In vivo enhancement of general and specific transcription in Escherichia coli by DNA gyrase activity

Gene, 7 (1979) 153--171 153 © Elsevier/North-Holland Biomedical Press, Amsterdam --Printed in The Netherlands

IN VIVO ENHANCEMENT OF GENERAL AND SPECIFIC TRANSCRIPTION IN Escherichia coli BY DNA GYRASE ACTIVITY (Recombinant plasmid; ), transducing phage; nalidixic acid; oxolinic acid; coumermycin; RNA synthesis) MEGUMI KUBO, YASUNOBU KANO, HARUJI NAKAMURA, AKIHISA NAGATA and FUMIO IMAMOTO*

Department of Microbial Genetics, Research Institute for Microbial Diseases, Osaka University, Yamada-kami, Suita, Osaka (Japan) (Received June 6th, 1979) (Accepted July 2nd, 1979)

SUMMARY

The effect of drugs which inhibit DNA gyrase, including nalidixic acid, oxolinic acid and coume~ycin, on transcription of Escherichia coil bacteria, phage and plasmid genomes was studied. Quantitative estimates of the synthesis of RNA under drug-treatment conditions showed that synthesis of many RNA species, including trp mRNA, was subject to inhibition by the drug. Transcription directed by the ), promoter PR was selectively less sensitive to the drug action than transcription initiated at the ~ promoter Pw.. Evidence was obtained showing that diminished transcription resulted from less frequent RNA chain initiation rather than a premature arrest of the chain elongation. Inhibition of transcription by these DNA gyrase inhibitors was observed even in the absence of DNA replication. The inhibition by oxolinic acid or coumermycin was not observed in an £. coil strain bearing a nalA r mutation or a cour mutation, respectively. The reduction of trp mRNA synthesis in oxolinic acid-treated cells cannot be attributed to the increase in the rate of nascent mRNA degradation. These results indicate that DNA gyrase is generally required for intracellular RNA synthesis, and suggest that the supercofling of DNA by this winding enzyme enhances the initiation of transcription. INTRODUCTION

The intracellular DNA of bacteria, bacteriophages and plasmids is subject to topological constraints that function to maintain it in a supercofled con*To whom correspondence should be addreaged and the reprint requests sent.

154 formation (for review, see Champoux, 1978). Recent observations have indicated that supercoiling of DNA duplexes is required for both DNA replication (for review, see Wickner, 1978) and recombination (for review, see Radding, 1978). The degree of supe~oiling of circular double-helical bacteriophage DNA also affects its transcription in vitro; i.e., the presence of negative supercoils stimulates the ability of DNA to act as a template for E. coU RNA polymerase (Hayashi and Hayashi, 1971; Botchan et al., 1973; Wang, 1974; Richardson, 1975; Botchan, 1976; Seeburg et al., 1977). In these studies, it was speculated that supercoiling fac'flitates unwinding of the DNA duplex, thus enhancing binding of DNA polymerase to open DNA. Nalidixic acid, oxolinic acid, coumermycin and novobiocin inhibit DNA replication in K ¢oli. Recently, the p~mary target enzyme for these drugs has been identified as DNA gyrase (Gellert et al., 1976b; 1977; Sugino et al., 1977), which introduces negative superhelical turns into topologically closed DNAs (Gellert et al., 1976a). Thus, these inhibitors would be valuable tools in testing the supercoiling requirement of transcription. Drlica and Snyder (1978) have indicated that coumermycin produces a loss of ~Jpercoiling of E. coil DNA in vivo. This suggests that the DNA is in a mechanically strained state inside the cell and that inhibition of DNA gyrase leads to relaxation of the DNA by a DNA nicking-closing [topoisomerase (Lin and Wang, 1978)] activity. Several reports have appeared which indicate that the synthesis of RNA is affected by those drugs. Nalidixic acid exerts an inhibitory effect on total RNA synthesis in nongrowing E. ¢oli cells starved for the amino acid or treated with chloramphenicol (Javor, 1974). In contrast, RNA synthesis is not significantly affected by the drug in exponentially growing cells (Goss et al., 1965; Winshell and Rosenkranz, 1970). In plasmolysed E. ¢oli cells, nalidixic acid and oxolinic acid partially inhibit RNA synthesis (Staudenbauer, 1976). In E. ¢oli cells infected with bacteriophage $13, synthesis of phage4pecific mRNA is inhibited by nalidixic acid, although bacterial RNA synthesis is unaffected by the drug (Puga and Tessman, 1973). A recent observation, that in vivo RNA synthesis of E. coil bacteriophage N4 by the phage~oded RNA polymerase is inhibited by coumermycin, has suggested a novel requirement of the activity of DNA gyrase for N4 transcription (Falco et al., 1978). Another observation which points to specific participation of DNA gyrase in viral transcription is the in vivo inhibition of the phage-promoter
155 MATERIALS AND METHODS

