichia coli in VariousAssay Systems: Viability Determined in

Liquid MediumHIROAKI NAKAYAMA AND JOHN L. COUCH

Department of Biological Sciences, Stanford University, Stanford, California 94305

Volume 114, number 1, p. 230, Fig. 1: The caption along the vertical axis should read "Numberof colony-forming units/ml."

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