Proc. Natl. Acad. Sci. USAVol. 89, pp. 7924-7928, September 1992Microbiology

Multicellular oxidant defense in unicellular organismsMUCHOU MA AND JOHN W. EATON*Division of Experimental Pathology, Department of Pathology and Laboratory Medicine, Albany Medical College, A-81, 47 New Scotland Avenue,Albany, NY 12208

Communicated by David W. Talmage, May 8, 1992

ABSTRACT Although catalase is thought to be a majordefense against hydrogen peroxide (H202), the catalase activitywithin individual Escherichia coil fails to protect against ex-ogenous H202. Contrary to earlier reports, we find that dilutesuspensions, of wild-type and catalase-deficient E. colt areidentical in their sensitivity to H202, perhaps because evenwild-type, catalase-positive E. colU cannot maintain an inter-nal/externail concentration gradient of this highly diffusibleoxidant. However, concentrated suspensions or colonies ofcatalase-positive E. colt do preferentially survive H202 chal-lenge and can even cross-protect adjacent catalase-deficientorganisms. Furthermore, high-density catalase-positive-butnot catalase-negative-E. colt can survive and multiply in thepresence of competitive, peroxide-generating streptococci.These observations support the concept that bacterial catalasemay defend colonial, but not individual, E. colU against envi-ronmental H202. Group protection by the activity of enzymesthat mitigate oxidative stress may have been a driving force inthe evolution of multicellular organisms.

Biologically hazardous reactive oxygen species, such ashydrogen peroxide (H202), arise from a variety of chemical(1-3) and metabolic (4-7) reactions. Accordingly, all aerobicorganisms possess enzymatic systems that protect againstH202 cytotoxicity. These systems include reduced glu-tathione and associated enzymes and catalases. The relativeimportance of glutathione-associated enzymes versus cata-lases may depend on the source and extent of H202 exposure(8-11). For many kinds of bacteria, catalase may be mostimportant in protection against high H202 concentrationsbecause catalases generally have very high turnover numbers(12). Furthermore, many bacteria such as Escherichia colilack glutathione peroxidase (13) and cannot use glutathionefor enzymatic catabolism of H202. In fact, glutathione ap-pears dispensable in the defense of E. coli against H202 (14).Although other metabolic pathways for H202 detoxificationmay exist in E. coli (15, 16), the quantitative importance ofthese alternative mechanisms is unclear.

Therefore, that microbial catalase is reported to be aneffective defense against relatively high concentrations ofexogenous H202 is not surprising (17-24). These earlier find-ings are, however, puzzling when one compares the readydiffusibility ofH202 with the small cellular dimensions ofmostmicrobes. That is, H202 should so rapidly permeate smallorganisms that even very high intracellular catalase activitymight not produce an outside/inside concentration gradient.To investigate this apparent ambiguity, we have carefullytested the susceptibility of catalase-deficient E. coli to killingby H202. The results indicate that bacterial catalase does notprotect isolated organisms against H202 challenge but doesfavor the survival of high-density and colonial E. coli. Suchgroup enzymatic defense in unicellular organisms may havebeen a driving force in the evolution of multicellularity.

MATERIALS AND METHODSReagents. Brain heart infusion broth, Todd-Hewitt broth,

Lennox L agar (LB agar), and Bactoagar were obtained fromGIBCO/BRL. The bicinchoninic acid protein microassaywas from Pierce. All other enzymes and chemicals werepurchased from Sigma.

Bacterial Strains and Culture Conditions. A catalase-deficient mutant strain of E. coli K-12 [UM1, hereafter,cat(-)] and its parent wild-type [CSH7, hereafter cat(+)] (17)were provided by P. C. Loewen (University ofManitoba). E.coli were grown statically in brain heart infusion broth or M9minimal salts medium supplemented with 10 mM glucose(M9/glucose) (25) at 370C in room air overnight (18-20 hr) toearly stationary-phase (OD600 = 2.2-2.4). Thiamin was notadded to the growth medium because these strains of E. coliwere phenotypically Thi+, probably from a spontaneous Thi-- Thi+ reversion. Bacteria in midexponential growth wereobtained by diluting these stationary-phase cultures 1:100 inprewarmed fresh brain heart infusion broth and incubatedfurther until the OD6(o of the culture reached 0.5-1.0. Cellswere harvested from the culture by centrifugation (10,000 xg for 10 min at 20C), washed thrice in cold phosphate-bufferedsaline (140mM NaCI/10mM Na2HPO4/10 mM KH2PO4, pH7.2) or in M9/glucose before being resuspended in theindicated buffers, and the absorbance at 600 nm was mea-sured. The correlation of OD6(, with bacterial concentrationwas affirmed by direct microscopic counts and by colony-forming units obtained by growth on LB agar. Bacterialstocks were maintained on LB agar plates, stored at 2-4C.The catalase status of colonies was constantly monitored bydirect catalase assay and by H202 drop tests.

