Vol. 140, No. 2

JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 424435

0021-9193/79/11-0424/12$02.00/0

Escherichia coli Mutants Thermosensitive for Deoxyribonucleic Acid Gyrase Subunit A: Effects on Deoxyribonucleic Acid Replication, Transcription, and Bacteriophage Growth KENNETH N. KREUZER"2 AND NICHOLAS R. COZZARELLI`3* Committee on Genetics' and the Departments of Biochemistry' and Biophysics and Theoretical Biology,3 The University of Chicago, Chicago, Illinois 60637

Received for publication 10 August 1979

Temperature-sensitive nalA mutants of Escherichia coli have been used to investigate the structure and functions of deoxyribonucleic acid (DNA) gyrase. Extracts of one such mutant (naUA43) had thermosensitive DNA gyrase subunit A activity but normal gyrase subunit B activity, proving definitively that nalA is the structural gene for subunit A. Extracts of a second nalA (Ts) mutant (naLA45) had a 50-fold deficiency of gyrase subunit A activity. The residual DNA supertwisting was catalyzed by the mutant DNA gyrase rather than by a novel supertwisting enzyme. The naLA45(Ts) extract was also deficient in the nalidixic acid target, which is defined as the protein necessary to confer drug sensitivity to in vitro DNA replication directed by a nalidixic acid-resistant mutant extract. Thus, gyrase subunit A and the nalidixic acid target are one and the same protein, the naA gene product. Shift of the naLA43(Ts) mutant to a nonpermissive temperature resulted in a precipitous decline in the rate of [3H]thymidine incorporation, demonstrating an obligatory role of the nalA gene product in DNA replication. The rates of incorporation of [3H]uridine pulses and continuously administered [3H]uracil were quickly reduced approximately twofold upon temperature shift of the naLA43(Ts) mutant, and therefore some but not all transcription requires the naA gene product. The thermosensitive growth of bacteriophages 4X174 and T4 in the naLA43(Ts) host shows that these phages depend on the host naA gene product. In contrast, the growth of phage T7 was strongly inhibited by nalidixic acid but essentially unaffected by the naLA43(Ts) mutation. The inhibition of T7 growth by nalidixic acid was, however, eliminated by temperature inactivation of the nalA43 gene product. Therefore, nalidixic acid may block T7 growth by a corruption rather than a simple elimination of the nalidixic acid target. Possible mechanisms for such a corruption are considered, and their relevance to the puzzling dominance of drug sensitivity is discussed.

Escherichia coli DNA gyrase (Eco DNA topoisomerase II), an enzyme which introduces negative supercoils into covalently closed duplex DNA, was discovered as a result of its role in phage A integrative recombination in vitro (12). The energy required by gyrase to drive the DNA into the supercoiled form is provided by ATP, as indicated by the requirement for ATP in the supertwisting reaction (12) and the intrinsic DNA-dependent ATP hydrolyzing activity of gyrase (25, 39). The closely related nalidixic and oxolinic acids block DNA gyrase activity, and drug-resistant mutations in the nalA gene render the enzyme resistant to these two inhibitors (11, 40). These inhibitors appear to trap a gyrase reaction inter424

mediate in vitro, since detergent treatment of an inhibited reaction results in double-strand breakage at specific sites on the DNA, with protein remaining covalently attached to the 5' termini (11, 26, 28, 40). Novobiocin and coumermycin Al also inhibit DNA gyrase, and mutations of the cou gene can render gyrase insensitive to these two drugs (13). These inhibitors block gyrase activity by preventing the binding of ATP to the enzyme (25, 39). It has been proposed that DNA gyrase is composed of subunits, termed A and B, which are the products of the naU and cou genes respectively (11, 16, 25,,40). Each gyrase subunit has been separated and purified alone using the reconstitution of activity in the presence of the complementary

425

VOL. 140, 1979

DNA GYRASE SUBUNIT A MUTANTS

subunit as an assay (16). It is likely that DNA gyrase maintains intracellular DNA in a state of negative superhelical tension, since coumermycin A1 markedly reduces the supertwist density of phage X DNA and the folded E. coli chromosome (8, 13). The role of gyrase in E. coli macromolecule synthesis has also been tested using gyrase inhibitors (5, 14, 30, 34, 36, 37). Nalidixic and oxolinic acids as well as novobiocin and coumermycin Al are potent inhibitors of replicative DNA synthesis. The role of the enzyme in transcription is less clear, since gyrase inhibitors did not block the synthesis of RNA as effectively as that of DNA. However, some inhibition of overall RNA synthesis was generally found, particularly when higher doses were employed, and very recent reports suggest that gyrase inhibitors block the synthesis of only a subset of RNA species (9, 31, 33). Recently, we isolated temperature-sensitive mutations that were demonstrated to be alleles of nalA by mapping and complementation tests (20). The conditional lethality conferred by each of these nalA(Ts) mutations proved that the naA gene product is an essential E. coli protein. [Phenotypes conferred by classes of nalA mutations are indicated in parentheses following the gene symbol as follows: (Ts), thernolabile gene product, and (R), resistance to nalidixic and oxolinic acid.] In the present communication, we provide confirmation that nalA is the structural gene for subunit A of DNA gyrase by showing that subunit A activity from one of these mutants is thermosensitive and that that from another is grossly deficient. We also show that shift of a nalA(Ts) mutant to restrictive temperature results in a dramatic inhibition of both replicative DNA synthesis and transcription, thereby providing direct evidence for a role of DNA gyrase subunit A in both processes. This approach is complementary to the in vivo studies using gyrase inhibitors, because it bypasses problems that can be associated with drug permeation, metabolism, and secondary targets. The results presented here also implicate the naA gene product in the growth of bacteriophages 4X174 and T4, since both phages displayed thermosensitive growth in the nalA(Ts) host, consistent with previous reports of their sensitivity to nalidixic acid (2, 24, 43). An unexpected finding was that the growth of phage T7 in the naUA(Ts) host was essentially unimpaired at the nonpermissive temperature, despite its sensitivity to nalidixic acid at the permissive temperature. However, thermal inactivation of the naUA(Ts) gene product eliminated the nalidixic acid sensitivity of T7, and therefore we

propose that this drug sensitivity results from the formation of an inhibitory drug-enzyme complex (see Discussion). MATERIA4LS AND METHODS Bacteria. The Escherichia coli strains H560

(naLA), H560-1 [naLA48(R)], KNK402 [naUA43 (Ts)], and KNK404 [nalA45(Ts)] are isogenic except for nalA and have the following genotype: F+ polAl endA2 thyA rpsL tsx-79 phx (20). Strains KNK453 [naUA43(Ts)] and HF4704 (naLAU) are also isogenic except for nalA and have the following genotype: FpolA thyA uvrA phx.

