Vol. 141, No. 1JouRNAL OF BACTERIOLOGY, Jan. 1980, p. 156-1630021-9193/80/01-0156/08$02.00/0

Mechanism of the Antibiotic Action of PyocyanineH. MOUSTAFA HASSANt AND IRWIN FRIDOVICH*

Department ofBiochemistry, Duke University Medical Center, Durhamr, North Carolina 27710

Exposure of Escherichia coli growing in a rich medium to pyocyanine resultedin increased intracellular levels of superoxide dismnutase and of catalase. Whenthese adaptive enzyme syntheses were prevented by nutritional paucity, the toxicaction of pyocyanine was augmented. The antibiotic action of pyocyanine wasdependent upon oxygen and was dimiished by superoxide dismutase and bycatalase, added to the suspending medium. Pyocyanine slightly augmented therespiration of E. coli suspended in a rich medium, but greatly increased thecyanide-resistant respiration. Pyocyanine was able to cause the oxidation ofreduced nicotinamide adenine dinucleotide, with 02- production, in the absenceof enzymatic catalysis. It is concluded that pyocyanine diverts electron flow andthus increases the production of 02 and H202 and that the antibiotic action ofthis pigment is largely a reflection of the toxicity of these products of oxygenreduction.

Pseudomonas aerugunosa is a widely distrib-uted opportunistic pathogen. It secretes proteinswhich are toxic to a wide range of organisms(23), and, when grown aerobically in a phos-phate-poor medium, it also secretes copiousamounts of phenazine pigments (3, 8, 30). Phos-phate depletion within P. aeruginosa has beensuggested to be the initiator of phenazine bio-synthesis (8, 18, 19). The major phenazine pro-duced by this organism is pyocyanine (1-hy-droxy-5-methyl phenazine), and its physiologicalsignificance has long been a mystery (35).

Interest in pyocyanine derives from its intensecolor, which makes its presence so obvious, fromits antibiotic properties (22, 31, 34, 37), and fromthe correlation between its production and path-ogenicity (20). Furthermore, the antibiotic cy-anomycin from Streptomyces cyanoflavus (12)has been identified as pyocyanine (33). Pyocy-anine can accept a single electron, yielding arelatively stable anion radical (28, 29), and read-ily undergoes a redox cycle (6, 9, 11, 26, 28, 29,38). We have previously noted that pyocyanineinduced the biosynthesis of the manganese-con-taining superoxide dismutase (MnSOD) in Esch-erichia coli and we inferred that it caused en-hanced production of 02- (17, 17a; H. M. Hassanand I. Fridovich, in T. E. King, H. S. Mason,and M. Morrison, ed., Oxidases and RelatedRedox Systenm, in press). It appeared possiblethat the antibiotic action of pyocyanine mightactually be an expression of the toxicity of the02 and of H202 produced in increased amounts

t Present address: Department of Microbiology, McGillUniversity, Montreal, Quebec, Canada.

in its presence. The results reported belowstrongly suggest this possibility.

MATERIALS AND METHODSE. coli B BI-2 (ATCC 29682) was grown aerobically

at 200 rpm and at 370C in a glucose-salts medium ±0.5% yeast extract or in Trypticase-soy-yeast extract(TSY), as previously described (17). Growth was mon-itored turbidimetrically at 500 nm, rather than at 600nm, to minimize interference from the optical absorb-ance of pyocyanine. Anaerobic growth was performedin cuvettes which allowed the medium to be scrubbedfor 2 h with a stream of pure N2, prior to sealing andinoculation (17). Specific growth rates and generationtimes were calculated as described before (14). Anti-biotic effects were assessed by a filter paper diskmethod. Sterile Whatman no. 1 disks, loaded withgraded amounts of pyocyanine, were placed onto aglucose-minimal medium seeded with E. coli and so-lidified with 0.7% agar. This soft agar rested upon alayer of glucose-minimal medium solidified with 2%agar. Test cultures were incubated for 24 h at 37°C,either aerobically or in a Coy anaerobic chamber,whose atmosphere contained less than 5 id of dioxygenper liter.