Streptococcal strains were obtained from the AmericanType Culture Collection (Streptococcus sobrinus; 33478), G.Germaine (University of Minnesota) (Streptococcus mitis,9811), and E. L. Thomas (University of Tennessee) (Strep-tococcus mutans OMZ176 and S. mutans GS5). Organismswere cultured in Todd-Hewitt broth statically at 370C eitherin room air (S. mutans) or in a candle extinction jar (S.sobrinus and S. mitis) and washed as described for E. coli.H202 King ofE. cofi. Suspensions of E. coli were diluted

to the concentration of 5 x 107 cells per ml (concentrated) or500 cells per ml (dilute) in M9/glucose and prewarmed to370C before being exposed to H202 at the indicated concen-trations. Bacterial viability, or, more accurately, ability ofthebacteria to replicate, was assayed by pour-plating dilutedaliquots (in triplicate) of the H202/bacteria mixture in bovinecatalase-supplemented (100 units per ml) LB agar at differenttimes during incubation. Killing experiments were performednot only in pure cat(+) and pure cat(-) E. coli suspensionsbut also were carried out in mixtures of cat(+) and cat(-) E.coli as indicated. To differentiate cat(+) from cat(-) E. coliafter H202 exposure of such mixtures, bacteria ofboth strainswere made resistant to ampicillin by pUC118 (a derivation of

Abbreviations: LB agar, Lennox L agar; M9, minimal salts medium;M9/glucose, M9/10 mM glucose; cat(-), catalase-deficient mutant;cat(+), wild-type strain CSH7.*To whom reprint requests should be addressed.

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pUC18-carrying ampicillin resistance) transformation.Transformed cat(+) E. coli were admixed with nontrans-formed cat(-) organisms (or vice versa) before H202 expo-sure, and the surviving bacteria were plated in LB agar withor without ampicillin supplementation (30 ,ug/ml).For the preparation of E. coli colonies, -500 individual

organisms were pour-plated in 10 ml of molten LB agar andallowed to solidify on a precisely horizontal platform toensure equal thickness of the agar layer. After preincubationat 370C for various periods of time, the embedded microcol-onies were exposed to 1.0 mM H202 in an agar overlay (20 mlof 1.5% Bactoagar in M9/glucose). After solidification, thePetri dishes were further incubated for 48 hr at 370C beforeviable colony-forming units were enumerated. Control platescontaining bovine catalase (100 units per ml) were alsoexposed to H202 under identical conditions.

Coculture of E. coli and Streptococci. Stationary-phase S.mutans OMZ176 were resuspended to an OD650 of 1.0 in M9medium/50 mM glucose. These suspensions of streptococciwere incubated aerobically (10-12 ml in 50-ml Erlenmeyerflasks shaken on a rotary platform at 135 rpm) at 370C for 60min, and then E. coli were added at 5 x 107 organisms per ml.The fate of the cocultured E. coli was determined by pour-plating in LB agar supplemented with erythromycin (0.4,ug/ml), conditions that suppress streptococcal but not E. coligrowth. The H202 concentration ofthe coculture supernatantwas assayed in parallel. Interactions between colonies ofstreptococci and E. coli were also tested. S. mitis weresparsely inoculated on plates of presolidified Todd-Hewittagar (1.5%) and incubated in a candle extinction jar at 370Cfor 4-5 days, by which time each plate bore several strep-tococcal colonies of 3- to 4-mm diameter. E. coli weresuspended at 106 cells per ml in molten M9 agar (1.5%)supplemented with 50mM glucose and overlain onto the platebearing mature streptococcal colonies. After overnight incu-bation in air at 37°C, the diameter of growth-inhibitory zonessurrounding each streptococcal colony was determined.