Media and chemicals. Minimal medium contained

46 mM K2HPO4, 23 mM KH2PO4, 8 mM (NH4)2S04, 0.4 mM MgSO4, 6 ,tM FeCl3, 1 mM sodium citrate, 1 ,ug of thiamine per ml, and 5 mg of glucose per ml. Rich medium contained, per milliliter, 10 mg of tryptone (Difco), 5 mg of yeast extract (Difco), 10 mg of KCl, and 10 ,ug of thymine. Medium A contained 30 mM K2HPO4, 22 mM KH2PO4, 7.6 mM (NH4)2SO4, 0.4 mM MgSO4, 1.2 mM sodium citrate, 2 jig of thiamine per ml, 20 ,ug of thymine per ml, 10 mg of glucose per ml, and 10 mg of Casamino Acids (Difco) per ml. Agarose (type II) and nalidixic acid were from Sigma Chemical Co., DEAE-cellulose (DE52) was from Whatman, oxolinic acid was a gift from Warner-Lambert Research Institute, and coumermycin Al was a gift from Bristol Laboratories. 4X174 replicative form I (RFI) and ColEl DNA were prepared as described (38,42), and the ColEl DNA was relaxed with rat liver DNA-untwisting enzyme (4). [3H]thymidine (55 Ci/ mmol), [3H]uridine (27 Ci/mmol), and [3H]uracil (19 Ci/mmol) were purchased from Schwarz-Mann. Preparation of enzymes. Bacteria were grown in medium A, harvested late in the exponential phase, and lysed as described (44). The crude extracts were then clarified by centrifugation, nucleic acids were precipitated with streptomycin sulfate, and supernatant proteins were concentrated by precipitation with ammonium sulfate as described by Sugino et al. (40). The streptomycin sulfate supernatant is referred to as fraction I, and the dialyzed ammonium sulfate resuspension is called fraction II. For further purification of the strain KNK402 enzymes, fraction II, containing 54 mg of protein, was applied to a DE52 column (0.95 by 15 cm) equilibrated with 50 mM Tris.hydrochloride (pH 7.5), 10% glycerol, 10 mM 2-mercaptoethanol, 1 mM EDTA, and 25 mM NaCl. After a 25-ml buffer wash, the column was developed with a 70-ml linear gradient of 25 to 500 mM NaCl in column buffer. Subunit A and B activities were partially resolved by this column; the subunit B pool was relatively free of subunit A activity, whereas the subunit A pool contained substantial subunit B activity. Except where noted, subunits A and B from wild-type strain H560 were purified through the hydroxylapatite step as described by Higgins et al. (16). Protein concentrations of fraction I and DE52 pools were determined by the method of Lowry et al. (23), and those of fraction II

pools were determined by optical density measurements (21). Enzyme assays. The DNA gyrase assay measured

the introduction of negative supercoils into closed

426

J. BACTERIOL.

KREUZER AND COZZARELLI

duplex ColEl DNA (12). The reaction mixture (17 Id) contained 35 mM Tris -hydrochloride (pH 7.6), 18 mM potassium phosphate, 6 mM MgCl2, 5 mM spermidine. hydrochloride, 1.4 mM ATP, yeast tRNA at 90,ug/ml, 5 mM dithiothreitol, bovine serum albumin at 50 ftg/ ml, and 23 fmol of relaxed CoIEl DNA. The reaction was stopped by the addition of 1% sodium dodecyl sulfate, and the production of supercoiled DNA was monitored by agarose gel electrophoresis as described (40). One unit of gyrase catalyzes the supertwisting of 23 fmol of DNA in 30 min. The assays for gyrase subunit A and B activities were identical to the gyrase assays, except that an excess of the complementary subunit purified from strain H560 (naU+) was provided in the reaction mixture (16). The nalidixic acid target protein, Pnal, was measured using the in vitro complementation assay described by Sugino et al. (40). In this assay, 4X174 RFI replication directed by an extract from an nalA (R) strain (H560-1) is rendered sensitive to oxolinic acid (100,ug/ml) by the addition of protein from drug-sensitive cells. One unit of Pnal confers oxolinic acid sensitivity to 1 nmol of dTMP incorporation in 60 min at 300C. Temperature shift experiments. For pulse-labeling, cultures were incubated with shaking at 300C in minimal medium supplemented with thymine and uracil at 5 ,ug/ml and Casamino Acids at 500 jLg/ml. The turbidity of the cultures was monitored with a Klett photometric colorimeter using filter no. 66 (red). At a turbidity of approximately 25 Klett units, the cultures were transferred to 43°C. At times thereafter, 0.2 ml of culture was added to 2.5 ,Ci of [3H]thymidine, 2.5 ,LCi of [3H]uridine, or 25 ,uCi of [3H]isoleucine in 0.1 ml of growth medium. After 1 min at 430C the pulses were stopped by the addition of trichloroacetic acid (10%) at 00C, and acid-precipitable radioactivity was determined after filtration onto glass fiber filters (Whatman GF/C). For the experiments measuring continuous [3H]uracil labeling, the cells were first grown at 300C in minimal medium supplemented with thymine and uracil at 25 ,ug/ml and Casamino Acids at 500 Lg/ml. At a turbidity of 5 Klett units, [3H]uracil was added to 1.25 jtCi/ml, and 130 win later the temperature was increased to 43°C. At various times after temperature shift, acid-precipitable radioactivity in 0.5-ml samples of the cultures was determined as described above. Phage growth experiments. The bacteria were grown at 320C in rich medium; 2 mM CaC12 was added for adsorption of 4X174. At a turbidity of 30 Klett units (approximately 10' cells per ml), portions of the culture were transferred to 41, 42, or 430C, and 1 h later bacteriophage were added to 10' plaque-forming units per ml. After phage adsorption, the infected cells were diluted 106-fold and titrated for plaque-forming units with and without chloroform treatment. Strain H560 was used to titrate T4, and HF4704 was used for 4X174 and T7. The titer of infected cells is equal to the chloroform-sensitive phage titer. The burst sizes given are equal to the chloroform-insensitive phage titer 60 min later, divided by the initial titer of infected cells.