P. aeruginosa ATCC 9027 was grown at 370C andat 200 rpm in a phosphate-limited medium (19) whichwas modified to contain, per liter. DL-alanine, 8.0 g;L-leucine, 8.0 g; lithium lactate, 3.0 g; MgSO4, 2.0 g;K2HPO4, 0.1 g; FeSO4.7H20, 0.01 g; and Tris, 6.1 g.When phosphate excess was desired it was achievedby adding K2HPO4 to 2% (wt/vol). In all cases themedium was adjusted to pH 7.1.

Soluble extracts were prepared (13) and assayed forsuperoxide dismutase (SOD) (27) and for SOD isoen-zymes (13) as previously described (1). Catalase wasassayed by the method of Beers and Sizer (2), and E.coli hydroperoxidases were separated by polyacryl-amide gel electrophoresis and visualized as described

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PYOCYANINE AND OXYGEN TOXICITY 157

before (16). Protein was estimated by the Lowrymethod (24). Respiration of cell suspension was meas-ured polarographically with and without pyocyanine± cyanide (17a). Rates of 2- production were meas-ured in terms of SOD-inhibitable reduction of nitroblue tetrazolium (1). Pyocyanine from Phann-Eco-Labs, Inc. of Simi Valley, Calif., was twice recrystal-lized according to Frank and DeMoss (8) and was

assayed based upon EI"Cm - 117 at 520 nm in 0.2 NHCI (25).

RESULTSNutritional modification of pyocyanine

toxicity. Paraquat causes enhanced productionof 02 within E. coli, and the cells adapt byincreased synthesis of MnSOD (15, 17). Anycondition that interfered with rapid protein syn-

thesis prevented this adaptive enzyme inductionand enhanced the lethality of paraquat (17). E.coli cells were thus much more susceptible toparaquat lethality in a glucose-salts mediumthan they were in richer media (17). If pyocy-

anine action also depends upon increased pro-

duction of 02 and H202, then E. coli should bemore susceptible to its antibiotic action in a

minimal medium which limits the rate ofMnSOD synthesis. Figure 1 demonstrates thatthe growth of E. coli was suppressed by pyocy-

anine in a glucose-minimal medium and thataddition of yeast extract markedly diminishedthis growth suppression. The toxicity of pyocy-anine was similarly minimized in a TSY me-

dium. Waksman and Woodruff (34) had previ-ously noted that the antibiotic action of pyocy-anine was dependent upon the growth medium,but did not offer an explanation.The ability of pyocyanine to induce the syn-

thesis ofSOD and of catalase in E. coli growingin a glucose-miniimal medium ± 0.5% yeast ex-tract was explored. There was no induction ofSOD in the minimal medium (Table 1). Supple-mentation with yeast extract permitted markedinductions of both SOD and catalase activities,with the catalase level going up more dramati-cally than the SOD level at the higher levels ofpyocyanine. As expected, the effect of pyocy-anine on the generation time (G) was the inverseof its effects on SOD and catalase. Thus, wheninduction of these protective enzymes was pre-vented by the nutritional paucity ofthe medium,pyocyanine markedly increased the generationtime. In contrast, when enrichment of the me-

dium with yeast extract permitted large induc-tion of the protective enzymes, the growth ratewas much less affected by pyocyanine.Dioxygen dependence ofpyocyanine tox-

icity. Biological production of 02 and H202 ispossible only in the presence of dioxygen. If the

*v v

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GLUCOSE MINIMAL

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I

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FIG. 1. Effects of media composition on pyocy-anine toxicity. E. coli B cells, grown aerobically tolate logarithmic growth phase in a glucose-minimalmedium, were diluted to an initial absorbance of0.16at 500 nm into fresh media containing the indicatedconcentrations of pyocyanine. Yeast extract (0.5%)was added to the glucose-minimal medium whereindicated. The cultures were incubated at 370C on arotary water-bath shaker at 200 rpm. Growth wasmonitored in terms of absorbance at 500 nm. Absorb-ances greater than 1.0 were measured upon dilutedsamples with correction for the dilution factor.

