Vol. 160, No. 3

Relaxation of Supercoiled Plasmid DNA by Oxidative Stresses inEscherichia coli

HIROYUKI HORIUCHI, MASAMICHI TAKAGI,* AND KEIJI YANO

Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo, 113, Japan

Received 19 July 1984/Accepted 17 September 1984

The relaxation of plasmid DNA was observed after the visible light irradiation of Escherichia coli AB1157harboring plasmid pBR322 or some other plasmids in the presence of a photosensitizing dye, such as toluidineblue or acridine orange, and molecular oxygen. Treatment of the cells with hydroperoxides, such as tert-butylhydroperoxide, cumene hydroperoxide, and hydrogen peroxide, also caused the plasmid DNA relaxation invivo. Relaxation was not observed in these treatments of purified pBR322 DNA in vitro. Plasmid DNArelaxation was also detected after near-UV irradiation. Far-UV irradiation did not induce such relaxation.

Ionizing radiation, cellular respiration, some enzymaticreactions, degradation of lipid peroxides, and photodynamicaction all produce active oxygen molecules, such as singletoxygen, superoxide anion, hydrogen peroxide, and hydroxylradical, inside or outside the cell. In bacterial cells, it hasbeen reported that active oxygen molecules not only kill thecells but cause mutations (13), and that a family of adenylat-ed dinucleotides such as AppppA is accumulated by oxidativestresses induced by tert-butyl hydroperoxide and hydrogenperoxide (12). Active oxygen mnolecules cause various dam-ages, such as lipid peroxidation, protein oxidation, protein-DNA cross-linkage, and DNA strand breaks (1, 8), butwhich damage is most important in cell killing is unclear.

In our laboratory, cell killing in Escherichia coli has beenstudied in cells treated with visible light in the presence of aphotosensitizing dye, toluidine blue. Toluidine blue pluslight treatment of E. coli causes damage on the cell surface(26), and the dvl gene (which is located at 7.7 min on the E.coli chromosome map) confers resistance to the cells againstthis treatment (27).

In this paper, we describe the effects of photodynamicaction and hydroperoxides on plasmid DNA in vivo. Since ithas been suggested (19, 25) that the lethal effect of near-UVradiation (290 to 400 nm) is related to radicals derived fromoxygen, we examined the effects of near-UV and, as anegative control, far-UV (254 nm) on plasmid DNA in vivo.When the cells were exposed to photodynamic action,treated with hydroperoxides, or irradiated by near-UV,plasmid DNA relaxation was observed as the cell survivaldecreased.

MATERIALS AND METHODSBacterial strains and culture condition. The strains of E.

coli K-12 used in this study were AB1157 argE3 his4 leuB6proA2 thr-J rpsL31 galK2 lacY1 xyl-5 mtl-l ara-14 supE4 thi-1 and DM800 A(topA-cysB)204 acrA13 gyrB225, which was a

kind gift from Y. Hirota (National Institute of Genetics,Mishima, Japan) (5, 18, 20). Cells were grown in L medium(1% tryptone [Difco Laboratories], 0.5% yeast extract, 0.5%NaCl [pH 7.0]) at 37°C and were harvested at stationaryphase, washed twice with potassium phosphate buffer (50mM, pH 7.0), and suspended in the same buffer.

Plasmids. Multi-copy-number plasmids used were pBR322

* Corresponding author.

(2), pBR313 (2), or pACYC177 (2). Low-copy-number plas-mids used were pSC101 (2), pRK248 (10), or pRK2501 (10).Photodynamic action. Photodynamic action procedure

used was based on the method previously described (26)with some modifications. Toluidine blue 0 was added to thecell suspension (ca. 2 x 109 cells per ml) at a concentrationof 25 ,uM. The light intensity on the surface of the samplewas 103 lx as determined by an illumination photometer(Illumination meter IM-1, Tokyo Kogaku Kikai Co., Ltd.).In some cases, 50 mM NaN3 was added to the cell suspen-sion 30 min before visible light irradiation. In vitro treatmentwas performed as follows. Samples which contained CsCl-cenitrifugation-purified pBR322 DNA (ca. 1 pLg/ml) in TEbuffer (5 mM Tris, 2 mM EDTA [pH 7.0]) were mixed withtoluidine blue and irradiated on a porcelain plate which was