RESULTS

Thermosensitivity and deficiencies of DNA gyrase subunit A from naIA(Ts) mu-

tants. The activities of DNA gyrase and its constituent subunits from naU(Ts) mutants were compared with those from the wild type to characterize the mutants and test the assignment of gyrase subunit A as the product of the nalA gene. The DNA gyrase assay monitors the introduction of negative supercoils into relaxed, covalently closed ColEl DNA (12), whereas the assays for each of the gyrase subunits measure the same reaction, but in the presence of an excess of the complementary wild-type subunit (16). Gyrase subunit A activity in a crude extract (fraction I) of the nalA+ control strain (H560) was consistently several-fold higher than subunit B activity (Table 1). As expected, the gyrase activity of the extract in the absence of added subunits was equal to the activity of the limiting subunit B. The specific activity of gyrase subunit A in fraction I from the naLA43 mutant (strain KNK402) was about eightfold less than that of the naA + control, whereas the subunit B activities were approximately equal (Table 1) (the twofold difference in subunit B activities shown in Table 1 is within the error of gyrase assays when different crude extracts are used). The gyrase activity in the naLA43(Ts) mutant extract without added subunits was equal to the activity of subunit A, which is the limiting subunit in the mutant (Table 1). This contrasts with the subunit B limitation of the wild type. Partially purified subunit A from the naUA43(Ts) mutant was much more thermosensitive than subunit A from an otherwise isogenic naLAU control. Subunit A from the naA + strain H560 was equally active when assayed at 30 and 390C (Fig. la), whereas subunit A from the naLA43(Ts) strain KNK402, although quite active at 300C, was almost totally inactive at 390C (Fig. lb). The addition of the mutant extract did not block the wild-type activity at 390C (data not shown). This result correlates with the reTABLE 1.

Summary of activities in naUA+ and naU(Ts) extractsa

Sp act (U/mg of protein) Sub- DNA unit Pnal gyrage B 1.6 80 80 H560 (naU+) b 160 40 KNK402 [naL443(Ts)] 0.1 130 5C KNK404 [naUA45(Ts)] a DNA gyrase, Pnal, and gyrase subunit A and B activities were determined at 30°C as described in the text. The activities of gyrase and its subunits were routinely measured with fraction I, whereas Pnal activity was measured with fraction II. bNot measured. 'This value was obtained with fraction II since the activity was below the limit of detection (7 U/mg) with the more dilute fraction I. Strain

Subunit A 300 40 5C

DNA GYRASE SUBUNIT A MUTANTS

VOL. 140, 1979 1001

1

C

b.

2

3

427

4

Pt

0

010 20 0

10

20 30 400

10

Min

FIG. 1. Thermosensitivity of subunit A activity from naLA43(Ts) mutant. Assays for DNA gyrase subunit A (a and b) and B (c) were carried out for the indicated times at 30°C (0) or at 39°C (0), and fully supertwisted product was quantitated. The subunit A assays contained (a) 2 U of homogeneous subunit A (16) from strain H560 (naUA) and (b) 2 U of subunit A from strain KNK402 [naL443(Ts)]. The subunit B assays in (c) contained 3 U of subunit B from strain KNK402 [nalA43(Ts)J.

cessive nature of the naLA43(Ts) allele in vivo and excludes the possibility that the mutant extract contains a temperature-dependent inhibitor of gyrase activity. In a further control, it was shown that subunit B activity from the nalA43(Ts) mutant was insensitive to the temperature treatment which eliminated the mutant subunit A activity (Fig. lc). The temperature sensitivity of the nalA43 subunit A is impressive, since subunit B is the more thermolabile of gyrase subunits isolated from nalA+ cells. The activity of wild-type subunit B was halved by preincubation at 460C for 10 min (A. Sugino and N. R. Cozzarelli, submitted for publication), whereas the nalA + subunit A was stable at 58°C for over 2 h. Extracts of a second nalA (Ts) mutant, strain KNK404 [nalA45(Ts)], were grossly deficient in total DNA gyrase activity at 300C, with activity being detected only in the more concentrated fraction II (Table 1). The DNA gyrase deficiency of the naLA45(Ts) mutant extract results from a deficiency of subunit A activity, since the specific activity of subunit A in the mutant extract was about 50-fold less than in wild-type extracts, while the specific activity of subunit B was approximately normal (Table 1). Whereas the small amount of supertwisting observed with large amounts of the naLA45(Ts) mutant fraction II extract (Fig. 2, lane 2) could constitute a second E. coli DNA gyrase activity, the following results indicate that it represents residual levels of the mutant DNA gyrase. First, the activity was sensitive to coumermycin A1 (Fig. 2, lane 3) and oxolinic acid (Fig. 2, lane 4), showing a dependence on the products of the cou and naU genes. Second, the preparation was capable of the oxolinic acid-induced DNA cleavage characteristic of the standard E. coli DNA gyrase (Fig. 2, lane 4). This gyrase-de-

FIG. 2. Sensitivity of residual supercoiling in nalA45(Ts) extract to gyrase inhibitors. The products of the gyrase assays at 30°C offraction 1 (25 p2) from

strain KNK404 [nalA45(Ts)] are displayed in lanes 2, 3, and 4, and a control with no added extract is shown in lane 1. The reaction in lane 3 contained coumermycin Al at 20.ug/ml, and the reaction in lane 4 contained oxolinic acid at 30 pg/ml. A photograph of the ethidium bromide-stained gel is shown; electrophoresis was from top to bottom. Relaxed and nicked DNA is the band near the top of the figure, linear DNA is in the middle (in lane 4), and supercoiled DNA is near the bottom (in lane 2).

pendent cleavage is activated by detergent treatment and presumably reflects the abortion of a trapped reaction intermediate (11, 40). Third, when the mutant extract was chromatographed on DEAE-cellulose, a small amount of gyrase subunit A activity and a normal amount of gyrase subunit B activity were eluted at the same salt concentrations as the wild-type subunits (A. Sugino and K. N. Kreuzer, unpublished results). Thus, the residual activity in this extract is dependent on a protein which elutes like DNA gyrase subunit A and which reconstitutes DNA gyrase activity specifically upon the addition of subunit B. The nalA gene product was initially purified using an independent assay based on its role in 4X174 duplex DNA synthesis, and was designated Pnal (40). Pnal converts 4)X174 in vitro replication directed by nalA (R) extracts from oxolinic and nalidixic acid resistance to sensitivity. Pnal activity was deficient by over an order of magnitude in extracts of the naLU45(Ts) mutant (Table 1). We conclude that both DNA gyrase subunit A and Pnal are the product of the nalA gene; they are one and the same protein being measured by two equally specific assays.