TABLE 1. Correlation between growth of E. coli Bin different media in the presence ofpyocyanine

with SOD and catalase synthesisaGlucose-minimal me- Glucose-minimal me-

dium dium + YEPyocy-anine SOD Cata- SOD Cats-(M) 0 (U/ lawe G (U! lase

(min)m) (U! (minm n) (U/mg) mg)

0 43 6.9 12.0 28 9.6 20.11 X 10-6 49 4.6 9.7 - - -

5 X 10-6 60 3.4 12.3 33 11.2 22.41 X 105 145 4.1 26.4 38 13.0 27.15 x 10-5 c - - 42 20.8 257.81 x 10-4 00 - - 48 34.2 358.4

a CelLs were from the experiment reported in Fig. LCells were sonicated, and dialyzed cell-free extractswere assayed for SOD and catalase activities. YE,Yeast extract; G, generation time. -, No data.

antibiotic action of pyocyanine is due to in-creased production of 02- and H202, then itsadverse effects on E. coli should be dioxygen

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158 HASSAN AND FRIDOVICH

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FIG. 3. Effect of anaerobiosis on the antibiotic ac-tion ofpyocyanine. E. coli B cells from a 24-h culture(stationary-phase cells) growing in glucose-minimalmedium were incorporated into the soft agar overlayand used to seed the glucose minimal agar plates.Sterile filter paper disks containing (disk 1) 0, (disk2) 4.2, (disk 3) 8.4, or (disk 4) 21 pg ofpyocyanine wereplaced on the top of the solidified soft agar. One setofplates was incubated aerobicaly, and another set

4 5 ofplates was incubated anaerobically in a Coy cham-ber. The plates were incubated for 24 to 48 h at 37°Cbefore the results were recorded.

FIG. 2. Effects of anaerobiosis on the toxicity ofpyocyanine. Special anaerobic cuvettes, filled withthe glucose-minimal medium plus the indicated con-centrations ofpyocyanine, were flushed with a streamof oxygen-free nitrogen for 2 h prior to sealing andinoculation by tipping in the inoculum from the sidearm. The inocula were from a late-log culture grownanaerobically in glucose-minimal medium. The cu-vettes were incubated at 37°C, and growth was mon-itored at 5X nm. g, Generation time in minutes (').

dependent. That this was the case is docu-mented by the data in Fig. 2 and 3. Figure 2presents the growth of E. coli in anaerobic glu-cose-minimal medium in the absence and in thepresence of 0.1 mM pyocyanine. This level ofpyocyanine, which completely suppressedgrowth in aerobic minimal medium (Fig. 1),caused only a slight increase in the generationtime in the anaerobic medium. Indeed, most ofthe effect of pyocyanine seen in the anaerobiccuvettes (Fig. 2) was probably due to residualtraces of dioxygen. In Fig. 3 the antibiotic actionof pyocyanine, by the filter paper disk assay, wascompared aerobically and anaerobically. Theantibiotic activity, seen aerobically, vanished inthe absence of dioxygen.

Effects of pyocyanine on SOD and cata-lase isoenzymes. E. coli contains three formsof SOD (13) and two hydroperoxidases (16).Thus, there are superoxide dismutases basedupon iron (FeSOD) (36) and upon manganese

(MnSOD) (21), and there is a hybrid which,although containing iron, is composed of onesubunit from FeSOD and one subunit fromMnSOD (7). Previous work has shown that theFeSOD is constitutive, being present even in

anaerobically grown cells, whereas the MnSODis under repression control and is made in re-

sponse to intracellular 02 production. Both ofthe hydroperoxidases of E. coli are effectivecatalases, but hydroperoxidase I is also an activegeneral peroxidase (4). Aerobic growth in thepresence of pyocyanine led to a marked increasein MnSOD with a concomitant slight decreasein FeSOD (Fig. 4A). This is consistent withprevious work in which the intracellular O2

production was raised by a variety of conditions(17a). The slight decrease in FeSOD probablyresults from a partial depletion of FeSOD, dueto greater production of the hybrid SOD in thepresence of higher levels of the MnSOD. Bothhydroperoxidases were increased during aerobicgrowth in the presence of pyocyanine (Fig. 4B;Table 1). Pyocyanine was thus qualitatively sim-ilar to paraquat in its effects on SOD and cata-lase in E. coli. There were, however, quantitativedifferences. Paraquat caused a relatively greaterinduction of SOD (15), whereas pyocyaninecaused a relatively greater induction of hydro-peroxidases.