(a) Bacteria, phages and plasmids The strains of Escherichia coil K-12 used as the sources of RNA were trpC9941 (trpC missense mutant), W3110trpR~ n (tryptophan constitutive and prototroph), BT10266 polA-polB-polC ts (tryptophan prototroph). The following phages and strains of E. coil K-12 were used for infection experiments; non-defective transducing phage, khS°hattS°trp60-3 trpABCDEimmkQSR ~, which replaces the k genes to the left of N with the entire trp operon of E. coli, and host bacteria, trpAE1 (trpR ÷, a deletion of the entire trp operon) and trpAEl(~), lysogenic for k. The trp-transducing phage ~,hs°hattS°trp60-3t~pABCDEimm~QSR ~ was obtained by crossing kh~attS°trp60.3trpABCDEimmkQSR ~ (Y amamoto and Imamoto, 1975) with ~80hS°htrp190htrpABCDE, originally isolated by S. Deeb and B. Hall (see Imamoto and Yanofsky, 1967). The following strains were used as recipient for transformations of the recombinant plasmid DNA; trpAE1, trpAElsuI, trpR-amtrp~ E1 (tryptophan constitutive), NI708 cou s (coumermycin A1- and novobiocin-sensitive, tryptophan prototroph), NI741 cour (coumermycin- and novobiocin-resistant, tryptophan prototroph), 15124 nalA26 (nalidixic acid- and oxolinic acidresistant, tryptophan prototroph). Strains NI708 cou s and NI741 cour were kindly supplied by J. Tomizawa. 15124 nalA26 was donated by M. Oishi. The plasmids pMT60-3 and pMT48 were constructed in vitro as a chimera between mini-ColE1 DNA and the EcoRI.generated trp.N fragment of phage DNAs of ~h~attS°trp60-3trpABCDEimm~QSR ~ and ~h~trp48trpABCDtrp ~DC181Namclts2cro-imm~QSR~ , which have a short deletion internal to the trp operon with deleting the low-efficiency internal promoter, P2trp, distal to trpD (Nakamura et al., 1978; Ishfi and Imamoto, unpublished results). The following phages were used as sources of DNA for DNA~RNA hybridization assays: the non-transducing phages, ~80 and kcI90, as well as nondefective transducing phages, ~80trpED and ~80trpCBA. The genetic map of the phages and plasmids is represented in Fig. 1. (b) Preparation o f phage DNA DNAs of ~80, ~, and ¢80trp phages were prepared as described elsewhere (Yam~noto and Imamoto, 1975) and dissolved in a saline-citrate solution (1 × SSC; 0.15 M NaCI--0.015 M sodium citrate) after dialysis against the solution. (c) Preparation o f pulse-labeled RNA Bacteria or bacteria carrying plasmids were grown with aeration to 6.10 s cells/ml in an enriched medium (L-broth) (Lennox, 195S) supplemented with L-tryptophan (50/~g/ml). In derepression studies, the cells were

156


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Fig. 1. Schematic molecular maps of bacteriophages and plasmids. The genetic map of transducing phages, ~80trp and ~trp, is based on previous studies (Nakamura et al., 1978). The genetic structure of the Xbio phages is based on the data described elsewhere (Lozeron et al., 1977; 8zybalski and 8zybalski, 1979). The map showing the structure of recombinant plasmids is taken from the data of Kano et al. (unpublished results). For the gem symbols see 8zybelski and 8zybalski (1979). Dashed, solid, zigzag, double dashed, double solid and double zigzag lines indicate the region of ~80 genome, ~ genome, 434 genome, miniColE1 genome, bacterial trp genes and bacterial b/o genes, respectively.

collected by centrifugation, washed twice with cold minimal medium (Vogel and Bonnet, 1956), and suspended in the same medium to give a final density of 3-10 z° cens/ml. A p o t i o n (0.2 ml) of the cell suspension was transferred quickly to prewarmed (30°C) minimal medium (3 ml) supplemented with 19 amino acids (each 0.5 mM), but without tryptophan, ~nd the cell suspension was shaken vigorously in a water bath at 30°C. L-Tryptophan (50 ~g/ml) was added to permit studies of transcription uniquely initiated at ~, promoters, P2, PR and p~ (or Pre), of the plasmid. At a suitable time during incubation, the cell suspension was pulse-labeled as indicated with 150 to 300 #Ci of tritiated uridine (19.0 Ci/mmol). Labeling was stopped by rapidly pouring the suspension onto 35 ml of crushed frozen medium containing 1-10-2M Tris- HCI buffer at pH 7.3, 5-10-3M MgCI2, 1-10-2M NaN3 and 250 #gofchloramphenicol/ml, RNA was prepared by the procedure reported previously (lm~moto e t ial., 1965). The !RNA obtained was filtered through a Millipore filter, precipitated ethanol, and dissolved

157

in 1-10-2M Tris- HCI buffer, pH 7.3, containing 0.5 M KC1 and 1-103M NazEDTA or in water (for sedimentation analysis). For infection experiments, bacteria grown to 6-10 s cells/ml in an enriched medium were collected by centrifugation, washed twice with cold T1dilution buffer [6-10~M MgSO4, 5-10-4M CaCl2, 1-10-3% gelatin and 6-10-3M Tris- HCI buffer (pH 7.3)1 and resuspended in the same buffer to give a final density of about 6-10 ~° cells/ml. About 6-109 cells of bacteria were infected with the Xtrp phage at a multiplicity of about 5 in 1.5 ml T1dilution buffer containing 1-10-3M KCN by incubating for 10 min at 30°C. The cells were collected by centri~gation and suspended in 0.1 ml of cold minimal medium (Vogel and Bonnet, 1956) and restored at 0°C less than an hour until labeling. The cell suspension was transfered to 2.8 ml of prewarmed (30°C) minimal medium supplemented with 19 amino acids (each 0.5 mM) excluding tryptophan, and the cell suspension was shaken vigorously in a water bath at 30°C. I~Tryptophan (50 pg/ml) was added in experiments to demonstrate uniquely pL-promoted synthesis of trp mRNA. Pulselabeling and RNA extraction were carried out as described above.