kept at 5°C.Treatment with hydroperoxides. Hydroperoxides, such as

tert-butyl hydroperoxide (Nakarai Kagaku Yakuhin Co.,Ltd.), cumene hydroperoxide (Nakarai Kagaku YakuhinCo., Ltd.), and hydrogen peroxide (Mitsubishi Gas KagakuCo., Ltd.) were added to the cell suspension (ca. 2 x 109cells per ml) with continuous shaking at 37°C.UV irradiation. For near-UV irradiation, UVGL-25 light

(Funakoshi Yakuhin Co., Ltd.) which has a range of emis-sion from 290 to 400 nm with a maximum emission at 366 nmwas situated at a distance of 4 cm from the cell suspension(9.2 J/m2 per s at 365 nm). The cell suspension (ca. 2 x 108cells per ml) was irradiated in a 50-ml beaker kept at 5°C andwas continuously stirred during irradiation.The far-UV irradiation source used was a 15-W Hitachi

germicidal lamp (model GL 15). The cell suspension (ca. 2 x

i08 cells per ml) was irradiated at room temperature in a petridish (inner diameter, 9 cm) with stirring. The dose rate was1.13 J/m2 per s.

Irradiated cell suspensions were diluted appropriately andplated on L medium in petri dishes. After overnight incuba-tion at 37°C in the dark, colonies were counted.Plasmid isolation. Plasmid isolation was performed in two

ways. (i) Samples (10 ml) taken after photodynamic action orhydroperoxide treatments were washed with the phosphatebuffer twice, and plasmid isolation was performed by themethod of Guerry et al. (7) with slight modifications. (ii)After near-UV or far-UV irradiation, the method of Kadoand Liu (9) was used with some modifications.Agarose gel electrophoresis. DNA samples were subjected

to electrophoresis through vertical 0.8% agarose gel in Tris-

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1018 HORIUCHI ET AL.

1 2 3 4 5 6 7 8

I a,,,,,,,,,, ,,......... ... .... ._.

DimerForm U

Form I-

FIG. 1. Effect of toluidine blue plus light treatment with orwithout azide on plasmid DNA in vivo: Toluidine blue plus lighttreatment was performed in the absence (lanes 1 through 4) orpresence (lanes 5 through 8) of 50 mM NaN3. Visible light irradiationwas for 5 min (lanes 2 and 6), 10 min (lanes 3 and 7), or 15 min (lanes4 and 8). Lanes 1 and 5 were controls (no visible light irradiation).

acetate buffer (40 mM Tris-acetate, 2 mM EDTA [pH 7.5]).The DNA samples loaded in each lane contained about 1 p.gof DNA each and were run at 2 to 3 V/cm at roomtemperature for 8 to 10 h. The gels were stained withethidium bromide, and fluorescent bands of DNA werephotographed with UV illumination with Polaroid type 667film or type 665 film for densitometric analysis.

Electron microscopy. DNA was extracted from the gels bythe method of Tabak and Flavell (21) with a column ofCellulofine GH-25-m (Seikagaku Kogyo Co., Ltd.) instead ofSephadex G50. For electron microscopy, the modified meth-od described by Lang and Mitani (11) was used.

RESULTSEffect of photodynamic action on plasmid DNA. The effect

of toluidine blue plus light treatment on plasmid DNAstructure was monitored by agarose gel electrophoresis.

(A)I A

(B)

(C)

(D)

C-) t=-.