Effects of pyocyanine on cell respiration.E. coli, grown for 2 h in aerobic TSY medium,was collected by centrifugation, suspended in 50mM potassium phosphate-0.1 mM MgSO4 atpH 7.0, and then placed under a Clark oxygenelectrode. Such cells respired vigorously whenTSY medium was added, and the respirationwas largely inhibited by 2mM cyanide (Fig. 5A).Pyocyanine, added after the cyanide, relievedthat inhibition. Figure 5b shows the rate of

GLUCOSE - MINIMAL(anaerobic)

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PYOCYANINE AND OXYGEN TOXICITY 159

a A

a

I

bB

____---HP I

___H P U

FIG. 4. (A) Effect ofpyocyartine on the synthesis ofSOD isozymes. E. coli B was grown aerobically for 3 hin TSY medium with and without 0.1 mMpyocyanine. Dialyzed cell-free extracts wereprepared, and samplescontaining equal amounts ofproteins (250 pg) were applied to 10%polyacrylamide gels. After electrophoresisthe gels were stained for activity. Gel a was obtained from the extracts of cells grown in the absence ofpyocyanine, whereas gel b was from the corresponding culture in its presence. (B) Effect ofpyocyanine on thesynthesis of catalase isozymes. E. coli B was grown for 3 h in glucose-minimal medium containing 0.5% yeastextract with and without 0.1 mMpyocyanine. Gel a, 500 pug ofprotein from extracts of cells grown in absenceofpyocyanine; gel b, 250 pg ofprotein from the corresponding culture grown in its presence.

respiration, ±2 mM cyanide, as a function ofpyocyanine. Pyocyanine slightly augmented nor-mal respiration, but grossly increased the cya-

nide-resistant respiration. Pyocyanine has beenreported to increase the respiration of mamma-lian cells (10, 32) and to reverse the cyanideinhibition of respiration (5).Mechanism of 02- generation by pyocy-

anine. E. coli contains a soluble diaphorase,which catalyzes the reduction of paraquat byNADPH. This diaphorase is presumed to be thesite of diversion of intracellular electron flow byparaquat (17b). Paraquat has been shown togenerate O2 with NADPH as the electron donoronly if this diaphorase is present (17b). In con-

trast, pyocyanine appears capable of direct in-teraction with NADH. Thus, pyocyanine cata-lyzed the reduction of nitro blue tetrazolium byNADH, and this reduction was largely inhibitedby SOD (Fig. 6). Phenazine methosulfate hasbeen reported to exert a similar effect (30).Extracellular events in the antibiotic ac-

tion of pyocyanine. Most of the paraquat re-

duced within E. coli reacts with dioxygen togenerate O2 before it can diffuse from the cell.This is a consequence of the fact that its reactionwith dioxygens is extremely rapid, i.e., k = 7.7

x 108 M-1 sec'. Some paraquat radical does,however, diffuse from the cell, especially whenrapid respiration depletes intracellular dioxygen.Under such conditions extracellular 02 produc-tion occurs by reoxidation of the effused para-quat radical (17b). The E. coli cell envelopeappears to be impermeable to °2, and althoughextracellular 02- can damage the cell, it cannotcause induction of MnSOD synthesis (17b). Ifreduced pyocyanine were to react with dioxygenmore slowly than does the paraquat radical, itwould escape from the cell to a greater degreeand thus lead to more extracellular 02 produc-tion. Extracellular 02 would there dismute, giv-ing rise to H202, which could enter the cell andstimulate the synthesis of catalase. This couldexplain the observation that pyocyanine inducescatalase more than SOD, whereas paraquat in-duces SOD more than catalase. Extracellular02 and H202 could also make a large contribu-tion to the lethality of pyocyanine. Either SODor catalase added to the medium was able topartially eliminate the antibiotic effect of pyo-cyanine, and SOD plus catalase was even moreeffective (Fig. 7). These protective effects byexogenous SOD and catalase indicate that extra-cellular O2 and H202 were important factors in