Catalase Assay. Catalase activity was assayed polarograph-ically according to the method of Rorth and Jensen (26) withbacterial suspensions or lysates as samples. Lysis of E. coliwas achieved by sonication (55-60 W for 3 min at 4°C). In theabsence of added H202, the oxygen tension of bacterialsuspensions, either cat(+) or cat(-), remained the same inroom-air-equilibrated buffer for at least 10 min. Therefore,oxygen consumption by intact cells in the catalase assaysystem did not compromise the results. Bacterial suspensionsor lysates were mixed with H202 (starting concentration = 10mM) in nitrogen-purged buffer supplemented with 10 mMglucose, and the initial (1-3 min) rates of oxygen evolutionwere measured and recorded with a modified Clark 02electrode at 24°C. Appropriate dilutions of bovine livercatalase were used as a standard.H202 Assay. H202 was determined by horseradish perox-

idase-catalyzed oxidation ofphenol red (27). Briefly, sampleswere mixed with phosphate-buffered saline containing horse-radish peroxidase (8.5 purpurogallin units per ml) and phenolred (0.28 mM) and incubated at 24°C for 10 min before beingalkalinized by adding 1.0 M NaOH. Absorbance of thereaction mixture at 610 nm was measured and correlated withvalues obtained by using appropriate dilutions of reagentH202. The concentration of reagent H202 was determined byUV absorbance at 230 nm (molar absorptivity = 81M-1cm-1). The accumulation of H202 by streptococci inaerobic culture was estimated by inoculating stationary-phase organisms into M9 medium/50 mM glucose at aconcentration of 5 x 109 colony-forming units per ml (OD650= 1.0). The culture was aerated (12 ml in 50-ml Erlenmeyerflasks shaken on a rotatory platform at 135 rpm) and incu-bated at 370C. At the indicated time points, H202 in theculture supernatant was determined.

Protein Assay. Protein concentrations were determined byusing the bicinchoninic acid protein microassay (28), usingbovine serum albumin as the standard.

RESULTSCatalase Activity of Intact and Lysed E. coli. In agreement

with previous reports (29, 30), our own direct measurementson intact and lysed E. coli indicate no significant differencein internal versus external H202 concentrations at millimolarperoxide concentrations. The apparent catalase activities ofcat(+) E. coli suspensions before (intact) and after sonication(lysate; >99.9o of the cells lysed as judged microscopically)were bacterial protein at 8.71 ± 0.97 and 9.67 ± 1.34 units permg (n = 4), respectively. Thus, it appears that, at higher H202concentrations, E. coli catalases cannot maintain an appre-ciable H202 gradient across the cell envelope. It should benoted, however, that a H202 permeability barrier at the levelof the cell envelope has been proposed for other microbialspecies (31-33).H202 Sensitivity of Suspensions of cat(+) and cat(-) E. coli.

If wild-type E. coli are, indeed, incapable of sustaining aninternal/external gradient of peroxide, then, contrary toearlier reports (17-24), cat(+) and cat(-) E. coli should beequally susceptible to H202-mediated cytotoxicity. To testthis hypothesis, we exposed dilute (500 organisms per ml)suspensions of stationary-phase cat(+) and cat(-) E. coli to1.0 mM H202 and followed the kinetics of replication inac-tivation (hereafter referred to as killing). Under these con-ditions, there is no detectable difference in the killing ofcat(+) versus cat(-) organisms by exogenous H202 (Fig.1A). As shown in Fig. 1A, Inset, at such low bacterialnumbers neither cat(+) nor cat(-) E. coli suspensions clearappreciable amounts of the exogenous H202. Similarly, brief(15 min) exposure of dilute E. coli suspensions to variousconcentrations (1o-7 M to 4 x 10-2 M) of H202 revealsidentical sensitivity of cat(+) and cat(-) organisms to H202,the LD50 for both strains being -2.5 mM (data not shown).Because bacterial catalase activity is known to vary withdifferent growth and metabolic states (34, 35), we alsoexamined H202 sensitivity of exponential-phase and mini-mal-medium-growth (M9/glucose) organisms. Again, H202killing in both strains is identical (data not shown). Therefore,intracellular catalase does not protect dilute suspensions ofE. coli against external H202.However, entirely different results are obtained when

