Gene, 13(1981) 173-184 173 Elsevier/North-tlolland Biomedical Press

In vivo cor re la t ion be tween DNA supercoi l ing and t ranscr ip t ion

(E. coli; recombinant DNA plasmid; DNA gyrase inhibitor; DNA supercoiling; DNA relaxation: RNA synthesis)

Yasunobu Kano, Tomoyuki Miyashita, Haruji Nakamura, Kazuyuki Kuroki, Akihisa Nagata and Fumio lmamoto

Department of Microbial Genetics, Research Institute for Microbial Diseases, Osaka University, Yamada-Kami, Suita, Osaka (Japan)

(Received October 7th, 1980) (Accepted January 5th, 1981)

SUMMARY

The superhelical density of pMT plasmid DNA in Escherichia coli cells was measured as a function of the trans- criptional activity, which was reduced by treatment with coumermycin or oxolinic acid. Superhelicity was assayed by agarose gel electrophoresis of DNA extracted from cells gently lysed by sarkosyl. Coumermycin treat- ment reduced the proportion of supercoiled plasmid DNA in concert with a decrease in the rate of plasmid-coded synthesis of trp mRNA, implying a correlation between supercoiling of DNA and its suitability for transcription. On the other hand, the oxolinic acid-induced loss of supercoiled plasmid DNA was relatively small, while con- comitant inhibition of trp mRNA synthesis was very severe. Treatment of the cells with these two drugs never removed all of the supertwists from the pMT plasmids carried.

INTRODUCTION

DNA gyrase is believed to be responsible for the maintenance of negatively supercoiled conformation in intracellular DNA. The enzyme is composed of two subunits (Gellert et al., 1976; 1977. Sugino et al., 1977; Higgins et al., 1978). One subunit, Pnal, has a nicking-closing activity and is thought to relax positive superhelicity in the intermediate DNA in the super- coiling reaction. This action is'inhibited by oxolinic

Abbreviations: DMSO, dimethylsulfoxide; Md, megadaltons; SDS, sodium dodecyl sulfate; [ ], brackets indicate plasmid carrier.

acid and nalidixic acid. The other subunit, Pcou, is possibly involved in energy transduction during the supercoiling reaction. This activity is inhibited by coumermycin and novobiocin (Peebles et al., 1978; Liu and Wang, 1978a, b; Brown and Cozzarelli, 1979).

Recent observations have indicated that DNA gyrase is an essential component for the transcription (Puga and Tessman, 1973; Javor, 1974; Stauden- bauer, 1976; Falco et al., 1978; Smith et al., 1978; Kubo et al., 1979; Sanzey, 1979), as well as for the replication of cellular DNA (for review, see Wickner, 1978) and recombination (for review, see Radding, 1978). Those experiments have demonstrated that the rate of in vivo transcription is generally reduced

0378-1119/81/0000-0000/$02.50 ©Elsevier/North-HoUand Biomedical Press

174

by specific inhibitors of DNA gyrase, such as nalidixic acid, oxolinic acid and coumennycin. Although the change in superhelicity of intracellular DNA in the drug-treated bacterium has not yet been directly mea-

sured, a loss of supercoiling was suggested to occur by

measuring superhelical density of isolated E. coli DNA (Drlica and Snyder, 1978) or X DNA (Gellert et

al., 1976; Kikuchi and Nash, 1979) after inhibiting DNA gyrase by coumennycin. On the other hand, the

inhibition of DNA gyrase activity by oxolinic acid, as

well as nalidixic acid, produces in vitro a relaxation- type complex between DNA gyrase and the super- coiled ColE1 DNA (Sugino et al., 1977: Gellert et al., 1977; Morrison and Cozzarelli, 1979). This complex is converted to linear DNA by treatment with SDS and protease. It is not clear, however, whether these facts reflect some similarities to the in vivo processes that produce the drug-induced relaxed DNA.

In vitro studies employing bacteriophage DNAs, such as qSX174 replicative allomorphic DNA (Hayashi and Hayashi, 1971), PM2 DNA (Wang, 1974; Richardson, 1975), X DNA (Botchan et al., 1973; Botchan, 1976), fd replicafive-form DNA (Seeburg et al., 1977), and plasmid DNAs (Yang et al., 1979) have demonstrated that either the rate of transcrip- tion or the rate of formation of initiation complexes with E. coli RNA polymerase is enhanced with super- coiled DNA.