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160 HASSAN AND FRIDOVICH

22 MjI min.

A

TI ME0 0-02 004 0(06 008 010

(PYOCYANINE), mM

FIG. 5. (A) Effect ofpyocyanine on the rate of cyanide-resistant respiration in E. coli. Cells were grownaerobically for 2 h at 37°C in TSY medium, collected by centrifugation, and suspended in 0.05 Mpotassiumphosphate buffer (pH 7.0) containing I mM MgSO4. The reaction mixture contained 0.05 ml of ceUs (0.56mg[dry weight] of cells), 0.1 ml offresh TSY, and 50mMpotassium phosphate (pH 7.0) to a final volume of2 ml.Where indicated, cyanide was added to a 2.0 mM final concentration. Pyocyanine was added to 0.05 mMafter inhibition by CN was established. (B) Effects ofpyocyanine on cyanide-sensitive and cyanide-resistantrespiration. Conditions were the same as in (A) except that different concentrations ofpyocyanine were tested.Oxygen uptake is presented in the ordinate as nanoatoms of 02 consumed per minute per milligram (dryweight) of cells.

the antibiotic action of pyocyanine.Atother way to account for the relatively

greater induction of catalase than of SOD bypyocyanine would be to propose that it under-goes a predominantly divalent redox cyclewithin the cell. Although this remains possible,it could not account for the protective effects ofextracellular SOD and of catalase, shown in Fig.7.Pyocyanine and P. aeruginosa If P.

aeruginosa uses pyocyanine production to itsadvantage in competing with other bacteria inthe same ecological habitat, it must thereforehave a mechanism to insure its own protectionor immunity against the bactericidal agent itproduces. This immunity could be via higherconcentrations of SOD and catalase or by lack

of permeability. We tested these possibilities. P.aeruguinsa made 62% higher catalase whengrown under conditions conducive for pyocy-anine production (Table 2). However, the levelof SOD was slightly lower. We also checked theeffect of pyocyanine on the rate of cyanide-re-sistant respiration in P. aeruginosa and found itto be nonresponsive to pyocyanine. The respi-ration of P. aeruginosa was generally resistantto cyanide; thus, 8 to 10 mM was required toinhibit the respiration by 91.3%, and 0.134 mMpyocyanine caused this cyanide-resistant respi-ration to rise from 8.7 to 14.8%. This increase isvery moderate compared to that seen in E. coli(Fig. 5). These results tentatively indicate thatP. aeruginosa is not as permeable to pyocyanineas is E. coli, and they show that the organism

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PYOCYANINE AND OXYGEN TOXICITY 161

E0 o.0i

2 min

TIME

FIG. 6. Pyocyanine and the generation of super-oxide radical (02-). The ability ofpyocyanine to gen-erate 02- was estimated in terms ofSOD-inhibitablereduction of nitro blue tetrazolium. Reaction mix-tures contained 0.44 mM NADH, 0.16mM nitro bluetetrazolium, 40 uMpyocyanine, ±10upg of the bovinecopper-zinc SOD, and 50 mM potassium phosphate(pH 7.8) containing 0.1 mM EDTA, to a total volumeof3 ml. nitro blue tetrazolium reduction was foUowedat 560nm and 25°C in a double-beam spectrophotom-eter in cuvettes with 1-cm path.

makes higher catalase to protect against H202that might be generated outside the cells viaextracellular autooxidation of pyocyanine. Wepresume that P. aeruginosa actively secretespyocyanine while keeping its intracellular levellow by a combination of low permeability andactive extrusion.