more concentrated (5 x 107 organisms per ml) suspensions ofcat(+) and cat(-) E. coli are similarly exposed to 1.0 mMH202. In this case, organismal catalase clearly confers sur-vival advantage (Fig. 1B). Unlike dilute suspensions ofE. colithat cannot clear H202 (Fig. 1A Inset), concentrated cat(+)E. coli efficiently catabolize added H202 (Fig. 1B Inset) and,therefore, survive. This concentration-dependent resistanceto H202 cytoxicity is not observed in cat(-) E. coli (Fig. 1B).Furthermore, when equal numbers of cat(+) and cat(-) E.coli are admixed in dilute suspensions and then exposed toH202, once again, there is no difference in the rate or extentof H202-dependent killing of the two strains (Fig. 2A).However, concentrated cat(+) E. coli will actually cross-protect cat(-) organisms in the same incubation mixture (Fig.2B).H202 Sensitivity of Colonial cat(+) and cat(-) E. coli. The

preceding results suggested that colonies of cat(+) E. colimight survive challenge by exogenous H202. We, therefore,grew individual bacterial colonies for increased periods oftime (during which the colonies progressively enlarge as aresult ofbacterial multiplication) before challenge with H202.As microcolonies of cat(+) E. coli increase in size, substan-tial resistance to H202 develops, whereas cat(-) microcolo-nies are killed by H202, regardless of their size (Fig. 3). If thegeneration time of E. coli embedded in such an artificial

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FIG. 1. Kinetics of replication inactivation ("killing") of dilute

(A) and concentrated (B) suspensions of cat(+) (0) and cat(-) (o) E.coli. Early stationary-phase bacteria were suspended to -5 x 102cells per ml (A) or -5 x 107 cells per ml (B) in M9/glucose. At zerotime, H202 was added to a final concentration of 1.0 mM, and thebacterial suspensions were then incubated at 37C. Surviving orga-nisms were quantitated by pour-plating appropriate dilutions in LBagar supplemented with bovine liver catalase at 100 units per ml.After 24-48 hr ofgrowth at 37"C, colonies were counted. Each pointrepresents the mean of triplicate determinations, and vertical linesshow ± 1 SD. (Insets) Suspension-phase H202 concentration duringincubations of cat(+) (0) and cat(-) (o) E. coli as measured by theperoxidase/phenol red reaction (27).

environment is 30 m* (37), the cat(+) microcolonies acquiresignificant resistance to H202 (survival increasing to >90% at6 hr) after =12 generations. The population of each micro-colony during this growth period should reach ""4000 bacte-ria. In contrast to the small dimensions of an individualorganism, a spherical colony of 4000 organisms should havea diameter of m40 pum.

Competitive Interations of cat(+) and cat(-) E. col withPeri genic S ooci. The above results suggested that,in an environment contaminated by H202 generated by othermicroorganisms, caaase might be critical for the survival ofE. coli. Indeed, when an agar overlay containing E. coli ispoured onto established colonies of H202-releasing S. miltis,large zones ofE. coli growth inhibition are observed for cat(-)E. coli, whereas the cat(+) bacteria grow in close proximity tothe streptococcal colonies (Fig. 4). Even in liquid culture,there is aprofound difference in the ability ofcat(-) and cat(+)organisms to compete with these streptococci. Under theseconditions, cat(+) E. coli proliferate after a short lag period,despite the presence of numerous H202-producing strepto-cocci. By contrast, cat(-) E. coli fail to grow and eventuallydie (Fig. 5). This competitive advantage of cat(+) E. coli is