428

KREUZER AND

J. BACTERIOL. COZZARELLIJ.BTEOL

lepend on the ature shift the rate of incorporation had dropped five independ- over 30-fold. In contrast, the nalA control culthat we isolated ture showed only a slight depression in incorporation after temperature shift, followed by a conditional lethal phenotype, and resulted in logarithmic increase in [3H]thymidine incorpotherefore the nalA gene product, gyrase subunit ration (Fig. 4b), consistent with the increase in essential E. coli proteir ,i(20). We have A, is rlby cell mass (Fig. 4a). The initial depression of a,sents of this the investigated romolecule syn- incorporation in the wild type presumably remeasuring cell growth and flects some physiological adjustment to the inthesis after shift of the naL443,I(Ts) Mutant to crease in temperature, such as an alteration in restrictive temperature. When strain KNK453 nucleotide pool size. The rapid and complete 30 to fr transferred !om 4200, [naL443(Ts)] inhibition in the rate of [3H]thymidine incorpomonitored by tuLirbidity, ceased growth, ration in the nalA43 (Ts) mutant directly demafter about 100 mmn (Fig. 3a). A x aore rapid effect onstrates a requirement for the nalA gene prodfound: imn mediately after viability. uct in E. coli DNA replication. The rapid shutin the increase of rate the shift, temperature off of [3H]thymidine incorporation excludes the number of viable colony-forminj units was conthat the nalA (Ts) mutation only has the connalA' possibility to reduced compared siderably DNA and RNA

synthesieis

nalA gene product. Each of thoe ent conditional nalA mutations I a

an

nature

e

maci

was

as

cell

on

was

cell

(Fig. 3b). After about 150 mm, mass had stopped viability started to decay exp trol

when

tl4e

an

effect on initiation (or termination) of chro-

increasing, the mosome replication, and therefore the nalA onentially, and gene product is required for the elongation phase ils at restrictive of DNA synthesis. of the eventually Treatment of the mutant or wild-type strain 'ig. 3b). temperature became inviable at 3000 with nalidixic acid at 25 ,ug/ml resulted The effect of temperature shifFt on macromolin a 20-fold reduction in the rate of incorporation ecule synthesis in the nalA43('] rs) mutant was of [3H]thymidine within 1 min, a much faster examined using pulse-labeling )nly during the effect than was seen upon temperature shift of first 100 mmn after shift to 430 C, to minimize the naLA43(Ts) mutant. The decaying incorposecondary consequences whicl i might ensue ration of [3H]thymidine seen after temperature after the cells had stopped gro-owing. Within a ictive tempera- shift of the mutant remained almost as drug few minutes after shift sensitive (data not shown). Therefore, this inture, rapid decline in the irncorporation of corporation depends on residual nalA gene prod[3H]thymidine by the naLA (Ts5) mutant comuct, with the time course of DNA synthesis menced (Fig. 4b). The initial half-life of the decay apparently reflecting the time course of abc)ut 10 min (0.2 decay in incorporation thermnal inactivation of the nalA (Ts) gene prodgeneration times), and by 100 in after temperuct. The incorporation of [3H]uridine was also clearly reduced after temperature shift of the ip~naLA43 (Ts) mutant (Fig. 4c). The rate of incorip iK 'I in the mutant dropped by about two~~poration AJ fold within the first 5 mmn and then slowly de/l to / ~~~~cayed by another twofold thereafter. The initial overall cell

mutant

most

ce

to restr

a

was

m:

b.

250

30

m

I

41

decline occurred even faster than the inhibition of[H]thymidine incorporation. There was a -e moderate depression followed by a substantial increase in the rate of [3H]uridine incorporation seen after temperature shift of the nalA + control strain (Fig. 4c), simiilar to the pattern of [3H]30 ~ 4 100 00 30 Min thymiAdine incorporation. The depression of FIG. 3. Growth of the wild type and a naLA43(Ts) [3H]uridine incorporation in the wild type was restrictive temperature. The nalA4+ strain less dramatic than that seen in the nalA43 (Ts) HF4704 (0) and the naLA43(T) st,rain KNK453 (10) mutant. The inhibition in the rate of [3H]uridine 30"C in minimal medium suPPlegrown incorporation in the nalA43 (Ts) mutant mented with thymine and uracil at 5 jig/ml and suggests that the nalA gene product is strongly 500 pg/mI. WA'hen the turbidity Casamino Acids also involved in transcription. reached 20 Klett units, the cultures iwere The incorporation of [3H]isoleucine was not 420C. At the indicated times th viabe cel inhibited by temperature shift of the measured optically (a), nalA43 (Ts) mutant until after a lag of about 30 determined bypllating on the same forming units 30'C (b). min (Fig. 4d). By this timne, the rate of growth of growth medium .e

2.

50

:2

25

0

-

0

100

3 00

200

/

400

mutant at

were

at

at

to

a

mass was

were

at

tedl tafterr ndeftr

VOL. 140, 1979

DNA GYRASE SUBUNIT A MUTANTS

429

d.

30 00

,0-°.O-:l

_ a.