0 (DE3 -i0

(cl-n00=

CD 3

-

('DXI

0

Q

0-CD-0

0-5

FIG. 3. Densitometric analysis of the effect of toluidine blue pluslight treatment on plasmid DNA. Samples containing ladder patternDNA were electrophoresed and were photographed with Polaroidtype 665 positive-negative film, and the negative film was scannedwith a Zeineh soft laser scanning densitometer (Biomed Instrui-ments, Inc.). Samples were irradiated with visible light for 0 min(A), 5 min (B), 10 min (C), or 15 min (D).

FIG. 2. Electron micrographs of pBR322 DNA observed after

toluidine blue plus light treatment. Negative supercotling decreases

from A to E.

Unexpectedly, a "ladder pattern" was observed betweennegative supercoiled (Form I) and open circular (Form II)DNA as the cell survival decreased (Fig. 1). The samepattern was reproducibly obtained if plasmid DNA extrac-tion was initiated within an hour after the treatment of thecells. When a singlet oxygen quencher, azide, was added tothe cell suspension, its protective effect was observed not

TABLE 1. Relation between DNA relaxation and DNA single-strand breaks after toluidine blue plus light treatment in vivo

Ratio of peak areas of the following DNATreatment forms':time (min)

0 0.10 0.00 0.105 0.25 0.15 0.11

10 0.36 0.22 0.1415 0.47 0.29 0.18

a Peak areas were determined from densitometric tracings of photographsof agarose gels as described in the legend to Fig. 3. llr, Peak area of form ItDNA relaxed by heat treatment; lIn, peak area of form II DNA before heattreatment minus IIr.

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PHOTODYNAMIC ACTION OF HYDROPEROXIDES AND NEAR-UV

Forin IIS

Form I

FIG. 4. Effectplasmid DNA in Xin vitro were elec(lanes 2, 4, 6, andexposure was for(lanes 5 and 6), o

only for the seipattern (Fig. 1).acridine orangethe plasmid inpSC101, pRK24the ladder pattewith a photoserTo determine

form the laddeelectron microserved are shoresistant to heathat these DN

I 2 3 14 5 6 7 8 changes of plasmid DNA. Figure 3 shows that the peakbands of the ladder pattern migrated in the direction fromForm I to Form II as the irradiation time increased. Wewished to determine whether the nicked circular DNAspecies increases in the Form II peak by the treatment invivo. DNA samples identical to those analyzed in Fig. 1were exposed to heat denaturation and, after agarose gelelectrophoresis, scanned as described in the legend to Fig. 3.The whole Form II peak was regarded as relaxed circularDNA, and it was compared with that of non-heat-treatedsample. The results indicate that nicked circular DNAappears as the amount of relaxed circular DNA increases(Table 1). Therefore, it is concluded that the damage oftoluidine blue plus light treatment on plasmid DNA in vivoconsists of two classes of events: relaxing and nicking. As

of toluidine blue plus light treatment on purified shown in Table 1, nicking is a minor event. It was alsovitro. Samples treated with toluidine blue plus light observed that strains AB1886 (uvrA) and AB2463 (recA):trophoresed before (lanes 1, 3, 5, and 7) or after showed almost the same sensitivity as AB1157 (wild type) to8) heat treatment at 100°C for 3 min. Visible light the inactivation by toluidine blue plus light treatment, and0 min (lanes 1 and 2), 5 min (lanes 3 and 4), 10 min that toluidine blue plus light treatment did not induce Xir 15 min (lanes 7 and 8). prophage in E. coli (unpublished data).

Effect of toluidine blue plus light treatment on purifiedplasmid DNA. Purified pBR322 DNA was mixed with tolu-

nsitized inactivation but also for the ladder idine blue and irradiated with visible light in the presence ofThis ladder pattern was also observed when oxygen. Figure 4 shows that the Form II DNA band in-was used as a photosensitizing dye or when creases as irradiated time extends. However, as this Form II

i E. coli cells was pBR313, pACYC177, DNA band disappeared with additional heat treatment, this48, or pRK2501. Neither the lethal effect nor DNA band must be composed of nicked circular DNA-rn was caused by the treatment of the cells produced by toluidine blue plus light treatment. It is suggest-isitizing dye or visible light alone. ed that in vivo toluidine blue plus light treatment inducese the structure of the DNA species which lesions resulting in DNA relaxation mediated by somer pattern, we examined a DNA sample by cellular component(s) as well as nicks that are probablyscopy. Some typical DNA structures ob- equivalent to those detected in vitro.Iwn in Fig. 2. As these DNA species were Effect of hydroperoxides. The survival curve of stationary-I denaturation (100°C, 3 min), it is suggested phase cells of E. coli AB1157 harboring pBR322 after tert-[A species are formed by the topological butyl hydroperoxide treatment is shown in Fig. 5A. The