(d ) DNA-RNA hybridization The hybridization procedure was as follows. DNA of ~80, ), or ~80trp was diluted to a concentration of 100 pg/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 concentration of 8 pg/ml in 3 × SSC. 5 ml of the DNA solution was filtered through a Millipore filter (type HA, 0.45 pm 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 [3HI RNA was appropriately diluted, divided into 100/~1 fractions, and annealed to a segment of Millipore filter bearing 5/~g of immobilized DNA from ~80, X and ~80trp phsges, for 18 hm at 66°C. Afterwards, the filter was treated with RNase (5 pg/ml) in 1 X SSC at 37°C for 30 min, washed with 1 × SSC, dried and counted in toluene-based scintillation fluid. The total [3H] uridine incorporated into RNA ([3H] bulk RNA) was measured as the radioactive material precipitable by cold trichloroacetic acid.

(e) Sucrose density-gradient analysis RNA preparation was sedimented in 5 to 30% linear sucrose gradients containing 2-10-ZM Tris-HCI buffer (pH 7.3), 0.1 M l~'aCl, 0.5% sodium dodecyl sulfate and 5-10-3M Na~EDTA for 120 min at 60 000 rev./min in an SW65 rotor at 15°C. After centrifugation the bottom of the tube was punctured and appropriate fractions were collected.

(f) Chemicals Oxolinic acid samples were giftsfrom J.D. Stein, Jr. (Warner-Lambert Research Institute,M o n ~ Plains,NJ). Coumennycin A1 (referred to as coumermycin throughout this paper) was kindly supplied by M. Oishi.

158

Other m t e r i a k and methods used in the present experiment were as described elsewhere (Nakamura et al., I978). RESULTS

(a) Inhibition of general transcription by the drugs acting on DNA gyrase In this section, we show that in vivo transcription directed by the genomes of bacteria, phages and plasm__idsis generally affected by nalidixic acid, oxolinic acid and coumermycin. The effect of these DNA gyrase inhibitors on RNA synthesis in the bacterial strains W3110trpR*trpC9941 and W3110trpR- in the absence and presence of tryptophan, respectively, was investigated. The rate of total RNA and trp mRNA synthesis (pulse-labeled with [3H] uridme at a steady transcription) in the presence or absence of the inhibitors is represented in Table I (lines a and b). The data are expressed relative to the results obtained with untreated control cells. The most pronounced effect of the inhibitors on RNA synthesis in a strain trpC9941 (Table la) was that oxolinic acid and coumermycin caused a substantial diminution of the synthesis of both total RNA and trp mRNA, while nalidixic acid did not significantly affect RNA synthesis even at a dose (100 pg/ml) which was sufficient to block nearly all DNA synthesis. This may be rumply due to less sensitivity of the cells to naUdixic acid than the other two drugs. Oxolinic acid is shown to be about 20-fold more effective in inhibiting DNA gyrase than nalidixic acid (Sugino et al., 1977), although the mode of action of these two agents appears to be identical (Gellert et al., 1977), and coumermycin is also a potent inhibitor of DNA gyrase (Geilert et al., 1976b). It is also noted that synthesis of trp mRNA is selectively and more severely affected by the drugs than the overall synthesis of total RNA. Prefer. ential reduction in the rate of trio mRNA synthesis was observed also in a trpR- constitutive strain growing in the presence of excess tryptophan (Table Ib). Therefore, the observed reduction cannot represent repression of trp mRNA synthesis by an accumulation of tryptophan which might have been some secondary effect of high drug concentrations. The effect of the DNA gyrase inhibitors on the phage
159 TABLE I EFFECT OF DNA GYRASE INHIBITORS ON THE RELATIVE RATES OF THE TOTAL RNA SYNTHESIS OR OF THE trp RNA DIRECTED BY THE PL OR P~p PROMOTERS Strain trpC9941 or W3110trpR- was incubated at 30°C in the absence or presence of L-tryptophan (50 ~g/ml), respectively. Nalidixic acid (hal), oxolinic acid (oxo) or coumermycin (cou) was added at start of incubation at a concentration of 100 pg/ml. Pulse-labeling was carried out with 150 ~Ci of [3H]uridine for I min starting at the 14.5th rain of incubation. Strain trpAEl(z)or trpAE1, infected with ktrp60-3trpABCDE, was incubated at 3O°C in the absence or presence of L-tryptophan, respective!y. Nalidixic avid, oxolinie acid or coumermycin was added at the 8th min of incubation at a concentration of 40 ~g/ml, 40 ~g/ml or 50 izg/ml, respectively. The cultures were pulselabeled for I min starting at the 20th min of incubation. The RNA values are expressed as [3H]RNA/10 ~g RNA (total RNA) or [3H]RNA hybridized with ~80trp DNA/10 pg RNA (trp mRNA) and normalized to 100% for the value of drug-untreated control. The value for trp mRNA is the sum of trpED mRNA and trpCBA m R N A ([3H]RNA hybridized specifically with ~80trpED DNA and ¢~80trpCBA DNA, respectively). The background values for ~80 DNA (7--60 cpm/10 ~g RNA) were subtracted from each hybrid wlue. Values represented are the average of duplicate determinations. The 100% points for total RNA and trp mRNA represented about 7.1-10 + and 2 . 0 103 (a), 2.5.10 s and 7.7-102 (b), 2.4-105 and 3.7-102 (c), and 2.7.. 10 s and 3.9-102 (d) cpm/10 pg RNA, respectively. The other conditions were as described in MATERIALS AND METHODS. Strain