These earlier findings support the possibility of existence of a correlation between superhelicity and

transcription in vivo. A reduction of negative super-

coiling of the intracellular DNA upon inhibition of DNA gyrase may directly result in a loss of ability of the DNA template to be transcribed.

This paper describes our results which indicated that indeed the over-all rate of transcription was

reduced co-ordinately with the decrease in superheli- cal density of the intracellular DNA, when the Pcou subunit of DNA gyrase was inhibited by coumermy- cin. In contrast, under the inhibition of the Pnal sub- unit by oxolinic acid, the reduction in the rate of transcription was much more drastic than the extent of loss of supercoiling DNA.

MATERIALS AND MF, THODS

(a) Bacteria, phages and plasmids

Derivatives orE. coli K-12 used as the recipient for the transformations with the recombinant plasmid were: W3110 trpAE1 (deletion mutant of entire trp operon), W3110 trpAElsuI, NI708(g),rA+B+), NI741- ~vrB) and M015124(x, yrA) (see Kubo et al., 1979). Strains N1708 and NI741 were kindly supplied by J. Tomizawa. M015124 was kindly donated by M. Oishi.

The plasmids pMT60-3 and pMT48 were con- structed in vitro as chimeras between mini-ColE1 DNA and the EcoRI-generated trp-N DNA fragments of phage XhXtrp60-3trpABCDE imm x QSR x and

XhXtrp48trpABCDtrpDC181Nalnclts2cro -immXQRS x (see Fig. 1).

The following phages were used as sources of DNA for DNA-RNA hybridization assays: the nontrans- ducing phage ~b80, as well as the nondefective trans- ducing phage, (~80trpED (see Kubo et al., 1979).

The genetic map of the plasmids and the direction of transcription of plasmid DNA are illustrated in Fig. 1.

(b) Preparation of phage DNA

DNAs of 480 and 080trpKD phages were prepared as described elsewhere (Kano et al., 1976) and dis-

solved in a saline-citrate solution (1 ×SSC: 0.15 M NaC1-0.015 M sodium citrate) after dialysis against the solution.

(c) Preparation of pulse-labeled RNA

Bacterial strain trpAE1 [pMT60-3] was grown at 37°C for 3 h with aeration to 5 X 108 cells/ml in an enriched medium (L-broth) (Lennox, 1955) supple- mented with L-tryptophan (50/~g/ml). The cells were collected by centrifugation, washed twice with a cold minimal medium (Vogel and Bonner, 1956), and sus- pended in the same medium to give a final density of 3 X 101° cells/ml. A portion (0.2 ml) of the cell sus- pension was transferred quickly to prewarmed (30°C) minimal medium (3 ml, adjusted to pH 8 with NaOH) containing 19 amino acids (each 0.5 mM except tryp- tophan, and the cell suspension was shaken vigorously in a water bath at 30°C. Oxolinic acid or coumermy-

/ pMT 48

175

Fig. 1. Schematic molecular maps of plasmids. The maps, showing the structure of recombinant plasmids pMT60-3 and pMT48, are based on the previous data (Segawa and Imamoto, 1974; Fiandt et al., 1974; Kubo et al., 1979; Y. Kano, H. Nakamura and F. Imamoto, unpublished results). The relative size of the E. coli trp genes (A, B, C, D, and E), the h genome and the miniColE1 genome is estimated from the data reported previously (Imamoto and Yanofsky, 1967; Szybalski and Szybalski, 1979; Tomizawa et al., 1977). For the symbols and functions of h genes (N, cl and tof) and k promoters (PL, PR and PE) see Szybalski and Szy- balski (1979). The pMT48 plasmid has a deletion of the beginning portion of the trp operon, including the region of the trp promoter and trpE, and a short deletion within the trp operon that removes the low-efficiency internal promotor, P2trp, distal to trpD (Nakamura et al., 1978; Ishii et al., 1980). The arrows indicate the direction of transcription. The molecular weights of pMT60-3 DNA and pMT48 DNA are 13 and 11 Md, respectively.

cin was added at the 15th min of incubation. The cell

suspension was pulse-labeled for 2 min with 150/~Ci

of [3H]uridine (19.0 Ci/mmol) at t imes indicated in

Fig. 5 after addit ion of the drug. Labeling was

stopped by rapidly pouring the cell suspension onto

35 ml of crushed frozen medium containing 1 X 10 -2

M Tris-HC1 buffer at pH 7.3, 5 X 10-3M MgCtz,

1 × 10 -2 M NaN3 and 150 ~g of chloramphenicol/ml.