500

E

E 0

~~~~~o.~~~~~.

I

0 .

IC

9',

250 .

If

8 100.

I

.s

0

/K

0.

.1

Ea1

0

._

I

25

0

100

100

Min

FIG. 4. Pulse-labeling with the wild type and a naUA43(Ts) mutant. The nalA + strain HF4704 (0) and the naLA43(Ts) strain KNK453 (0) were grown at 300C and then shifted to 430C at time zero. The cell mass was monitored optically (a), and DNA, RNA, and protein syntheses were measured by pulse-labeling with [3H]thymidine (b), [3H]uridine (c), and [3Hlisoleucine (d), respectively.

the mutant

was diminishing (Fig. 4a), and the apparent rates of DNA and RNA synthesis had already dropped by about six- and threefold, respectively. The delayed inhibition of protein synthesis is presumably a secondary consequence of the inhibition of RNA synthesis. A limitation of pulse-labeling experiments is that fluctuations in the size of the internal nucleotide pools as well as an alteration in the synthetic rate will change the incorporation of

labeled precursor. Since, in the pulse-labeling studies described above, [3H]uridine incorporation in the nalA (Ts) mutant was not as severely inhibited as [3H]thymidine incorporation, and the wild-type control showed some depression in [3H]uridine incorporation, we measured RNA synthesis after shift to 430C using continuously administered [3H]uracil to minimize the influence of nucleotide pool fluctuations. In the naA+ control, the accumulation of [3H]uracil in RNA exactly paralleled the increase in turbidity (Fig. 5a). However, in the naLA43(Ts) mutant, there was an immediate diminution in the rate of increase of incorporated [3H]uracil such that the accumulation of label did not keep up with the accumulation of cell mass (Fig. 5b). The initial rate of [3H]uracil accumulation after temperature shift of the mutant was about twofold less than that of the wild type or that of the mutant before temperature shift. After 100 min, when the growth of the cells had ceased, RNA was being degraded faster than it was synthe-

Min

FIG. 5. Continuous [3H]uracil labeling with the wild type and a nalA43(Ts) mutant. The nalA + strain HF4704 (a) and the nalA43(Ts) strain KNK453 (b) were grown at 30°C, and then, at 130 min before temperature shift, [3H]uracil was added to both cultures at 1.25 ,iCi/ml. At the indicated times after shift to 43°C, the turbidity (0) was measured, and 0.5-ml samples were tested for acid-precipitable radioactivity (0). The turbidity corresponding to 100% is 20 Klett units for both cultures, and the acid-precipitable radioactivity corresponding to 100% is 2,677 cpm for strain HF4704 and 3,562 cpm for strain KNK453.

sized, as evidenced by a decline in the amount of acid-insoluble [3H]uracil. The inhibition of RNA synthesis seen in this mutant as measured by either continuous or pulse-labeling directly implicates the nalA, gene product in transcrip-

430

J. BACTERIOL.

KREUZER AND COZZARELLI

tion, but the failure to completely inhibit transcription indicates that there is a substantial component of RNA synthesis for which gyrase subunit A is dispensable or required in only minute amounts. Effect of the nalA43(Ts) mutation on bacteriophage growth. We measured the effect of the nalA43 (Ts) mutation on the growth of bacteriophages 4X174, T7, and T4. These DNAcontaining bacteriophages differ in their sensitivity to nalidixic acid; 4X174 and T7 are very sensitive, and T4 is partially resistant (2, 43). Two sets of isogenic strains were used in these studies, since phage T4 failed to grow in the HF4704 genetic background for unknown reasons and phage T7 did not grow in the H560 background because of the presence of the F+ factor. Phage burst sizes in a nalA43(Ts) host and its isogenic nalA+ control are shown in Table 2. For ease of comparison, the burst size in the nalA (Ts) mutant is also given as a fraction relative to that in the wild type at the same temperature. All phage burst sizes at permissive temperature were slightly reduced in the mutant compared to the wild type. This reduction could result from a partial defect in the nalA43 (Ts) TABLE 2. Effect of naLA43(Ts) mutation on bacteriophage growtha Burst size Bacteriophage

Temp (OC)

4X174

32 41 43

600 230 58

280 3.6 0.2

0.47 0.016 0.003

T4

32 42

300 81

260 9.4

0.87 0.12

nalA+ nalA43(Ts) nalA43/ nalA

150 0.56 100 0.48 0.35 38 The growth of bacteriophage T4 was tested in the isogenic strains H560 (naA +) and KNK402

T7

32 41 43

270 210 110

a

[naLA43(Ts)], and the growth of phages 4¢X174 and T7

was tested in the isogenic strains HF4704 (naUA+) and KNK453 [natA43(Ts)]. The cultures were grown at the temperatures indicated for 1 h before phage infection. The times allowed for phage adsorption were 2 min for 4X174, 10 min for T4, and 3 min for T7. These times were chosen for satisfactory phage adsorption; each culture had greater than 75% phage adsorption for 4X174, greater than 85% for T4, and between 40 and 70% for T7. The burst sizes after 60 min were calculated as described in the text, and the ratio of the burst size in the naUA43(Ts) mutant to that in the nalA + control at the same temperature is given in the last column. Each experiment was repeated at least once with very similar results.

gene product even at the permissive temperature; this could explain also the reduced activity of the nalA43 subunit A in vitro and the reduced rate of incorporation of [3H]thymidine and [3H]uridine in the mutant at permissive temperature (data at time zero in Fig. 4; 28). The growth of phage pX174 was completely blocked in the nalA43(Ts) mutant derivative of strain HF4704 at a restrictive temperature of either 41 or 43°C (Table 2), demonstrating that this phage has an absolute requirement for the host nalA gene product. This result is consistent with the total inhibition of 4X174 growth by nalidixic acid (43). We observed a similar temperature inhibition of 4X174 growth with the nalA43 (Ts) mutation in the H560 genetic background (data not shown). The burst size of phage T4 at the restrictive temperature of 42°C in the nalA43(Ts) host was about 12% of that in the nalA+ control (Table 2). This is about the same magnitude of inhibition observed after treatment with a high concentration of nalidixic acid (100 jLg/ml) (24) but more than obtained in other studies with lower drug levels (2). The inhibition of T4 growth in the mutant indicates that this phage does utilize the host nalA gene product, but barring leakiness of the mutation, the residual burst implies that some T4 DNA replication can occur without the host gyrase subunit A. In contrast to the inhibition of 4X174 and T4 growth, the burst size of phage T7 was not significantly reduced in the nalA43(Ts) mutant compared to the nalA+ control by growth at restrictive temperature (Table 2). However, the growth of phage T7 is quite sensitive to nalidixic acid, and the drug markedly inhibits phage DNA synthesis (2). This discrepancy between the effects of nalidixic acid and the nalA (Ts) mutation does not result from strain differences, since the growth of T7 in the nalA43 (Ts) mutant at permissive temperature and in the isogenic wildtype strain at both temperatures was sensitive to nalidixic acid (Table 3). It is possible that the nalA43 (Ts) mutant has a reduced level of the nalA gene product that is sufficient for T7 but not for host or phage 4X174 growth. If this were the case, the growth of T7 at the restrictive temperature should be completely sensitive to nalidixic acid, since it would be dependent on residual levels of the nalidixic acid-sensitive nalA43 gene product. The crucial observation is that T7 growth became completely insensitive to the inhibitor in the nalA43(Ts) mutant at the restrictive temperature (Table 3). Thus, temperature inactivation of the nalA gene product protected T7 from the inhibitory effect of nalidixic acid. These results