A100

10_

1

0.10 50 100

Concentration (mM)

B2 3 4

DimerFormI-j-

FormI-t

FIG. 5. Effect of tert-butyl hydroperoxide on plasmid DNA andon survival curve of E. coli AB1157. (A) Survival curve with tert-butyl hydroperoxide. (B) Electrophoretic analysis of the effect oftert-butyl hydroperoxide on plasmid DNA in vivo. tert-butyl hydro-peroxide was added to lanes to the following concentrations: noaddition (lane 1; control), 10 mM (lane 2), 50 mM (lane 3), and 100mM (lane 4).

150

0-1

a0

L-

Cr)

0

0

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1020 HORIUCHI ET AL.

A12 3 4

Fo rmi 11-

Form I -

Form II

Forni I -

iB12 3 4

FIG. 6. Effect of near-UV radiation on plasmid DNA and on

survival curve of E. coli AB1157. (A) Electrophoretic analysis of the

effect of near-UV irradiation on plasmid DNA in vivo. DNA was

irradiated for 0 min (no irradiation) (lane 1); 60 min (lane 2), 120 min(lane 3), and 180 min (lane 4). (B) Electrophoretic analysis of the

effect of far-UV irradiation on plasmid DNA in vivo. DNA was

irradiated with 0J/m2 (lane 1), 67.8 J/m2 (lane 2), 101.7J/m2 (lane 3),

and 135.6 J/m2 (lane 4).

effect on plasmid DNA structure was monitored by agarosegel electrophoresis and the result shown in Fig. 5 indicates

that plasmid DNA is relaxed in a manner similar to thatcaused by toluidine blue plus light treatment. This physiolog-

ical effect was also observed when cells were treated with

hydrogen peroxide or cumene hydroperoxide or when the

plasmid in E. coli cells was pACYC177 or pSC101. Plasmid

DNA relaxation was not observed, but DNA strand breaks

were detected to some extent when purified pBR322 DNA

was mixed with tert-butyl hydroperoxide in vitro (data not

shown). In this case too, some cellular component(s) may

mediate the effect of hydroperoxides on plasmid DNA

structure.Effect of near-UV and far-UV. The effect of near-UV and

far-UV on the pBR322 DNA pattern from stationary-phaseE. coli AB1157 is shown in Fig. 6. With near-UV irradiationin vivo, plasmid DNA relaxation was observed (Fig. 6A),

while no plasmid DNA relaxation was detected after far-UV

irradiation in vivo even when the survival was reduced to

about 0.1% (Fig. 6B). As expected, the irradiation of near-

UV or far-UV on purified pBR322 DNA in vitro induced no

change on the plasmid DNA band pattern after agarose gel

electrophoresis (data not shown).

DISCUSSIONPlasmid DNA relaxation was observed to accompany the

decrease of the cell survival when E. coli was treated with

toluidine blue plus light or hydroperoxides, and it is suggest-

ed that this physiological effect requires some cellular com-

ponent(s).In E. coli, it is thought that the superhelicity ofDNA in the

cell is maintained by the equilibrium between the supercoil-ing activity of DNA gyrase and the relaxing activity of DNA

topoisomeraseI,II', and III (3, 15). ATP is required and

hydrolyzed in the supercoiling reaction ofDNA gyrase but is

not required in the relaxation reaction of these DNA topoiso-

merases. Therefore, it is presumed that ATP concentrationin the cell affects greatly the maintenance of the equilibrium