Functioning promoter for trp transcription

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Relative rate of RNA synthesis in presence of none

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mRNA synthesis to 25--64% or 10--28%, respectively, of the level observed in the absence of the drug. Consistent with the previous finding (Smith et al., 1978) is the observation that under these conditions the P~rppromoterdependent transcription of the translocated trp operon is less sensitive to the drug action than transcription derived from the phage promoter. The role of the DNA gyrase as an essential component for the replication of the circular duplex plasmid DNAs has been studied in detail (for review, see CozzareUi, 1977). The drugs such as nalidixic acid, oxolinic acid, coumermycin and novobiocin inhibit the supercofling of these extrachrom-

160

ceomal DNA (Gellert et al., 1976b; Sugino et al., 1977). Because of their physical properties and small size, the plasmid DNAs m useful for studying the mechanism c.f action of DNA gyra~ inhibitors for transcription. The trp DNA fragments obtained from the EcoRI digestion of bacteriophsges ~trp60-3 and ),trp48 (kN-clts2cro-) were closed into the mini-Co[E1 plasmid. In the absence of tryptophan, strain trpAE1 eanying the recombinant plssmid with the tvp60..3 DNA frqment (pMT60-3) exhibited about a 2D-fold increase in the rate of trp mRNA synthesis at 30°C over that of the tzyptophan prototrophic strain not containing pMT60-3. Cells containing the recombimmt plumid with the ~p48 DNA fragment (pMT48) had highly elevated rates of trp mRNA synthesis at 42°C but only repressed levels of the trp mRNA at 30°C (Nakamura et al., unpublished results). Under the above conditions, transcription of the trp genes in the pMT60-3 plasmid is reduced by the trp repressor, while in the pMT48 plasmid is under control of the ~. repressor. Fig. 2, a and b, shows the effect of oxolinic acid and coumermycin, respectively, on the amount of plmunid-specific mRNA synthesized by the strain trpAEl carrying pMT60-3 during a short pulse of [3H] uridine, in comparison with the effect on the total RNA synthesis directed by the host

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Fig. 2. RNA synthesis of bacteria carrying pMT60-3 plmmid at various doses of oxolinic acid (oxo) or ¢oumermycia (eou}. Strain trpAE1 emzying pMT60-3 was incubated in the absence of tryptophan at 30°C at indicated doses of (s) oxolinie acid or (b) eoumermycin, The drug was added at the 8th rain (a) or at the start (b) of incubat/on. Pulse-labeling was carried out with 150 ~Ci of [3H]uridine for I mln starting at the 14.5th rain of incubation. The ordln.te represents values relative to those obtained in the drug-untreated control culture. The values with @80 DNA baeksround (10--60 c p m l l 0 ~g RNA) were subtracted from each hybrid velue. The 100% points for total RNA, trp mRNA and ~, mRNA ([~H]RNA hybridized with ~,c190 DNA)represent about 1.5.10 s, 7.9-103 and 6.2-10 = (a). and 1.8,10 s. 8,6- I0 z and 9.9-10 z (b) cpml10 #g RNA, respectively. The other conditions were as described in Table [ and MAI[MRIALS AND M E T H O D S . . , total RNA; e , / r p mRNA; o, x mRNA.

161

bacteriL The maximum inhibitory effect of these drugs on RNA synthesis was observed at 100 pg/ml and an increase to 200 pg/ml for oxolinic acid or 400 pg/ml for coumermycin did not give any further inhibition. In the presence of the high doses of oxolinic acid or coumermycin, the overall rate of plasmid~iirected trp mRNA synthesis decreased to approx. 14% or 8%, respectively, of that found in the untreated control. The inhibition curves indicated the lesser inhibition o f synthesis for the total RNA and plasmid~iirected ~, mRNA, thereby suggesting that a certain fraction of these transcripts may be resistant to the inhibitors.

(b) Evidence for direct participation of DATA gyrase in transcription It is possible that diminution of transcription in the drug-treated cells is simply due to a reduction in the number of DNA templates available for transcription or results from some indirect effect caused by inhibition of DNA replication. Therefore a control experiment was carried out employing a mutant bearing a temperature.sensitive DNA polymerase III and lacking DNA polymerase I and II (polA-polB-polC~S). Addition of oxolinic acid to the cells incubated at the restrictive temperature (42°C) led to inhibition of the synthesis of both trp mRNA and total ENA (Fig. 3b) as was observed with the control cells incubated at the permissive temperature (Fig. 3a). Under these conditions, the overall rate of [3H] thymidine incorporation into DNA was reduced at the restricted temperature to about 5% of the

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DNA replication. Strain BTlO266polA-polB-poIC t" was incubated in the absence of tryptophan at 30°C (a) or 42°C (b) at indicated concentrations of oxolinic acid (oxo), which was added at start of incubation. Pulse-labeling was carried out with 150 ~Ci of [~H]uridine for I rain starting at the 14.5th rain (a) or 9.5th rain (b) of incubation. The 100% points for total RNA, trpED mRNA and trpC.BA mRNA represent about 1.6-10 s, 1.1-103 and 7.6-102 (a), and 7.8.104, 7.9-102 and 5.6-102 (b) cpm/10 ~g RNA, respectively. The other conditions and representations were as described in Fig. 2 and MATERIALS AND METHODS. s, total RNA; e, trpED mRNA; o, trpCBA mRNA.