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FiG. 2. H202 killing of heterogeneous dilute (A) or concentrated(B) suspensions ofcat(+) and cat(-) E. coli. The two strains ofE. coliwere made resistant to ampicillin by pUC118 (Ampr) transformation.(A) Dilute suspensions (-5 x 102 cells per ml) of untransformedcat(+) and transformed cat(-) bacteria, or vice versa, were preparedin M9/glucose, and equal volumes of these suspensions were ad-mixed immediately before adding 1.0mM H202. After incubation at3rC for the indicated times, cat(+) and cat(-) survivors weredifferentiated by pour-plating in LB agar containing 100 units ofcatalase per ml with or without ampicillin (30 pg/ml). Identity of thecolonies was further confirmed by the H202 drop test (36). *,Untransformed cat(+); a, transformed cat(+); o, untransformedcat(-); o, transformed cat(-). (B) Survival of concentrated ("'5 x107 cells per ml) Ampr-transformed cat(-) E. coli alone (-) oradmixed with equal numbers of cat(+) E. coli (---). Each pointrepresents the mean of triplicate determinations, and vertical linesshow + 1 SD.

clearly related to catalase-dependent protection from environ-mental H202. When bovine catalase is added to the liquidcoculture system, even the cat(-) organisms survive andmultiply (Fig. 5). Furthermore, the ability of different speciesand strains of streptococci to suppress and kill coculturedcat(-) E. coli is directly correlated with their various H202-accumulating potentials (data not shown).

DISCUSSIONIt is difficult to imagine that the catalase activity within smallunicellular organisms might be an effective protection againstexogenous H202. In view of the highly diffusible nature ofthis oxidant, it is unlikely that intraorganismal catalase couldsustain an internal/external concentration gradient. Indeed,direct measurements of the "apparent" catalase activitywithin intact and lysed E. ccli yield very similar values.

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FIG. 3. Enhanced resistance of colonial cat(+) E. coli to H202killing. Known numbers (-500 colony-forming units per plate) ofcat(+) (o) or cat(-) (o) E. coli were pour-plated in 10 ml ofLB agar

and incubated at 370C. After various periods of preincubation, 20 mlof molten 1.5% Bactoagar containing freshly added H202 (finalconcentration, 1.0 mM) was overlaid on the LB agar-embeddedbacterial microcolonies. Surviving colonies were counted after 48 hrof incubation and expressed as percentage survival of control plateseither not exposed to an H202-containing overlay or supplementedwith catalase (100 units per ml). Data from one representativeexperiment are shown with error bars of + 1 SD from counts oftriplicate plates. The growth rates of cat(+) and cat(-) E. coli, as

measured by either turbidometry in brain heart infusion broth or

visually as colonies in LB agar, were identical.

Because the rate of reaction of catalase with H202 (at leastbetween 1 and 40 mM) is an almost linear function ofsubstrate concentration (12), this result indicates that thecatalase activity within intact E. coli cannot lower the effec-tive intracellular concentrations of H202 and, therefore,cannot prevent H202-mediated damage to the organism. Indirect support of this conclusion [but contrary to a number ofearlier reports (17-24)], we find that dilute suspensions ofcat(+) E. coli are as readily killed by bulk-phase H202 as are

FIG. 4. Preferential inhibition of the growth of cat(-) E. coli byH202-generating colonies of S. mitis. S. mitis was first culturedovernight in Todd-Hewitt broth at 370C in a candle extinction jar,then sparsely inoculated on presolidified Todd-Hewitt agar (1.5%)plates, and allowed to grow for 4 to -5 days, by which time thestreptococcal colonies were -3-4 mm in diameter. E. coli wereprepared as described in the legend for Fig. 1 and resuspended at=106 organisms per ml in molten M9 agar (1.5%) supplemented with50 mM glucose. Ten milliliters of E. coli-containing top agar wasoverlaid onto the plate bearing the streptococcal colonies. Afterovernight incubation at 370C in room air, clear growth-inhibitoryzones appear surrounding each streptococcal colony on the cat(-)plate (Left). In contrast, the plate overlaid with cat(+) E. coli (Right)shows no such zone of inhibition of E. coli. The important role ofH202 in this experimental system was confirmed by addition ofcatalase (100 units per ml) in the top agar, which prevented devel-opment of any detectable zone of inhibition (data not shown).