of the activities of these enzymes. Treatment of E. coli

harboring pBR322 with 2,4-dinitrophenol, an uncoupler of

the oxidative phosphorylation, caused plasmid DNA relax-

ation; the supercoiled plasmid DNA was recovered after

glucose addition (14). In bacterial cells, cellular respiration

and oxidative phosphorylation are carried out in the cell

membrane. Therefore, it is plausible that the normal function

of the cell membrane is also important to maintain DNAsuperhelicity in the cell. Active oxygen is known to causemembrane damages, such as lipid peroxidation and proteinoxidation. Thus, there are two possibilities that can explainthe plasmid DNA relaxation observed in the experiments.These are (i) that the cellular components involved in themaintenance of DNA superhelicity are oxidized and inacti-vated by radical reactions caused by active oxygen, and (ii)that lipids, proteins, or both of the cell membrane areoxidized, resulting in loss of membrane functions (decreaseof ATP concentration in the cell, etc.), and the equilibrium isbroken. In vitro systems in which purified plasmid DNA,hydroperoxide, and some specific cellular components areincubated may be useful for determining which of thesepossibilities is correct. Analysis of changes in ATP concen-tration in the cell after the treatments may also be helpful todistinguish between these possibilities. Since DNA relax-ation is the repeat of breaking and rejoining of DNA, it ishighly probable that DNA topoisomerases are involved inthis reaction. However, plasmid DNA relaxation was alsoobserved when DM800 (topA), a DNA topoisomerase I-deficient strain (5, 18, 20), was treated with toluidine blueplus light or tert-butyl hydroperoxide (data not shown),suggesting that DNA topoisomerase I is not necessary forplasmid DNA relaxation. Although the superhelical changesof chromosomal DNA have not been detected directly, it ishighly probable that some parts of chromosomal DNA areaffected, as is plasmid DNA, leading to impairment ofreplication, transcription, and recombination (6) of DNA.These superhelical changes of DNA caused by the treatmentof toluidine blue plus light and hydroperoxides may result inbacterial cell death.The lethal effect of near-UV irradiation on E. coli is

different from those of far-UV irradiation in the followingrespects: (i) it shows strong oxygen dependence (29), (ii)stationary-phase cells of uvrA and recA show similar sensi-tivity to wild-type strain (23, 24), (iii) membrane damage isobserved (16), (iv) SOS function is not induced (22), and (v)photoreactivation is not detected against the cell death afternear-UV irradiation (28). It was reported that induciblerepair different from SOS function exists against near-UVirradiation (17). Tuveson and March described that a nurstrain, a near-UV-sensitive strain, is as sensitive as the wild-type strain is to far-UV irradiation, whereas a nur strain ismore sensitive than the wild-type strain is to acridine orangeplus light treatment (25). A mutant (xthA) which is sensitiveto hydrogen peroxide (4) is sensitive to near-UV irradiationalso, although it is as resistant as the wild-type strain to far-UV irradiation (19). Therefore, it has been proposed that thecellular damage by near-UV irradiation is different from thatby far-UV irradiation and that active oxygen is involved innear-UV irradiation damage. Our data in this report supportthis hypothesis with respect to the plasmid DNA relaxationeffect. On the other hand, the fact that plasmid DNArelaxation is not induced by far-UV irradiation suggests thatplasmid DNA relaxation is not the common physiologicaleffect in bacterial cell death and that it can be used as anindex to classify the types of the cell damage. Recently, itwas reported that oxidative stress, heat shock stress, andethanol treatment all induce accumulation of adenylateddinucleotides in bacterial cells (12); thus, it will be veryinteresting to examine whether plasmid DNA relaxation iscaused by these stresses and to determine whether adenylat-ed dinucleotides such as AppppA are accumulated aftertreatment of the cells with a photosensitizing dye plus visiblelight and near-UV.

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PHOTODYNAMIC ACTION OF HYDROPEROXIDES AND NEAR-UV 1021

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