162

rate observed at 30°C (data n o t shown). Therefore, the observed inhib'm'on o f transcription by the d r ~ arisas independently of DNA replication. In order to clarify the p o i n t that the d r u p inhibit transcription through their effect on host bacterial DNAsyrase , the sensitivity to oxolinic acid and coumermycin o f R N A oynthesis drug-resistsnt DNA gyrme was invastJl~ted. £ . coil DNA Consists of two subunits identiffed a s t h e products of t w o genes, ~ and ¢ou, ~ determine resistance t o nalidix/c acid or oxolinic acid, and t o coumermycin or novobiocin, respectively (Gellert et al., 1976b; 1977; S t q ~ o et al., 1977; ~ et al., 1978). Fig. 4 shows results o f experiments employing the coumermycinresistant m u t a n t (N1741) ~ the pMT48 plasmid. Less inhibition by coumermycin of either total RNA synthesis and plasmid-directed trp m R N A synthesis was observed (Fig. 4a) even under conditions where 90% of transcription was inhibited in the isogenic coumermycin-sensitive (N1708) containing the plasmid (Fig. 4b). In contrast, oxolinic acid severely inhibited transcription in the coumermycin-resistant strain containing pMT48 (Fig. 4c) as well as the drug-sensitive cells e.~u~ying the plasmid

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Fig. 4. Effect of eoumermyein and oxolinie acid on RNA synthesis in the eoumermyeinsensitive or resistant pMT48 carriers. Strain N1741cour (a and e)mad NlT08eou s (b and d), carrying pMT48 plasmid, were incubated at 42°C in the presence of L-tryptophan (50 ~g/nd) at indicated concentration of eoumermyein (a and b) or oxolinie acid (e and d). The drugs were added at the ~ of incubation. Pulse4abelini| was carded out with 150 ~Ci of [SH]uridine for I rain starting at the 10th rain of incubation. The 100% points for total RNA and trp mRNA represent about 1.6.10 s and 9.2-10' (a and e), and 2.9-10 s and 8.9° 103 (b and d) cpm/10 Isg RNA, ~ i v e l y , The other conditions and representations were as described in Fig. 2 and M A T E R ~ AND METHODS. s, total RNA; o, trp mRNA.

163

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Fig. 5. Effect of oxolinie acid and coumermycin on RNA synthesis in the oxolinic acidsensitive (na/A s) or resistant (na/A r) pMT48-plasmid carriers. Strain 15124na/A r (a and b) and trpAElsul (c and d), carrying pMT4S plasmid, were incubated at 42°C in the presence of L-tryptophan (50 ~g/ml) at indicated concentration of oxolinie acid (a and c) or coumermycin (b and d). The drugs were added at start of incubation. Pulse-labeling was carried out with 150 ~Ci of [~H]uridine for I rain starting at the 10th min of ineubation. The 100% points for total RNA and trp mRNA represent about 1.2.10 s and 6.8-103 (a and b), and 3.5-10 s and 2.0,10" (c and d) cpm/10 ~g RNA, respectively. The other conditions and representations were as described in Fig. 2 and MATERIALS AND METHODS. s, total RNA; e, trp mRNA. (Fig. 4d). In a nalidixic acid-resistant m u t a n t (15124r~/A26) containing pMT48, inhibition o f transcription by oxolinic acid was completely (in the case of bulk RNA synthesis) or partially (in the case of trp m R N A synthesis) alleviated (Fig. 5, a and b). Transcription directed by the nalidixic acid-sensitive strain t r p A E l s u f ( p M T 4 8 ) was severely reduced by either oxolinic acid and c o u m e n n y c i n (Fig. 5, c and d). These results indicate t h a t the function of D N A gyrsse is absolutely required for in vivo transcription in E. coli. An alternative to the hypothesis t h a t the antibiotics cause a direct inhibition o f transcription is the possibility t h a t they interfere indirectly with the overall rate of R N A synthesis by enhsncing the rate of R N A degradation. This possibility was examined by following the decay of preformed

164

trp mRNA of a trpR- constitutive strain in the presence or absence of oxolinic acid. The cells were exposed to a 2-min pulse of [3H] uridine during ~ ~ s t a t e tnmscription in the presence of excess t,tryptophan. Any further incorporation of [3H] uridine into mRNA was prevented by adding rifampicin and by diluting the label with an excess of unlabeled uridine. Oxolinic acid was added together with rifampicin. Under the corresponding condition, oxolinic acid e x e ~ quickly its effect on trp mRNA synthesis; it reduced the synthesis by about 60% within five minutes after the antibiotic addition (data not shown). The pattern of trp mRNA degradation, shown in Fig. 6, is consistent with the previous observation (Yamamoto and Imamoto, 1975). The trp£D mRNA decayed exponentially with a halflife of ~bout 3 min. The complete transcription of the trp£D genes and trp£DCBA genes requires about 3 and 6 min, respectively, under the conditions employed (Imamoto, 1969; 1970). Transcription of the trpCBA region was completed about 4 to 5 min after the addition of rifampicin. At that time trpCBA mRNA began to decay at slight]y dower rates as trpED mRNA. It can be seen that oxolinic acid did not significantly increase the rate of nascent trp mRNA degradation.