Time (hs)

FIG. 5. Competitive elimination qf cat(-) E. coli by S. mutansOMZ176. Early stationary-phase cultures of E. coli and S. mutanswere grown separately in M9/glucose and Todd-Hewitt broth, re-spectively, and used as inocula. Streptococci were inoculated intoprewarmed M9/50 mM glucose, at -5 x 109 colony-forming units(CFU) per ml (OD65o = 1.0), aerated (12 ml in 50-ml Erlenmeyerflasks shaken on rotatory platform at 135 rpm), and preincubated at37°C. After 60 min of preincubation, cat(+) or cat(-) E. coli wereinoculated at 5 x 107 organisms per ml, and their viability wasfollowed by pour-plating in LB agar supplemented with erythromy-cin (0.4 1ug/ml), which suppresses streptococcal but not E. coligrowth. e, cat(+) without catalase; o, cat(-) without catalase; ,

cat(+) with catalase; n, cat(-) with catalase. Each point representsthe results of triplicate determinations, and vertical lines show + 1SD. E. coli were inoculated at zero time when [H202] was 0.258 mM,which increased to 0.801 mM in cat(-) coculture in 9 hr. In thecontrol experiment, catalase (100 units per ml) was added immedi-ately before streptococcal inoculation, which completely preventedboth H202 accumulation and cytotoxicity to cat(-) E. coli.

their cat(-)-but otherwise isogenic-counterparts. Thisresult is very simply explained by the inability of organismalcatalase to (i) maintain an internal/external concentrationgradient and (ii) catabolically decrease the concentration ofthe bulk-phase H202.On the other hand, the catalase activity of cat(+) E. coli

does protect high-density or colonial bacteria from externalH202, whereas cat(-) E. coli show persistent sensitivity toH202, regardless of density. In these conditions, cat(+)organisms have high aggregate catalase activity, are ablerapidly to catabolize environmental peroxide, and, therefore,survive. Interestingly, this group protection can extend toneighboring cat(-) E. coli, lending further support to the ideathat catalase may function to protect groups of bacteria,rather than discrete, isolated organisms.

In the laboratory setting, bacteria are generally treated asunicellular organisms, whereas, in the natural world, manymicrobial species, including bacteria, live a more colonialexistence (38). Colonic E. coli, for example, are interspersedwith many other species of enteric flora in the form ofa densebiofilm adjacent the intestinal epithelial surface (39, 40). Innatural environments like this, one important source ofexogenous H202 might be that generated by competitivemicroorganisms of other genera (41-43). Streptococci andlactobacilli, found in significant numbers in human feces,generate substantial amounts of H202 (44). In fact, peroxi-dogenic streptococci, known to be frequent inhabitants oftheoral cavity, suppress growth of other microbial species(45-52).We have, therefore, studied interactions between cat(+)/

cat(-) E. coli and peroxidogenic streptococci. When an agaroverlay containing E. coli is poured onto established coloniesof H202-releasing S. mitis, large zones of E. coli growthinhibition are observed for cat(-) E. coli, whereas the cat(+)

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bacteria grow in close proximity to the streptococcal colo-nies. Even in liquid culture, there is a profound difference inthe ability of cat(-) and cat(+) organisms to compete withthese streptococci. Under these conditions, cat(+) E. coliproliferate after a short lag period, despite the presence ofnumerous H202-producing streptococci. By contrast, cat(-)E. coli fail to grow and eventually die. This competitiveadvantage of cat(+) E. coli is clearly related to catalase-dependent protection from environmental H202. When cat-alase is added to the liquid coculture system, even the cat(-)organisms survive and multiply (Fig. 5). Furthermore, theability of different species and strains of streptococci tosuppress and kill cocultured cat(-) E. coli is directly corre-lated with their various H202-accumulating potentials (datanot shown).

Unfortunately, the present work does not elucidate themechanism(s) whereby H202 is cytotoxic to E. coli. Earlierinvestigations suggest that low (1-3 mM) concentrations ofH202, such as those used iu our work, may cause lethality viatransition metal-mediated DNA damage (whereas this maynot be true of higher H202 concentrations) (53-55). If so,catalase-dependent group protection evidently worksthrough prevention of irreparable H202-mediated damage toorganismal DNA.