RNA was prepared by the procedure reported previ-

ously (hnamoto , 1969). The RNA obtained was

filtered through a Millipore f'dter, precipitated by

ethanol, and dissolved in 1 X 10 -2 M Tr is 'HC1

buffer, pH 7.3, containing 0.5 M KC1 and 1 X 10 -3 M

Na2EDTA.,

(d) DNA-RNA hybridization

The hybridizat ion procedure was as follows. DNA

of ~80 or 080trpED was diluted to a concentrat ion of

100 #g/ml in 1 × SSC and heated in boiling water for

10 min followed by rapid cooling in ice water. The

DNA was then further diluted to a concentrat ion of

8 #g/ml in 3 X SSC. 5 ml o f the DNA solution was

filtered through a Millipore filter '(type HA, 0.45 gun

pore size) of 25 mm diameter. The filter was washed

with 40 ml of 3 X SSC, cut into 8 sectors and dried at

80°C for 2 h. The solution of [3H]RNA was appro-

priately diluted, divided into 100 #1 fractions, and

annealed to a segment of Millipore fdter bearing 5/~g

of immobilized DNA from q~80 or ~80trp phage for

18 h at 66°C. Afterwards, the filter was treated with

RNase (5 #g/ml) in 1 X SSC at 37°C for 30 min,

washed with 1 X SSC, dried and counted in toluene-

based scintillation fluid.

(e) Preparation of labeled plasmid DNA

Bacteria carrying the plasmid were grown at 30°C

or 37°C for about 3 h to a density of 4 X 108-5 X

108 cells/ml in L-broth containing 10 ~zCi/ml o f [3H]-

thymidine (21.8 Ci/mmol). Cells were washed and

suspended in the medium supplemented with 19

176

amino acids except tryptophan or 20 amino acids as described in section (c). 3 ml of the cell suspension

was shaken vigorously in a water bath at 30°C. Oxo-

linic acid or coumermycin, when needed, was added

at a suitable tinle during aerobic incubation. The cell

suspension was poured onto 2 ml of iced medimn (20% sucrose, 5 X 10 -2 M potassium phosphate buffer

pH 7.5, 1 X 10 -2 M NaN3 and 0.1 M NaC1), pelleted

by centrifugation, washed and then resuspended with 1.25 ml of the same medium. After treatment of the

cell suspension with lysozyme {0.125 ml of 10 rag/ ml) for 5 min at 0°C, 0.45 ml of sarkosyl solution

(2% in water) was added. The suspension was mixed

by drawing it in and out of a pipette 10 times. The

sarkosyl lysate was treated with proteinase K (0.05

ml of 4 mg/ml) for 30 rain at 30°C, followed by addi-

tion of 0.183 ml of SDS (10% in water) and further incubation for 30 rain at 30°C. After mixing well by

drawing with a pipette, the lysate was shaken vigor-

ously with an equal volume of phenol saturated with

water (adjusted to pH 8 with Tris base) and a half

volume of chloroform-isoamyl alcohol (24 : 1, v/v),

then centrifuged at room temperature. Extraction

with phenol and chloroform-isoamyl alcohol was

repeated. The aqueous layer was then shaken with an

equal volume of chloroform-isoamyl alcohol (24 : 1, v/v). After centrifugation of the aqueous layer to

remove any precipitate, DNA was precipitated with

2 vols. of ethanol and 1/5 vol. of 1 M Na-acetate

buffer at pH 5.0 and 20°C. The precipitate was

washed with cold 70% ethanol and suspended with

0.5 ml of 1 × 10 .3 M Tris - HC1 buffer at pH 8.0 con-

taining 1 X 10 -3 M Na2EDTA. The DNA solution was

heated for 3 min in boiling water, followed by cool- ing rapidly in an ice bath. 0.5 ml of heat-denatured

DNA solution were mixed with 0.33 ml of 5 × 10 -2

M Tris • HC1 buffer at pH 7.3 containing 2.5 M KC1

and then filtered slowly through a Millipore filter

(type HA, 0.45 wxn pore size) of 25 mm diameter.

More than 98% of the amount of linear F.. coli DNA was trapped on the filter under these conditions (data not shown). The filtrate containing closed-circular

plasmid DNAs was diluted to 2-fold with water and

then precipitated with cold ethanol-Na-acetate in the presence of 100 #g/ml ofF.. coli tRNA as carrier. The

plasmid DNA precipitated was dissolved with 0.1 ml of 1 X 10 -2 M Tris • HC1 buffer (pH 8.0) containing 1 × 10 -3 M Na~EDTA, and then analyzed by agarose

gel electrophoresis.