(¢) Diminished tran~ription results from lesser frequency of RNA chain initiation The reduction of the overall rate of transcription by the antibiotics could result either from a decrease in the frequency of initiation of RNA synthesis or premature arrest of RNA chain elonption. In an effort to distinguish among these two alternatives, we have obtained three lines of evidence which favors the former possibility. First, oxolinic acid blocks preferent~ly initiation of mmseription of the tvp operon rather than transcription already in pzogrem along the operon. In bacterial operons, the cistron nearest to the operator is the f'mt to be transcribed, followed by the others in order. For example, mRNA sequences from the tvp£-t~D (trp£D) region are made before mRNA is synthesized from the tvpC-trpB-tvpA (tvpCBA) r e , o n when synthesis is initiated at the promoter of the trp operon (Imamoto, 1969). When further initiation of transcription is blocked at the promoter, the synthesis of different regions of trio mRNA ceases in the same order as the s~metural genes (Imamoto, 1970). As shown in Fig. 7, oxolinie acid added to a t~R-tvpAE1 strain carrying pMT60-3 during steady~state transcription of the tvp operon acted quickly to shut down initiation of trp mRNA synthesis; as RNA polymerase completed their final transit of the tvp operon after addition of oxolinie acid, [3H] uridine added at pro~:~ively later times was incorporated in pro~essively more 5'-distal portion of the tvp mRNA in higher proportion, thus exhibiting a characteristic sequential diminution of transcription in the order, trpED to trpCBA. Reduction in the overall rate of mRNA synthesis could result from frequent premature arrest of transcription in the antibiotic-treated cells,

165

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I

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I

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e

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J

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Time after additionof rtfampicin (rain)

Time

10

after addition of

lS

oxolimc acid

~mm,

Fig. 6. Effect of oxolinic acid on chemical stability of trp mRNA controlled by a bacterial PIrD promoter. Strain W3110trpR- was incubated in the presence of L-tryptophan (50 ~g/ml) at 30 C. Pulse-labeling (1 ram) camed out w,th 150 ~Cl of [ H]urndme at the 14.5th rain of incubation was immediately followed by addition of rifampicin (300 izg/ml) and unlabeled uridine (1 mg/ml) in the presence (o, v ) or absence (e, • ) of oxolinic acid (100/zg/ml). At the indicated times, eJiquots of the cultures (6-109 cells) were removed for RNA extraction. The b~ mRNA values are expressed as the amount of ['H]RNA hybridized per 10 ~g of RNA and normalized to 100% for the maximum value. The 100% points for trpED mRNA and trpCBA mRNA represent about 3.5-102 and 4.0-102 cpm/10 ~g RNA, respectively. These values represented the averages of duplicate determinations. The other conditions were as described in MATERIALS AND METHODS. o and e, trpED mRNA; v and • , trpCBA mRNA. -

"

O

"

"

"

"

"

3

"

"

Fig. 7. Sequential cessation of transcription within the trp operon after addition of oxolinic acid. Strain b~R'trpA£1 carrying pMT60-3 was incubeted in the presence of L4ryptophan (50 ~g/ml) at 30°C. Oxolinic acid (100 ~g/ml) was added at the 15th min of incubation, and immediately thereafter (zero time) and at successive times indicated, aliquots of the culture (6-10' ceils) were pulse-labeled for I min with 150 ~Ci of [ 3H]uridine. The amount of ['HI RNA hybridized/10 ~g of RNA are normalized to 100% of the maximum wdue and plotted at the mid-point of each pui~-period. The 100% points for trpED mRNA and i~pCBA mRNA represent about 6.3-10 s and 3.5-103 cpm/10/zg RNA, respectively. The other conditions were as described in MATERIALS AND METHODS. e, trpED mI~NA; o, trpCBA mRNA. T o test this possibility, we investigated t h e molecular sizes o f R N A synthesized by a strain t r p A £ 1 carrying p M T 4 8 in the presence of relati,lely low doses of oxolinic acid, such as 50 ~ g / m l and 100 ~g/ml, which inhibited the synthesis of trp m R N A o r bulk R N A by 61% and 88% or 47% and 74%, respectively. Fig. 8 shows the sed!_mentation profdes o f these RNAs labeled f o r a s h o r t period during steady.state ~ m s c r i p t i o n in the cells at 420(:. Such profiles generally e x h i b i t a heterogeneous distribution, presumably because a p o p u l a t i o n o f nascent R N A molecules o f varying length is labeled ( I m a m o t o e t al., 1970). The trp m R N A molecules bearing sequences for the p r o m o t e r -

166 8

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16s

I,s

Csnt~

b

i'_

T?

J 4=

¢

23s

o.o

I I ~ Iqllml)

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ISs ~,

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Fig. 8. Sedimentation prorde of RN& synthesized in cells carrying pM'I'48 in the presence or abunce of ozolinie ~id. 8L~ain L~rpA£1 ~Tyinll p]~'48 w ~ ineubsted in the presence of L-tryptoplum (60 ~81ml) at 42°C with (b and c) or without (s) oxolinic acid. Oxolinic acid was added at the concentration of 50 ~fJlml (b) or 100 ~fj,Ind (e), at start of incubation. Pulse4abelinfJ was carried out with 160 ~Ci of [ SH]uridine for I rain starting at the 10th min of incubation. The [3H]RNA was eosedimented with [ " C ] R N A ( 2 - 1 0 4 epml•g), prepared from strain RF8622 as described previously (Imamoto, 19"/3), which served as a source of 238 rRNA, 168 rRNA and 48 tRNA markers. After centrifugation, 29--30 fractions were collected, and a 10-~I portion from each fraetion was used for the determination of [ " C ] R N A and [SH]total RNA. Each fraction was diluted to a volume of about 0.4 m| with 4.6 x 88C to attain a concentration of 3 x 88C. An 100 ~I portion from eaeb fraction was used for trp mRNA assay by hybridization with DNA from ~80, ~80trp~D or e80trpOBA phafJes. The m o u n t of [~H] RNA sedhnented was 2.1-10' (a), 1.1-10" (b) or 5.4- I0 s (e) epm. The other eonditio~ were as described in MATERIALS AND METHODS. e, trp£D mRNA; o, trpCBAmRNA; o, total RNA. Arrows in the figure indicate position of 238 rRNA, 168 rRNA and 48 tRNA.