DNA GYRASE SUBUNIT A MUTANTS

VOL. 140, 1979

TABLE 3. Effect of nalidixic acid on phage T7 growth' Host

naU+

Temp (00)

32 43

Burst size No addi- Nalidixic acid tion

198 63

1.9 0.3

3.5 130 61 80 aThe experiment was identical to that described in Table 2 except that, where indicated, nalidixic acid at 25 iLg/ml was added to the bacterial cultures 2 min before the addition of phage and was present throughout the period of phage growth. All cultures had between 40 and 70% phage adsorption at the start of phage growth. Repetition of this experiment gave essentially identical results.

naL443(Ts)

32

43

suggest that phage T7 does not require the nalA gene product but, rather, that the nalA gene product in combination with nalidixic acid prevents T7 growth. A possible mechanism is considered in the Discussion.

DISCUSSION The evidence that nalA is the structural gene for subunit A of DNA gyrase is now convincing. The thermosensitivity of gyrase subunit A activity from the naL443(Ts) mutant (Fig. 1) flfills a classical test for structural gene assignment. This result corroborates our earlier finding that radioactively labeled nalA gene product, induced uniquely by a specialized XnalA transducing phage, coelectrophoresed with gyrase subunit A (16, 20, 28). The fact that subunit A controls the nalidixic acid sensitivity of gyrase is perfectly consistent with this structural gene assignment (16). The naLA45(Ts) mutation results in a gross deficiency and the naLA43(Ts) mutation results in both a deficiency and thermosensitivity of gyrase subunit A activity. The reduced subunit A activities of these mutants could result from denaturation of the mutant protein during extraction, reduced intracellular levels of the nalA gene product at permissive temperature, or an alteration in the kinetic parameters of the mutant enzyme. In light of the essential nature of subunit A, it seems unlikely that the nalA45(Ts) mutant could have a 50-fold deficiency of activity in vivo, and therefore the low activity presumably reflects denaturation of the protein during extraction. This mutant has been of practical use, since extracts have provided an excellent source for the purification of subunit B totally free of subunit A activity (Sugino and Cozzarelli, submitted for publication).

431

Although the naA (Ts) mutants have defects in gyrase subunit A, both mutants have normal DNA gyrase subunit B activity. This result provides convincing genetic evidence that DNA gyrase is composed of heterologous subunits, since inactivation of one subunit caused by a nalA (Ts) mutation has no effect on another subunit. There is now strong evidence that gyrase subunit B is the product of the cou gene (16, 25; F. G. Hansen and K. von Meyenberg, Mol. Gen. Genet., in press), and Hansen and colleagues have proposed that the genetic symbols naU and cou be replaced by gyrA and gyrB, respectively, consistent with the biochemical function of the gene products. Transfer of the nalA43(Ts) mutant to restrictive temperature provides a straightforward method of inactivating DNA gyrase. We find that DNA synthesis, as monitored by pulse-labeling with [3H]thymidine, is dramatically inhibited upon temperature shift of this mutant. This is in agreement with the nalidixic acid sensitivity of DNA replication, but provides a more direct demonstration of an obligatory role of the naU gene product in DNA synthesis. The rapid shut-off of DNA synthesis (Fig. 4) shows that the naU gene product is required for elongation and not just initiation of chromosome replication. The naA gene product could provide the swivel for replication by relieving the positive supercoils introduced ahead of the replication fork as the DNA strands at the fork unwind, since gyrase can act on a positively supertwisted substrate (28). Alternatively, DNA gyrase maintains the negative superhelical tension of the DNA (8, 13), and this superhelicity could facilitate the binding, activity, or both, of replication proteins. Temperature shift of the naUA43(Ts) mutant results in a nearly immediate, but incomplete, inhibition of RNA synthesis, as measured by incorporation of either [3H]uridine administered during a pulse (Fig. 4) or [3H]uracil administered continuously (Fig. 5). Consistent with this, nalidixic acid and coumermycin A1 partially block transcription in the strains used here (unpublished data). These results clearly inplicate DNA gyrase subunit A in transcription. The observation that RNA synthesis directed by a phage N4 RNA polymerase is completely blocked at restrictive temperature in the nalA43(Ts) mutant (L. Rothman-Denes, personal communication) further supports a transcriptional role for the naU gene product. A role of DNA gyrase in transcription would not be surprising. Negative superhelicity increases initiation of RNA synthesis by E. coli RNA polymerase in vitro probably by facilitat-

432

KREUZER AND COZZARELLI

ing helix unwinding at the promoter site (3). Differences in ease of opening of various promoters could explain the operon-specific effects of gyrase inhibitors (9, 29, 31, 33). Gyrase could also aid RNA chain elongation by removing positive supertwists generated by transcription, or it could play a more direct role by coupling a rotation of the DNA double helix to transcription and thereby avoid tangling of the nascent RNA and template DNA and'a helical traverse of RNA polymerase. After temperature shift of the naLA43(Ts) mutant, protein synthesis is inhibited after about 30 min (Fig. 4d). This delayed inhibition is presumably a secondary consequence of the inhibition of RNA synthesis, but the lag might be expected to be shorter since the half-life of most E. coli mRNA is only a few minutes. Perhaps the synthesis of nontranslated RNA, including the stable rRNA and tRNA species, is preferentially inhibited. The utilization of a host enzyme by bacterial viruses often clarifies the physiological role of that enzyme. This approach should be particularly interesting for gyrase subunit A, because of the following reasons. (i) Treatment with nalidixic acid has a variable effect on bacteriophage growth; phages T7 and 4X174 are very sensitive, whereas T4 is partially resistant (2,43). (ii) DNA gyrase has been postulated to be involved in replication of both linear and circular doublestranded, but not of single-stranded, phage DNA