(f) Agarose-gel electrophoresis of plasmid [ 3 H ] DNA

A gel solution was prepared by dissolving agarose

(1.0-1.2% w/v by autoclaving for 5 rain in electro-

phoresis buffer containg 4 X l 0 -2 M Tris • HC1 buffer at pH 8.0, 5X10 -3 M Na-acetate and 1X10 -3M

Na2EDTA, and allowed to solidify in 10 X 13 X0.3

cm slab gel apparatus at room temperature. Electro-

phoresis of DNA samples {10-50 ;zl containing

0.01% bromophenol blue. 1% SDS and 8.3% glycerol)

was performed at 40-50 V (3 -4 V/cm) for 15 20 h

at room temperature just after running of the DNA at

100 V for 5 rain. After electrophoresis, the gels were

soaked for 15 rain in electrophoresis buffer contain-

ing 5/.tg/ml ethidium bromide, followed by washing

in the same buffer without ethidium bromide for 15

rain. 2.5 or 5 mm gel slices were then cut from the

tracks and dissolved in 0.9 ml of 55% formamide at

80°C for 60 min. After cooling, 9 ml of toluene-PPO- based scintillant containing 33% (v/v) polyethylene

glycol mono-p-iso-octylphenyl ether were added and radioactivity was counted.

(g) Chemicals

Oxolinic acid was a gift from J.D. Stein Jr.

(Warner-Lambert Research Institute, Morris Plains,

N J). Coumermycin A1 {referred to as coumermycin

throughout this paper) was kindly supplied by M. Oishi. [3H]uridine (19.0 Ci/mmol)and [3H]thymi-

dine (21.8 Ci/mmol), purchased from New England

Nuclear, Boston, were used without addition of

carrier. Millipore filters were purchased from Milli-

pore Filter Company, Bedford, MA. DNase and

RNase were obtained from Worthington Biochemical

Corporation. RNase was heated at 80°C for 20 rain in

0.15 M NaC1 to inactivate any contaminating DNase.

Agarose-I was purchased from Wako Pure Chemical

Industries, Ltd., Osaka. Polyethylene glycol mono- p-iso-octylphenyl ether was purchased from Nakarai

Chemicals Ltd., Kyoto. Proteinase K was purchased from E. Merck A.G.

RESULTS

(a) Decrease in superhelical density of pMT plasmid DNA as result of inhibition of DNA gyrase by coumermycin or oxolinic acid

An experiment to determine the superhelical den- sity of pMT plasmid DNA in E. coli cells consisted of plasmid labeling with [3H]thymidine throughout a period of cell growth, gentle extraction of intact plasmid DNA from cells, and analysis of the DNA in the agarose gels. During the agarose-gel electrophore- sis, the [3H]DNA formed three major bands contain-

ing linear E. coli DNA, supercoiled pMT DNA and relaxed pMT DNA, in order of increasing mobilities (Fig. 2). Most of the plasmid DNA was in supercoiled

177

form, which had a mobility distinct from that of the E. coli DNA fragments, which were present as a con- taminant. The amount of E. coli DNA contaminant was estimated to be less than 2% of the bulk DNA extracted in the sarkosyl lysate (data not shown).

As seen in Fig. 2, intracellularly supercoiled pMT DNA has roughly twice the mobility of relaxed pMT DNA. The amount of relaxed plasmid DNA in the sample was generally one-third of the amount of the supercoiled plasmid DNA. The relaxed plasmid DNA was assumed to be of covalently closed-circular form rather than open-circular form, because the latter DNA must be eliminated from the sample by proce- dures of purification by heat denaturation and filtra- tion through a Millipore filter (Mukai et al., 1973). In fact, about 80% of pMT plasmid [3H]DNA in the

0

x

E o2

a b

Su percoiled

' H o s t DNA ~'