DISCUSSIONPyocyanine increases the cyanide-resistant

respiration of E. coli and causes increases in thebiosynthesis of SOD and of catalase. We inferthat pyocyanine can divert the electron flowwithin this organism from the normal cyto-chrome pathway to an 02-- and H202-producingpathway. Since pyocyanine, per se, catalyzes theSOD-inhibitable reduction of nitro blue tetra-zolium by NADH, it appears that direct reduc-tion of pyocyanine by NADH provides themeans for this diversion of the electron flow.The antibiotic action of pyocyanine appears tobe largely due to the toxicity of the 02 andH202 it engenders. Thus, (i) molecular oxygen is

TABLE 2. SOD and catalase activity in P.aeruginosa ATCC 9027"

Growth condition SOD (U/ Catalanemg) (U/mg)

Phosphate limited 38.1 541.8Phosphate unlimited 43.3 333.8

a p. aeru,ginosa were grown for 36 h in minimalmedium (4) at 37°C and 200 rpm.

essential for expression of this antibiotic action;(ii) rapid induction of SOD and catalase in richmedia markedly decreases the toxicity ofaerobicpyocyanine; and (iii) addition of SOD and cata-lase to the suspending medium provides protec-tion against the antibiotic effect.The quantitative differences between pyocy-

anine and paraquat can be explained in terms ofdifferences in rates of reaction of their univa-lently reduced forms with dioxygen. If the pyo-cyanine radical reacted more slowly than theparaquat radical it would, to a greater extent,escape from the cell before giving rise to 02.Hence a greater fraction of02 production wouldbe extracellular with pyocyanine than was thecase with paraquat. Since E. coli appears to beimpermeable to 02, this would have the conse-quence that H202, generated by dismutation of02 in the medium, would reenter the cell andcause induction of catalase. In toto, one wouldthen see greater induction of catalase than ofSOD with pyocyanine and the reverse with pa-raquat. Because the paraquat radical reacts with02 at an almost diffusion-limited rate, it is easyto imagine that the corresponding reaction ofthe pyocyanine radical is slower. Altematively,one could propose that the autooxidation ofreduced paraquat gives rise mostly to 02,whereas the autooxidation of reduced pyocy-anine gives rise mostly to H202. We do not, atpresent, have the data for definitely distinguish-ing between the explanations, but the markedprotection offered by exogenous SOD and cata-lase certainly supports the first proposal.What advantages are gained for P. aeruginosa

by the secretion of pyocyanine? When growingin the presence of other cells this organism couldgain nutrients by eliminating competition andcould even gain access to the nutrients alreadylocked up inside the other cells, by killing them.The fact that starvation for phosphate triggerspyocyanine production supports this interpre-tation. Dioxygen, which is essential for themechanism of antibiotic action supported by ourwork, is also needed for production of pyocy-anine. P. aeruginosa thus makes pyocyaninewhen phosphate deficiency indicates the need toeliminate competing cells and when dioxygen is

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162 HASSAN AND FRIDOVICH

FIG. 7. Effects ofexogenously added SOD and catalase on the toxicity and antibiotic action ofpyocyanine.E. coli B cells from 24-h culture (stationary-phase cells) growing in glucose-minimal medium were used toseed the glucose-minimal agar plates. Catalase (0.21 mg per plate) and copper-zinc SOD (0.5 mg per plate)were incorporated with E. coli cells into the soft agar overlay. Sterile filter-paper disks containing differentconcentrations ofpyocyanine were placed on the top of the solidified soft agar. Plates were incubated in airat 37°C for 24 to 48 h before recording the results. (Disk 1) 0, (disk 2) 8.4, (disk 3) 21, and (disk 4) 42 pg ofpyocyanine. (A) Control; (B) SOD; (C) catalase; (D) SODplus catalase.

present to support the antibiotic action of thisdye.

ACKNOWLEDGMENTSThis work was supported by research grants from the

National Institutes of Health (Public Health Service grantGM-10287) and from the United States Army Research Office,Research Triangle Park, N.C. (DAAG 29-78-6-0088).

LlTERATURE CIMD1. Beauchamp, C., and I. Fridovich. 1971. Superoxide

dismutase: improved assays and an assay applicable topolyacrylamide gels. Anal. Biochem. 44:276-287.