templates (7, 18, 24, 41, 42). (iii) The nalidixic acid sensitivity of in vitro phage DNA replication is generally lower than that of in vivo replication (5). Since each of these statements depends on the effects of gyrase inhibitors, we have taken the complementary approach of analyzing the effect of a nalA (Ts) mutation on phage growth; the results have been illuminating. The complete inhibition of the growth of phage 4)X174 in the naU443(Ts) mutant at restrictive temperature (Table 2) demonstrates an obligatory role of the naA gene product and is consistent with the nalidixic acid sensitivity of 4)X174 growth (43). Stages II and III, but not I, of 4X174 DNA replication are probably the affected sites, since they are sensitive to gyrase inhibitors (41, 42). It is also possible that transcription of OX174 is dependent on DNA gyrase, since transcription of the related phage S13 is sensitive to nalidixic acid (29). Temperature shift of the naLA43(Ts) mutant or nalidixic acid treatment of the wild type both result in a substantial reduction of the burst of phage T4 (Table 2; 2, 24), and therefore this phage also utilizes the host nalA gene product. Baird et al. (2) first reported that the nalidixic

J. BACTERIOL.

acid inhibition of phage T4 was only partial, but could not eliminate the possibility that the residual burst was a drug-related artifact-for example, that the cellular permeability to nalidixic acid was decreased after T4 infection. Our finding of a similar residual T4 burst in the naUA43(Ts) mutant at restrictive temperature (Table 2) implies that some T4 replication can indeed occur without the host nalA gene product. Recently, it has been found that certain T4 DNA-delay mutants are hypersensitive to gyrase inhibitors and are deficient in a T4-induced topoisomerase (24; W. M. Huang, personal communication; L. Liu, C. Liu, and B. Alberts, personal communication). The involvement of topoisomerases in phage T4 replication may therefore be analogous to homologous recombination of phage A, which can occur either by host enzymes (the rec pathway) or phage-induced enzymes (the red pathway) (32). The involvement of DNA gyrase in phage T7 growth and replication is particularly interesting, since T7 DNA is linear and no circular replication intermediates have been found. It has been postulated that topological constraints imposed by the binding of proteins, RNA, or membranes might prevent the expected free rotation of linear DNA about the helix axis and introduce a dependency on a topoisomerase such as DNA gyrase (7, 18). However, we see essentially no inhibitory effect of the naLA43(Ts) mutation on phage T7 growth at the restrictive temperature, in striking contrast to the 100-fold inhibition of phage 4X174 growth in the same strain under the same conditions (Tables 2 and 3). The failure to block T7 growth could be explained if T7 requires the nalA gene product, but in smaller amounts than the host or phage 4X174, and if the nalA43 mutation is leaky enough at the restrictive temperature to provide this level of activity. The finding that T7 growth becomes nalidixic acid resistant after temperature shift of the mutant requires the further ad hoc assumption that the residual subunit A activity has become drug insensitive. However, this assumption seems inconsistent with the nalidixic acid sensitivity of the residual host DNA synthesis. These considerations lead us to believe that phage T7 does not require the host nalA gene product. Rather, we propose that the nalidixic acid inhibition of T7 may result from a corruption rather than a simple elimination of the nalidixic acid target protein. According to this model, a poison is formed upon interaction of nalidixic acid with nalA gene product, and thermal inactivation of the naLA43 gene product eliminates the drug sensitivity of T7 by preventing the formation of this poison. The poison is

VOL. 140, 1979

DNA GYRASE SUBUNIT A MUTANTS

possibly a complex of gyrase and DNA analogous to that induced by nalidixic or oxolinic acid in vitro (11, 40). This complex is thought to be a trapped reaction intermediate consisting of DNA gyrase attached stably and perhaps covalently to both DNA strands (28). Evidence for formation of a similar complex in vivo is provided by the finding that detergent treatment of DNA isolated from oxolinic acid-treated cells results in DNA fragmentation, as it does with oxolinic acid-inhibited gyrase reactions (34a). Although the corruption model remains to be established, it could explain some puzzling observations. First, the dominance of naA+ over naA(R) (15) would be expected. It is difficult to explain this dominance if nalidixic acid just inhibits the drug-sensitive protein, since the drug-resistant protein should remain active. Second, T7 DNA replication in vitro is not very sensitive to nalidixic acid, with partial inhibition seen only at very high doses (17). This relatively poor inhibition could be due to formation of the complex rather than elimination of supertwisting activity. Nalidixic acid sensitivity in any in vitro replication system in which a supertwisted template is provided or in which no circular form is present might be caused by complex formation. Indeed, extracts of the nalA45(Ts) mutant, which have negligible subunit A activity, are not deficient in supporting 4X174 RF replication (where a supertwisted template is provided), but rather support synthesis that is relatively drug insensitive (data not shown). If phage T7 does not require the host gyrase, then why are its growth and DNA replication also selsitive to the inhibitor coumermycin Al, which inMhibits gyrase subunit B (7, 18)? As with nalidixic acid, drug sensitivity may be dominant to resistance for the coumermycin A1 target protein, the product of the cou gene, and in vitro T7 DNA replication is coumermycin Al insensitive (7). Therefore, the coumermycin sensitivity of phage T7 might also be anomalous and not reflect a dependence on the cou gene product. It would be informative to measure T7 growth and its sensitivity to coumermycin Al in a temperature-sensitive cou mutant. Our recent results provide still another complexity in understanding the bacterial physiology of the nalA gene product. This subunit interacts not only with subunit B to form gyrase but also with a third subunit, v, to form a DNArelaxing enzyme (Brown, Peebles, and Cozzarefli, Proc. Natl. Acad. Sci. U.S.A., in press). In general, drug-sensitive alleles of the gene for a target protein involved in macromolecule synthesis are dominant to their drug-resistant counterparts. Examples are provided by the fol-