F r a c t i o n n u m b e r

Fig. 2. Analysis of intracellular superhelicity of pMT plasmid DNA by agarose gel electrophoresis. Cultures of strain trpAE1- [pMT60-3] (a) and trpAElsuI[pMT48] (b) were grown at 37°C and 30°C, respectively, for about 3 h to a density of 5 × 108 cells/ml in a medium (L-broth) containing 10 uCi [3H] thymidine/ml (21.8 Ci/mmol). Ceils were washed twice with cold minimal medium (Vogel and Bonner, 1956) and resuspended in medium supplemented with 19 amino acids (each 0.5 mM) except trypto- phan (a) or with all 20 amino acids (b) to give a final density of 1.8 × 109-1.9 × 109 cells/ml. 3 ml of the cell suspension was shaken vigorously for 60 min (a) or 45 min (b) in a water bath at 30°C, followed by addition of 2 ml of ice-cold medium (20% sucrose, 5 × 10 -2 M potassium phosphate buffer pH 7.5, 1 X 10 -2 M NaN 3 and 0.1 M NaC1). The cells were pelleted by centrifu- gation, washed and then resuspended with 1.25 ml of the same medium. DNA was extracted and electrophoresed on 1% (a) or 1.2% (b) agarose gels. After 18 h (a) or 20.5 h (b) of electrophoresis at 3.6 v/cm or 3.0 v/cm, respectively, the gels were sliced, and [3H]DNA was determined by counting in a liquid scintillation counter. The peaks of the [3tI]DNA are, from the left, relaxed plasmid, supercoiled plasmid, and linear E. coli DNA molecules. The other conditions were as described in MATERIALS AND

METHODS.

178

sarkosyl lysa te cons i s ted o f cova len t ly c losed-circular

molecu les , as m e a s u r e d b y s e d i m e n t a t i o n analysis in a

CsC1/e th id ium b r o m i d e dens i ty gradient . Of these

closed-circular DNA molecu les , at least 90% was

recovered in the f inal p lasmid DNA sample o b t a i n e d

af te r h e a t - t r e a t m e n t o f the lysate and f i l t ra t ion

t h r o u g h a Mill ipore f i l ter . No s ignif icant a m o u n t o f

pMT ol igomers was de t ec t ed in the lysa te , e i the r by

A

211

..~41

Super a coiled

Relaxed

b

1 2

20

5 10 15 F r a c t i o n

e

h

n u m b e r

Fig. 3. Changes in superhelical density of pMT plasmid DNA in ceils treated for various times with oxolinic acid or coumermycin. Strain trpAE1 [pMT60-3], labeled with [3H]thymidine, was aerobically incubated at 30°C in the presence or absence of oxolinic acid (a -d) or coumermycin (e -h) . The drugs (each 200 /sg/ml) were added at the 45th (b and O, 30th (c and g) or 0 (d and h) rain of incubation, followed by further incubation for 15, 30 or 60 min, respectively, before DNA extraction. At the start of incubation of control cultures, 4 X 10 -4 M KOH (a) or 0.5% DMSO (e) was added, which were employed as solvents for oxolinic

acid or coumermycin, respectively, and the cultures were incubated for 60 rain before DNA extraction. For other conditions see Fig. 2 and MATERIALS AND METHODS. The 100% value represents total radioactivity of [3H]plasmid DNA analyzed.

179

611

40

20

Super coiled

a

i

4~ b

<~ Z

E u~

~ , , i i i i J

041] C

> ,m

0

f9 ._>

"~ d

4 6 8 10 12 14 F r a c t i o n

e

i | i

g

~ 8 10 12 ~1 n u m b e r

Fig. 4. Effect of oxolinic acid or coumermycin concentrations on the superhelical density of the intraceUular plasmid DNA. Strain

trpAE1 [pMT60-3], labeled with [3H]thymidine, was aerobically incubated at 30°C in the presence or absence of oxolinic acid

( a - d ) or coumermycin (e-g) . Oxolinic acid was added at the start of incubation at a concentration of 50 pg (b), 100/ag (c) or

200 ~tg (d) /ml. At the start of incubation of the control culture (a), 4 × 10 -4 M KOH was added. Coumermycin was added at the

start of incubation at a dose of 100 ~tg (e), 300 ag (f) or 501)/lg (g)/ml. After 60 rain incubation, DNA was extracted and elec-

trophoresed. For other conditions see Fig. 3 and MATERIALS AND METHODS.