In summary, we find that one particular oxidant-defenseenzyme-catalase-does not protect individual E. coliagainst bulk-phase H202. However, highly concentrated orcolonial cat(+) E. coli are resistant to external H202, whereascat(-) E. coli show persistent sensitivity to H202, regardlessof density. From an evolutionary perspective, it is probablyimportant that the catalase status of E. coli determines theability of aggregates of these organisms to compete withH202-generating microbial competitors, such as strepto-cocci. Furthermore, rival bacteria are not the only source ofconcentrated exogenous H202 that may be encountered innature. When unicellular prokaryotes first populated thepristine surface water on precambrian Earth, the thin atmo-sphere likely allowed massive x-ray and UV radiation toinfiltrate these aquatic systems (56). Such intense irradiation,especially in the presence of green photoautotrophs andorganic molecules, may rapidly generate millimolar H202concentrations (1, 57-59). The catalase elaborated by smallunicellular organisms may protect the group-rather than theindividual-against attack by such environmental H202. Wespeculate that this enzymatic "safety in numbers" may havebeen an important driving force in the evolution of multicel-lular organisms.We thank Drs. 0. R. Brown, G. Germaine, P. C. Loewen, W. S.

Sauerbier, J. Haldane, and E. L. Thomas for providing materialsused in these studies and for offering helpful advice and also thankMs. Diane Konzen for manuscript preparation. This work wassupported by National Institutes of Health Grant AI-25625.

1. Frey, H. E. & Pollard, E. C. (1966) Radiat. Res. 28, 668-676.2. Peyton, G. R. & Glaze, W. H. (1987) in Photochemistry of Envi-

ronmentalAquatic Systems, eds. Zika, R. G. & Cooper, W. J. (Am.Chem. Soc., Washington), pp. 76-88.

3. Cooper, W. J. & Zika, R. G. (1983) Science 220, 711-712.4. Boveris, A. & Chance, B. (1973) Biochem. J. 134, 707-716.5. Foerder, C. A., Klebanoff, S. J. & Shapiro, B. M. (1978) Proc.

Nat!. Acad. Sci. USA 75, 3183-3187.6. Weiss, S. J. & LoBuglio, A. F. (1982) Lab. Invest. 47, 5-18.7. Dupuy, C., Virion, A., Ohayon, R., Kaniewski, J., Deme, D. &

Pommier, J. (1991) J. Biol. Chem. 266, 3739-3743.8. Jones, D. P., Eklow, L., Thor, H. & Orrenius, S. (1981) Arch.

Biochem. Biophys. 210, 505-516.9. Starke, P. E. & Farber, J. L. (1985) J. Biol. Chem. 260, 10099-

10104.10. Gaetani, G. F., Galiano, S., Canepa, L., Ferraris, A. M. & Kirk-

man, H. N. (1989) Blood 73, 334-339.11. Eaton, J. W. (1991) J. Lab. Clin. Med. 118, 3-4.12. Chance, B. (1949) J. Biol. Chem. 179, 1311-1330.

13. Penninckx, M. J. & Jaspers, C. J. (1982) Bull. Inst. Pasteur 80,291-301.

14. Greenberg, J. T. & Demple, B. (1986) J. Bacteriol. 168, 1026-1029.15. Jacobson, F. S., Morgan, R. W., Christman, M. F. & Ames, B. N.

(1989) J. Biol. Chem. 264, 1488-1496.16. Sample, A. K. & Czprynski, C. J. (1991) Infect. Immun. 59, 2239-

2244.17. Yoshpe-Purer, Y. & Henis, Y. (1976) Appl. Environ. Microbiol. 32,

465-469.18. Filice, G. A. (1983) J. Infect. Dis. 148, 861-867.19. Barbado, C., Ramirez, M., Blanco, M. A., Lopez-Barea, J. &

Pueyo, C. (1983) Curr. Microbiol. 8, 251-253.20. Gregory, E. M. & Fanning, D. D. (1983) J. Bacteriol. 156, 1012-

1018.21. Loewen, P. C. (1984) J. Bacteriol. 157, 622-626.22. Meir, E. & Yagil, E. (1984) Curr. Microbiol. 11, 13-18.23. Mauel, J. (1990) J. Leukocyte Biol. 47, 187-193.24. Abril, N. & Pueyo, C. (1990) Environ. Mol. Mutagen. 15, 184-189.25. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular

Cloning: A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, NY), 2nd Ed., Vol. 3, p. A3.