distal tvpCBA region o f t h e o p e r o n s e d i m e n t e d faster t h a n t h o s e e n c o d e d b y t h e p r o x i m a l typED r e g i o n . T h i s c o n f i r m s t h e 3'-distal l o c a t i o n o f tvpCBA in t h e p o l y c i s t r o n i c transcripts. T h e trpCBA m R N A species f r o m antibiotict r e a t e d cells also s e d i m e n t e d fester t h a n t h e t r p £ D m R N A species (Fig. 8, b a n d c), thus, e.y~hibiting nearly t h e s a m e size d i s t r i b u t i o n as t r p m R N A

167

from untreated control cells (Fig. 8a). Pulse-labeled bulk RNA from oxolinic acid-treated cells did n o t exhibit any significant difference in the sedimentation profiles from that of the RNA from the untreated control. Therefore, the observed inhibitory effect of the drug appears to result primarily in a block in initiation of transcription. In pMT48, three types of major transcription can be identified. These are pL-promoted transcription of the N-trp operon, pR-promoted transcription of the tof region and probably p~-promoted transcription of the cI region (see Fig. 1). The transcripts arising from the promoter p a or p~ were assayed by employing the r-strand or the/-strand, respectively, of ~bio3h-1 DNA as a DNA probe in DNA-RNA hybridization. In ~bio3h-1 most segments of the leftward genome located downstream from the PL promoter are replaced by heterolo~;ous E. ¢oU DNA (Blattner et al., 1974). The PL-RNA was determined as a difference of hybridiz.ed RNA fractions with the/-strands of ~bio256 DNA and Xb/o3h-1 DNA (Lozeron et al., 1977). The pL-directed trp mRNA was assayed here by using the/-strands of q~8OtrpED and ~80trpCBA DNA. Fig. 9 compares the rates of transcription at various doses of oxolinic acid or coumermycin added to the cultures at 42°C of a strain a

b

I0

i

! v

100 oxo /uglmll

o

~o

~

~

~o

Fig. 9. Oxolinic acid and coumennycin sensitivities of RNA synthesis directed by various promoters in pMT48-plasmid carriers. Strain trpAElsul carrying pMT48 was incubated in the presence of L-tryptophan (50 ~g/ml) at 42°C at indicated concentrations of oxolinic acid (a) or coumermyein (b). The d r u b were added at the start of incubation. Pulselabeling was earried out with 150 uCi of [3H]uridine for I rain starting at the 10th rain of incubation. DNA strands of bacteriophages were separated according to Szybalski et ai. (1971). DNA-RNA hybridization assay with the single-stranded DNAs was earried out as described previously (Tani and Imamoto, 1975). The 100% points for total RNA, trp mRNA, pL-N mRNA, Plt mRI~A and p~ mRNA represent about 1.4-10 s, 1.5-104, 2.2.103, 1.1.103 and 2.2.103 (a), and ~..6.10 s, 1.7-10", 1.7-103, 1.4-103 and 9.2.103 (b) cpm/4 ug RNA, respectively. The other conditions and representations were as described in Fig. 2, MATERLAL8 ~ D METHODS and text. m, total R N A ; . , PL-trp mRNA; o, pL-N mRNA; a, Pit mRNA; ~,, PE mRNA.

trpAElsul c ~ pMT48. There was more than 85% reduction in the rates ofpLdirected synthesis of N mRNA and trp mRNA at relatively high concenWations of the drugs employed. In contrast, transcription initiated at the PR promoter was inhibited to a lesser extent; the rate of Pa mRNA synthesis was reduced by only about 50% at concentrations of 50--200 pg oxolinic acid per ml or by about 2 0 - - 5 ~ at concentrations of I00--400 pg coumermycin per m!. The inhibition of ps-directed transcription appears to be slightly, yet discernibly, less than pw-directed transcription. A lesser inhibition observed with the effect of the drugs on bulk RNA synthesis is consistent with the data represented in the foregoing sections. Thus, these antibiotics exhibit different degrees of inhibition with different promoters on the same piece of DNA. D~U~ON

E. coli DNA gyrase is composed of two subunits. One of the subunits is

th~ product of the na/A gene, which controls sensitivity to oxolinic acid and nalidixic acid (Gellert et al., 1977; Sugino et al., 1977). The other subunit is the product of the cou gene, which specifies sensitivity to comnermycin and novobiocin (Gellert et al., 1976b; Higgins et al., 1978). The n d A subunit is believed to have a nicking~losing activity that is capable of relaxing supercoiled DNA and the cou subunit is possibly involved in energy transduction during the mpercoiling reaction. DNA gyrase is assumed to be responsible for the maintenance of negative mpercoiling in the cellular DNA (Drlica and Snyder, 1978). Phage ~, DNA superinfecting a ~-lysogenic bacterium treated with coumermycin or oxolinic acid is recovered in the relaxed closed~ircular form (Getlert et al., 1976b; 1977). Inhibition of DNA gyrase activity by coumermycin, as well as nalidixic acid, produces in vitro a re!aation-type complex between DNA gyrase and the supertwisted ColE1 DNA. This complex is converted to linear DNA by treatment with sodium dodecyl sulfate and protease (Sugino et al., 1977; Gellert et al., 1977). It is not evident however whether these reflect some similarities to Ln vivo processes that produce the drug-induced relaxed DNA. The most straightforward interpretation fro" the present findings is that DNA gyrase is involved in modifying the structure of topologically closed circular DNA. Several reports have appeared, which indicate that the superhelical turns of the closed~irculsr duplex DNA influence its properties as a template for RNA synthesis. In in vitro studies employing bacteriophage DNAs, such as ~X174 replicative allomorphic DNA (Hayashi and Hayashi, 1971), ~ DNA (Botchan et al., 1973; Botchan, 1976), PM2 DNA (Wang, 1974; Richardson, 1975) and ¢.d replicative form DNA (Seeburg et al., 1977), it has been shown that either the rate of t r ~ p t i o n or the rate of formation of initiation complexes with E. ¢oli RNA polymerase is enhanced with supercoiled DNA. These observations ~ the possibility that supercoiling in the DNA template facilitates the opening of base p B in DNA,