180

gel electrophoresis or by sedimentation in sucrose gradients (data not shown). Similarly, no contaminat- ing linear pMT DNA, which can migrate faster than

the linear E. coli DNA under the conditions of gel

electrophoresis employed, was detected in tile lysate. To estimate the effect of inhibition of DNA gyrase

on the reduction in superhelical density of intracellu- larly supercoiled plasmid DNA, the change in electro- phoretic mobility of pMT DNA was followed. A reduction in superhelicity of plasmid DNA is known to reduce electrophoretic mobility of that molecule in gel electrophoresis. Treatment of bacteria carrying

pMT plasmids with oxolinic acid or counlermycin

produced significant changes in the plasmid DNA

supertwisting. The time course of such a reaction with pMT DNA has been monitored by electrophore-

sis, and the data (Fig. 3) indicate a loss of DNA super- helical density following treatment with the drugs. The level of supercoiled pMT DNA decreased to about 62, 47 and 29%, of that found in the untreated control after treatment for 15, 30 and 60 rain, respectively, with oxolinic acid, and to about 38, 36 and 33% of that of the untreated control after treat- ment for 15, 30 and 60 min, respectively, with coumermycin. During the initial treatment for 30 rain, the change in DNA superhelical density induced

by oxolinic acid was generally smaller than that found after treatment with coumermycin (see also Fig. 5). On the agarose gel pattern of pMT plasmid DNA from drug-treated cells, some discernible amount of the DNA was distributed in the region between both peaks of supercoiled and relaxed DNA. This is probably due to production of partially relaxed DNA which migrated more slowly than intra- cellularly supercoiled DNA. The pMT plasmid DNA contained in the mutant strain having an oxolinic acid-resistant (M015124) or coumennycin-resistant (NI741) DNA gyrase did not show any significant change in DNA supertwisting following the drug treatment, suggesting that the inhibition of DNA gyrase is responsible for the reduction in superhelical density of intracellularly supercoiled DNA (data not shown).

Fig. 4 shows the effect of increasing concentra- tions of oxolinic acid and coumermycin on the super- helical density of intracellular plasmid DNA. The effect of oxolinic acid treatment for 60 min at 30°C on the level of supercoiled pMT DNA was to reduce it by approx. 51, 55 and 63% at concentrations of 50,

100 and 200 #g/ml, respectively. Coumermycin treat- ment reduced the level by about 56, 48 and 45%. at concentrations of 100, 300 and 500 /~g/ml, respec- tively. The maximum inhibitory effect of these drugs on accumulation of supercoiled plasmid DNA in the cell was observed at 50 ~g/ml for oxolinic acid or

100 /.zg/ml for coumermycin. Increasing the drug con- centration did not significantly cause any further

decrease in the level of intracellularly supercoiled

plasmid DNA. Treatment for at least 60 min with oxolinic acid or coumermycin never removed all of

the plasmid DNA supercoils, and this residual intact supercoiled DNA was at a level 23-55% (or 35%, on an average) of that found in untrated cells (see also Figs. 3 and 5).

(b) Relationship between loss of DNA supereoiling and reduction in transcriptional activity

The drugs that inhibit DNA gyrase, including nali- dixic acid, oxolinic acid and coumermycin, reduced

the rate of in vivo RNA synthesis directed by pMT plasmid DNA (Kubo et al., 1979). In order to exam- ine the possibility of the dependence of transcrip- tion activity on the degree of superhelicity of the

DNA template, the rate of inhibition of RNA synthe- sis following the loss of DNA supercoiling was mea- sured. Fig. 5 compares changes in the level of super- coiled pMT DNA and the rate of trp mRNA synthesis directed by the plasmid DNA during a period of 30 rain of incubation of bacteria carrying pMY plasmid in the presence of oxolinic acid or coumermycin. Ill coumermycin-treated cells, the rate of trp mRNA synthesis decreased in parallel with the loss of super- coiled pMT DNA, implying a correlation between RNA

synthesis and supercoiling. After 20 rain of coumer- mycin treatment, trp mRNA synthesis was almost

totally blocked, while some residual supercoiled pMT DNA was still observed (see Fig. 5 ; a plateau at 23 to 26% of the level for untreated cells).

Oxolinic acid has been shown to be nmch more effective in inhibiting RNA synthesis at a relatively low dose than coumermycin (Kubo et al., 1979). As seen in Fig. 5 oxolinic acid treatment for 5 min caused more than 90% inhibition of trp mRNA syn- thesis, while treatment with the same dose of coumer- mycin reduced the RNA synthesis by only 20%,. In spite of the drastic inlfibition of trp mRNA synthe- sis by oxolinic acid, the drug-induced loss of super-