26. Rorth, M. & Jensen, P. K. (1967) Biochim. Biophys. Acta 139,171-173.

27. Pick, E. & Keisari, Y. J. (1980) Immunol. Methods 38, 161-170.28. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,

Gartner, F. H., Provenzana, M. D., Fujimoto, E. K., Goeke,N. M., Olson, B. J. & Klenck, D. C. (1985) Anal. Biochem. 150,76-85.

29. Engel, M. S. & Adler, H. I. (1961) Radiat. Res. 15, 269-275.30. White, D. C. (1962) J. Bacteriol. 83, 851.31. Herbert, D. & Pinsent, J. (1948) Biochem. J. 43, 193-202.32. Clayton, R. K. (1959) Biochim. Biophys. Acta 36, 35-39.33. Howard, D. H. (1983) Infect. Immun. 39, 1161-1166.34. Yoshpe-Purer, Y., Henis, Y. & Yashphe, J. (1977) Can. J. Micro-

biol. 23, 84-91.35. Hassan, H. M. & Fridovich, I. (1978) J. Biol. Chem. 253, 6445-

6450.36. McLeod, J. W. & Gordon, J. (1922) Biochem. J. 16, 499-506.37. Woldringh, C. L. & Nanninga, N. (1985) in Molecular Cytology of

Escherichia coli, ed. Nanning, N. (Academic, New York), pp.161-197.

38. Shapiro, J. A. (1988) Sci. Am. 259, 82-89.39. Savage, D. S. (1980) in Bacterial Adherence, ed. Beachey, E. H.

(Chapman & Hall, London), pp. 31-59.40. Hamilton, W. A. (1987) in Ecology ofMicrobial Communities, eds.

Fletcher, M., Gray, T. R. G. & Jones, J. G. (Cambridge Univ.Press, New York), pp. 361-385.

41. McLeod, J. W., Gordon, J. & Pyrah, L. N. (1923) J. Pathol.Bacteriol. 26, 127-128.

42. Cook, F. D. & Quadling, C. (1962) Can. J. Microbiol. 8, 933-935.43. Thomas, E. L. (1985) in The Lactoperoxidase System: Chemistry

and Biological Significance, eds. Pruitt, K. M. & Tenovuo, J. 0.(Dekker, New York), pp. 179-202.

44. Drasar, B. S. (1988) inRole ofthe Gut Flora in Toxicity and Cancer,ed. Rowland, I. R. (Academc, New York), pp. 23-38.

45. Kraus, F. W., Nickerson,J. F., Perry, W. I. & Walker, A. P. (1957)J. Bacteriol. 73, 727-735.

46. Holmberg, K. & Hollander, H. 0. (1973) Arch. Oral Biol. 18,423-434.

47. Germaine, G. & Tellefson, L. M. (1982) Infect. Immun. 38, 1060-1067.

48. Thomas, E. L. & Pera, K. A. (1983) J. Bacteriol. 154, 1236-1244.49. Donoghue, H. D., Perrons, C. J. & Hudson, D. E. (1985) Arch.

Oral Biol. 30, 519-523.50. Allen, B. W. (1985) J. Med. Microbiol. 19, 227-235.51. Carlsson, J., Edlund, M.-B. K. & Lundmark, S. K. E. (1987) Oral

Microbiol. 2, 15-20.52. Hillman, J. D. & Shivers, M. (1988) Arch. Oral Biol. 33, 395-401.53. Imlay, J. A. & Lin, S. (1987) J. Bacteriol. 169, 2967-2976.54. Greenberg, J. T. & Demple, B. (1988) EMBO J. 7, 2611-2617.55. Imlay, J. A. & Linn, S. (1968) Science 240, 1302-1309.56. Levine, J. S. (1985) in The Photochemistry ofAtmospheres, Earth,

the Other Planets, and Comets, ed. Levine, J. S. (Academic, NewYork), pp. 3-38.

57. McCormick, J. P., Fischer, J. R., Pachlatko, J. P. & Eisenstark, A.(1976) Science 191, 468-469.

58. Wang, R. J. & Nixon, B. T. (1978) In Vitro 14, 715-722.59. Zepp, R. G., Skurlatov, Y. I. & Pierce, J. T. (1987) in Photochem-

istry ofEnvironmentalAquatic Systems, eds. Zika, R. G. & Cooper,W. J. (Am. Chem. Soc., Washington), pp. 215-224.

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