169

allowing the formation of a productive open promoter complex with RNA polymerase bound to the DNA. As an alternative possibility, it is tempting to speculate that the degree of superhelicity influences the ability of RNA polymerase to recognize promoter sequences in DNA template. In view of this, interesting is the recent demonstration that hairpins or stem-and-loop structures of DNA can be favorably formed when the palindromic sequence is embedded in a supercoiled molecule (Gellert et al., 1978). In most cases, less inhibition by the antibiotics was observed with bacterial bulk RNA synthesis than with synthesis of trp mRNA. In bacteriophage Xtrp60-3, upon superinfection of a k-lysogenic bacterium, inhibition of the Pup
We thank Dr. Cassandra L. Smith for her critical reading of the manuscript. REFERENCES

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171 Lin, L.F. and Wang, J.C., Micrococcus lute~s DNA gyrase: Active components and a model for its supercofling of DNA, Proc. Natl. Acad. Sci. USA, 75 (1978) 2098--2102. Lozeron, H.A., Anevski, P.J. and Apirion, D., Antitermination and absence of processing of the leftward transcription of coliphage lambda in the RNAase IH-deficient host, J. Mol. Biol., 109 (1977) 359--365. Nakamura, H., Kano, Y. and Imamoto, F., Restoration of polarity by N-deficiency in iambda phage containing a translocated trp operon segment, Mol. Gen. Genet., 159 (1978) 13--20. Puga, A. and Tessman, I., Mechanism of transcription of bacteriophage $13, H. Inhibition of phage-specific transcription by nalidixic acid, J. Mol. Biol., 75 (1973) 99--108. Radding, C.M., Genetic recombination: Strand transfer and mismatch repair, Annu. Rev. Biochem., 47 (1978) 847--880. Richardson, J.P., Initiation of transcription by Escherichia coil RNA polymerase from supercofled and non~upercoiled bacteriophage PM2 DNA, J. Mol. Biol., 91 (1975) 477--487. 8eeburg, P.H., Niisslein, C. and Schaller, H., Interaction of RNA polymerase with promoters from bacteriophage fd, Eur. J. Biochem., 74 (1977) 107--113. Segawa, T. and Imamoto, F., Diversity of genetic transcription, II. Specific relaxation of polarity in read-through transcription of the translocated trp operon in bacteriophage lambda trp, J. Mol. Biol., 87 (1974) 741--754. Smith, C.L., Kubo, M. and Imamoto, F., Promoter-specific inhibition of transcription by antibiotics which act on DNA gyrase, Nature, 275 (1978) 420--423. Staudenbauer, W.L., Replication of Escherichia coli DNA in vitro: Inhibition of oxolinic acid, Eur. J. Biochem., 62 (1976) 491--497. $ugino, A., Peebles, C.L., Krenzer, K.N. and Cozzarelli, N.R., Mechanism of action of nalidixic acid: Purification of E. coli naiA gene product and its relationship to DNA gyrase and a novel nieking~losing enzyme, Proc. Natl. Acad. Sei. USA, 74 (1977) 4767--4771. 8zybalski, W., Kubinski, H., Hradccna, Z. and Summers, W.C., Analytical and preparative separation of the complementary DNA strands, in Grossman, L. and Moldave, K. (Eds.), Method in Enzymology, Vol. 21, Academic Press, New York, 1971, pp. 383-413. Igzybaiski, E.H. and Szybalski, W., A comprehensive molecular map of bacteriophage iambda, Gene, 7 (1979) in press. Tani, 8. and lmamoto, P., Fusions of the trp and N messagesynthesized originating at thePL promoter in lambda trp phage, J. Mol. Biol., 92 (1975) 305--309. Vogel, H.J. and Bonner, D.H., Acetylornithinase of £scherichia coli: Partial purification and some properties, J. Biol. Chem., 218 (1956) 97--106. Wang, J.C., Interaction between twisted DNAs and enzyme: The effeetors of superhelieal turns, J. Mol. Biol., 87 (1974) 797--816. Wickner, 8.H., DNA replication proteins of Escherichia ¢oli, Annu. Rev. Biochem., 47 (1978) 1163--1191. WinshelI, E.B. and Rosenkranz, H.S., Nalidixic acid and the metabolism of Escherichia ¢oli, J. Bacteriol., 104 (1970) 1168--1175. Yamamoto, T. and Imamoto, F., Differential stability of trp messenger RNA synthesized originating at the trp promoter and Pw. promoter of lambda trp phage, J. Mol. Biol., 92 (1975) 289--309. Communicated by W. Szybabki.