181

1 0 0

g

_R

[3

1'0 20 30 Time after addition of drug ( rain )

lOO

.-= o

50 "6

Fig. 5. Compar ison of changes in superhelical densi ty o f pMT

plasmid DNA and rate of transcript ion after inhibit ion of

DNA gyrase by oxolinic acid or coumermycin . Strain

trpAE1 [pMT60-3] was grown at 37°C to a densi ty o f 5 X 108

cells/ml in an enriched medium (L-broth). A port ion of the

culture was labeled with 10 ~zCi [3H] thymid ine /ml for 3 h

and then washed twice with cold minimal med ium and resus-

pended in the medium supplemented with 19 amino acids

except t ryp tophan to give a final densi ty of 2 × 109 cells/

ml. Oxolinic acid (200 ~tg/ml) or coumermycin (200 ~tg/ml)

was added to 3 ml of the culture at the 15th min o f incuba-

t ion at 30°C, followed by further incubat ion for 5, 10, 20 or

30 rain before DNA extract ion. For the control cultures, the

0 t ime in the Fig. 5 corresponds to 15 min incubation with-

out addit ion of the drug. The values of supercoiled plasmid

DNA were normalized to 100% of the value of drug-

untreated control. The 100% points for the relative amoun t

of supercoiled plasmid DNA in the ceils t reated with oxolinic

acid or coumermyc in represented 74.2% or 72.4% of the

total labeled plasmid DNA, respectively. For assay o f trp m R N A synthesis , bacteria grown to 5 X 108 cells/ml in a

enriched medium were collected by centrifugation, washed

twice with cold minimal medium and resuspended in mini-

real medium supplemented with 19 amino acids except trypto-

phan to give a final density o f 2 × 109 cells/ml. Oxolinic acid

(200 t~g/ml) or coumermycin (200 ~g/ml) was added to 3 ml

of culture at the 15th min of incubat ion at 30°C. After addi-

tional incubat ion for 5, 10, 20 or 30 rain in the presence o f

the drug, cells were pulse-labeled with [ 3 H] uridine for 2 rain.

The control cultures (0 t ime) were pulse-labeled for 2 min after

addit ion o f 1 × 10 -3 M KOH or 1% DMSO at the 15th rain o f

incubat ion. Presence of KOH or DMSO in the culture did not

significantly affect the transcript ion under the condit ions

employed (data not shown). After extract ion of RNA from

labeled cells, trp m R N A was analyzed by DNA-RNA hybridi-

zat ion with a probe of DNA from 080trpED phage. The RNA

values were expressed as [3H]RNA hybridized to 080trpED DNA (per ~tg RNA) and normalized to 100% tbr the value of

drug-untreated control. The background values for 080 DNA

(less than 50 cpm/~zg RNA) were subtracted from each

hybrid value. Values represented are the average of duplicate

TABLE I

Effect o f oxolinic acid and coumermycin on supercoiling o f intracellular plasmid DNA

Strain t rpAEl [pMT60-3] labeled with [3Hl thymid ine was

aerobically incubated at 30°C in the presence or absence of oxolinic acid and/or coumermycin . The drugs (each 200 ~zg/

ml) were added at the 45th rain of incubation, followed by

further incubat ion for 15 min before DNA extraction. Vor

control cultures, 4 × 10 -4 M KOH (a) or 0.57; DMSO (b) or

both (c) were added at the start of incubation, which was

then cont inued for 60 rain and lbllowed by DNA extraction.

For other condit ions see Fig. 2 and MATERIALS AND METHODS.

Antibiotics added Percent o f

supercoiled DNA

(1) none (control) 69 (100) a

coumermycin 26 (38)

(2) none (control) 68 (100)

oxolinic acid 42 (62)

(3) none (control) 65 (100)

coumermycin and oxolinic acid 39 (60)

a Numbers in parentheses are adjusted to controls normalized to 100.

coiled pMT DNA was only 33 to 45% of the level of the untreated control even after 10 to 30 rain of drug

treatment. A lesser reduction in the level of pMT DNA supercoils observed with oxolinic acid was not changed by the simultaneous addition of coumer- mycin (Table I).

DISCUSSION

When pMT plasmid-carrying bacteria were treated with increasing amounts of coumermycin or oxolinic acid, the negative supercoils in the plasmid DNA were removed and resulted in gradual accumulation of the relaxed form (Fig. 3). We observe that the loss of

determinat ions . The 100% point for oxolinic ac id-or coumer-

mycin-treated cells represented about 500 cpm/~zg RNA. The

other condit ions were as described in Fig. 2 and MATE-

RIALS AND METHODS. a, Supercoiled [3H]DNA in oxo-

linic acid-treated ceils; o, supercoiled [3H]DNA in coumer-

mycin-treated cells; =, trp m R N A synthesized in oxolinic

acid-treated cells; e, trp m R N A synthesized in eoumermycin- treated cells.