2. Beers, R. F., and I. W. Sizer. 1952. A spectrophotometricmethod for measuring the breakdown of hydrogen per-oxide by catalase. J. Biol. Chem. 195:133-140.

3. Chang, P. C., and A. C. Blackwood. 1969. Simultaneousproduction of three phenazine pigments by Pseudomo-nas aeruginosa Mac 436. Can. J. Microbiol. 15:439-444.

4. Claiborne, A., and L Fridovich. 1979. Purification ofthe o-dianisidine peroxidase from Escherichia coli B.J. Biol. Chem. 254:4245-4252.

5. DeMeio, R. H., M. Kissin, and K. S. G. Barron. 1934.Studies on biological oxidation. IV. On the mechanism

of the catalytic effect of reversible dyes on cellularrespiration. J. Biol. Chem. 107:579-590.

6. Dickens, F., and H. McIlwain. 1938. Phenazine com-pounds as carriers in the hexose monophosphate sys-tem. Biochem. J. 32:1615-1625.

7. Dougherty, H. W., S. J. Sadaowski, and E. E. Baker.1978. A new iron-containing superoxide dismutase fromEscherichia coli. J. Biol. Chem. 254:5220-5223.

8. Frank, L. H., and R. D. DeMoss. 1959. On the biosyn-thesis of pyocyanine. J. Bacteriol. 77:776-782.

9. Friedheim, E. A. H. 1931. Pyocyanine, an accessoryrespiratory enzyme. J. Exp. Med. 54:207-221.

10. Friedheim, E. A. H. 1934. The effect of pyocyanine onthe respiration of some normal tissues and tumors.Biochem. J. 28:173-179.

11. Friedheim, E., and L. Michaelis. 1931. Potentiometricstudy of pyocyanine. J. Biol. Chem. 91:355-368.

12. Funaki, M., F. Tsuchiya, K. Maeda, and T. Kamiya.1958. Cyanomycin, a new antibiotic. J. Antibiot. Ser. A11:143-149.

13. Hassan, H. M., and I. Fridovich. 1977. Enzymatic de-fenses against the toxicity of oxygen and ofstreptonigrinin Escherichia coli. J. Bacteriol. 129:1574-1583.

14. Hassan, H. M., and I. Fridovich. 1977. Physiologicalfunction of superoxide dismutase in glucose-limitedchemostat cultures of Escherichia coli. J. Bacteriol.130:805-811.

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PYOCYANINE AND OXYGEN TOXICITY 163

15. Hassan, H. M., and I. Fridovich. 1977. Regulation ofthe synthesis of superoxide dismutase in Escherichiacoli: induction by methyl viologen. J. Biol. Chem. 252:7667-7672.

16. Hassan, H. M., and I. Fridovich. 1978. Regulation ofthe synthesis of catalase and peroxidase in Escherichiacoli. J. Biol. Chem. 253:6445-6450.

17. Hassan, H. KL, and I. Fridovich. 1978. Superoxide rad-ical and the oxygen enhancement of the toxicity ofparaquat in Escherichia coli. J. Biol. Chem. 253:8143-8148.

17a.Hassan, H. KL, and I. Fridovich. 1979. Intracellularproduction of superoxide radical and of hydrogen per-

oxide by redox active compounds. Arch. Biochem. Bio-phys. 196:385-395.

17b.Hassan, H. M., and I. Fridovich. 1979. Paraquat andEscherichia coli: mechanism of production of extracel-lular superoxide radical. J. Biol. Chem. 254:10846-10852.

18. Ingledew, W., and J. J. RI CampbeLL 1969. A new

resuspension medium for pyocyanine production. Can.J. Microbiol. 15:595-598.

19. Ingram, J. M., and A. C. Blackwood. 1962. Studies onthe biosynthesis of pyocyanine. Can. J. Microbiol. 8:49-56.

20. Ingram, J. M., and A. C. Blackwood. 1970. Microbialproduction of phenazines. Adv. Appl. Microbiol. 18:267-282.

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