182

plasmid DNA supercoiling is more rapid in the coumermycin-treated cells than in tile oxolinic acid-

treated cells (Fig. 5). The coumermycin-induced loss of negative DNA supercoils must arise from the presence of the relaxing function of two topoiso- merases, I (co protein) (Wang, 1971) and 11 (the gyrA gene product of DNA gyrase) (Sugino et al., 1977; Gellert el al., 1977: Morrison and Cozzarelli, 1979). In contrast, under inhibition of topoisomerase activity of DNA gyrase by oxolinic acid, the DNA supercoils may be relaxed by a function of the co pro- tein. The extent of decreased level of supercoiled

plasmid DNA was comparable with both oxolinic acid- and coumennycin-treated cells after 60 rain of drug treatment (Fig. 3). This could be due to rela- tively weak activity of co protein.

Treatment of the plasmid-carrying bacteria with these two drugs never relaxed all the supertwisted

DNA molecules. The residual level of supertwisted pMT plasmid DNA was about 35% on average under the conditions used (see Figs. 3 and 4). Previous studies investigating changes in superhelical density of isolated DNA after inhibiting DNA gyrase in vivo have indicated that some of the supertwists are never removed; i.e., ~ DNA isolated from cells treated with coumermycin appears to have about 15% of the superhelical density found in the drug-untreated con- trol cells (Gellert et al., 1976), and the bacterial chromosomal DNA retains 25-30% of the normal superhelicity after treatment with coumermycin (Drtica and Snyder, 1978). The residual supertwisting could arise from inability of the nicking-closing enzymes to remove all of the negative DNA super- twists, due to structural features of the intracellular

DNA-protein complexes. In addition, some residual supercoiled plasmid DNA may reflect heterogeneity of the intracellular location of plasmids in cells, to which the nicking-closing enzymes are not accessible.

Comparing changes in trp mRNA synthesis with those in superhelicity of the template DNA after treatment with oxolinic acid, it was found that the inhibition of mRNA synthesis of the drug-treated cells was more drastic than the extent of loss of supercoiling (Fig. 5). One possible interpretation is that oxolinic acid seems to trap the DNA gyrase-DNA template complex in a covalently attached intermedi- ate form during a process of relaxation, which leads to site-specific cleavage of the DNA by deproteiniza-

tion (Morrison and Cozzarelli, 1979). Such a state of the tenrplate DNA may prevent strand separation and therefore on-going transcription. Alternatively, inabil- ity to remove positive-twisting stress that is generated by processes such as DNA replication and transcrip- tion by a unique topoisomerase II' (Brown et al., 1979; Gellert et al., 1979b), which is constructed with the Pnal subunit and a 50000 dalton protein called u and is inhibited by oxolinic acid, may result in strong inhibition of transcription.

Several reports appeared previously, which indi- cate that the supercoiled DNA enhances in vitro

transcription by purified E. coli RNA polymerase (Hayashi and Hayashi, 1971; Botchan et al., 1973; Botchan, 1976; Wang, 1974: Richardson, 1975: See-

burg et al., 1977). These observations support tile possibility that supertwisting of the DNA template facilitates the localized opening of base pairs in DNA,

allowing the formation of a productive open complex with RNA polymerase bound to the DNA. However,

the effect of loss of superhelicity on in vitro trans-

cription is generally less than that observed in in vivo systems. Transcription experiments which compare the dependency of trp mRNA synthesis on the super- helicity of the template pMT DNA in an h'. coli SIO0 crude extract with that in the system employing puri- fied RNA polymerase have indicated that decrease in

the overall rate of trp mRNA synthesis upon a loss of

superhelicity is more drastic in the S100 extract than in the pure system (K. Kuroki, S. lshii, Y. Kano and F. Imamoto, unpublished data).

Virtually all duplex DNA in the cell exists in a

negatively supercoiled form. This is an hnportant facet of the processes of DNA replication, recolnbina- tion and also transcription. Supercoiling in the tem- plate DNA may facilitate opening of the double helix in the promoter region. An alternative possibility is that the degree of superheticity influences the ability of RNA polymerase to recognize the promoter sequence in the DNA template. It has been suggested that hairpins or stem-and-loop structures of DNA can be favorably formed when the palindrome sequence is embedded in a supercoiled DNA (Gellert et al., 1979a). In the cell, superhelicity of the chromosomal DNA could also alter interaction of nuclear proteins and some protein factors for transcription with tem- plate DNA.

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Comnmnicated by Z. Hrade~na.