lowing inhibitors of DNA, RNA, and protein synthesis: (i) rifampin (1) and streptolydigin (19), which inhibit the ,8 subunit of RNA polymerase; (ii) streptomycin (22), spectinomycin (35), and erythromycin (27), inhibitors of ribosomal proteins S12, S5, and IA, respectively; (iii) kirromycin (10), which acts specifically on protein synthesis elongation factor Tu; and (iv) nalidixic acid (15), an inhibitor of DNA gyrase. Dominance of drug sensitivity could result from preferential expression of the wild-type gene product, either because the mutant protein is translated poorly or it is degraded more readily by proteases (35). Alternatively, corruption of a target protein by its inhibitor may be a general phenomenon, occurring by any one of at least three distinct mechanisms, as follows. (i) The target protein may be a multimer which can include both sensitive and resistant protomers and in which inactivation of any one protomer blocks the activity of the multimer. The potential importance of this mechanism increases rapidly with the number of protomers per multimer (n), since the active fraction of enzyme equals (0.5)n. However, there can be an effect on the cell only when the enzyme becomes limiting and dominance studies require multiple gene dosage, whereas one gene dose suffices for the wild type. Also, the wild-type state is expected to often have an excess of the target. (ii) The inhibited target protein itself actively prevents uninhibited protein from acting. In this model, the subversion is from within, since the inhibited target protein is a participant in the process in question. This type of mechanism has been invoked for those antibiotics (streptomycin, spectinomycin, and erythromycin) which trap a drugsensitive ribosome-mRNA initiation complex and thereby physically block initiation by a drug-resistant ribosome (6). (iii) The inhibited target protein becomes an active inhibitor of a process in which it nornally does not participate, and therefore the subversion is from without. The proposed inhibition of T7 growth by a DNA gyrase-nalidixic acid complex could provide an example of this mechanism.

433

ACKNOWLEDGMENTS We thank Valerie Lindgren for her participation in some of the phage growth experiments. This research was supported by Public Health Service grants GM-21397, CA-19265, and GM-07197 from the National Institutes of Health. LITERATURE CITED 1. Austin, S., and J. Scaife. 1970. A new method for selecting RNA polymerase mutants. J. Mol. Biol. 49: 263-267. 2. Baird, J. P., G. J. Bourguignon, and R. Sternglanz.

434

KREUZER AND COZZARELLI

1972. Effect of nalidixic acid on the growth of deoxyribonucleic acid bacteriophages. J. Virol. 9:17-21. 3. Botchan, P., J. C. Wang, and H. Echols. 1973. Effect of circularity and superhelicity on transcription from bacteriophage A DNA. Proc. Natl. Acad. Sci. U.S.A. 70: 3077-3081. 4. Champoux, J. J. 1977. Strand breakage by the DNA untwisting enzyme results in covalent attachment of the enzyme to DNA. Proc. Natl. Acad. Sci. U.S.A. 74: 3800-3804. 5. Cozzarelli, N. R. 1977. The mechanism of action of inhibitors of DNA synthesis. Annu. Rev. Biochem. 46: 641-668. 6. Davis, B. D., P.-C. Tai, and B. J. Wallace. 1974. Complex interactions of antibiotics with the ribosome, p. 771-790. In M. Nomura, A. Tissieres, and P. Lengyel (ed.), Ribosomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 7. DeWyngaert, M. A., and D. C. Hinkle. 1979. Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA. J. Virol. 29:529-539. 8. Drlica, K., and M. Snyder. 1978. Superhelical Escherichia coli DNA: relaxation by coumermycin. J. Mol. Biol. 120:145-154. 9. Falco, S. C., R. Zivin, and L. B. Rothman-Denes. 1978. Novel template requirements of N4 virion RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 75:3220-3224. 10. Fischer, E., H. Wolf, K. Hantke, and A. Parmeggiani. 1977. Elongation factor Tu resistant to kirromycin in an Escherichia coli mutant altered in both tuf genes. Proc. Natl. Acad. Sci. U.S.A. 74:4341-4345. 11. Gellert, M., K. Mizuuchi, M. H. O'Dea, T. Itoh, and J. Tomizawa. 1977. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc. Natl. Acad. Sci. U.S.A. 74:4772-4776. 12. Gellert, M., K. Mizuuchi, M. H. O'Dea, and H. A. Nash. 1976. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. Natl. Acad. Sci. U.S.A. 73:3872-3876. 13. Gellert, M., M. H. O'Dea, T. Itoh, and J. Tomizawa. 1976. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Natl. Acad. Sci. U.S.A. 73:4474-4478. 14. Goss, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on Escherichia coli II. Inhibition of deoxyribonucleic acid synthesis. J. Bacteriol. 89:1068-1074. 15. Hane, M. W., and T. H. Wood. 1969. Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J. Bacteriol. 99:238-241. 16. Higgins, N. P., C. L. Peebles, A. Sugino, and N. R. Cozzarelli. 1978. Purification of the subunits of Escherichia coli DNA gyrase and reconstitution of enzymatic activity. Proc. Natl. Acad. Sci. U.S.A. 75:17731777. 17. Hinkle, D. C., and C. C. Richardson. 1974. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Requirements for deoxyribonucleic acid synthesis and characterization of the product. J. Biol. Chem. 249: 2974-2984. 18. Itoh, T., and J. Tomizawa. 1977. Involvement of DNA gyrase in bacteriophage T7 DNA replication. Nature (London). 270:78-80. 19. Iwakura, Y., A. Ishihama, and T. Yura. 1973. RNA polymerase mutants of Escherichia coli. II. Streptolydigin resistance and its relationship to rifampicin resistance. Mol. Gen. Genet. 121:181-196. 20. Kreuzer, K. N., K. McEntee, A. P. Geballe, and N. R. Cozzarelli. 1978. Lambda transducing phages for the nalA gene of Escherichia coli and conditional lethal nalA mutations. Mol. Gen. Genet. 167:129-137. 21. Layne, E. 1957. Spectrophotometric and turbidimetric

J. BACTERIOL. methods for measuring proteins. Methods Enzymol. 3: 447-454. 22. Lederberg, J. 1951. Streptomycin resistance. A genetically recessive mutation. J. Bacteriol. 61:549-550. 23. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 24. McCarthy, D. 1979. Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products. J. Mol. Biol. 127:265284. 25. Mizuuchi, K., M. H. O'Dea, and M. Gellert. 1978. DNA gyrase: subunit structure and ATPase activity of the purified enzyme. Proc. Natl. Acad. Sci. U.S.A. 75:59605963. 26. Morrison, A., and N. R. Cozzarelli. 1979. Site-specific cleavage of DNA by E. coli DNA gyrase. Cell 17:175184. 27. Nomura, M., and F. Engbaek. 1972. Expression of ribosomal protein genes as analyzed by bacteriophage Mu-induced mutations. Proc. Natl. Acad. Sci. U.S.A. 69:1526-1530. 28. Peebles, C. L., N. P. Higgins, K. N. Kreuzer, A. Morrison, P. O. Brown, A. Sugino, and N. R. Cozzarelli. 1979. Structure and activities of Escherichia coli DNA gyrase. Cold Spring Harbor Symp. Quant. Biol. 43:4152. 29. Puga, A., and I. Tessman. 1973. Mechanism of transcription of bacteriophage S13. II. Inhibition of phage-specific transcription by nalidixic acid. J. Mol. Biol. 75:99108. 30. Ryan, M. J. 1976. Coumermycin Al: a preferential inhibitor of replicative DNA synthesis in Escherichia coli. I. In vivo characterization. Biochemistry 15:3769-3777. 31. Sanzey, B. 1979. Modulation of gene expression by drugs affecting deoxyribonucleic acid gyrase. J. Bacteriol. 138:40-47. 32. Signer, E. 1971. General recombination, p. 139-174. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 33. Smith, C. L., K. Kubo, and F. Imamoto. 1978. Promoter specific inhibition of transcription by antibiotics which act on DNA gyrase. Nature (London). 275:420-423. 34. Smith, D. H., and B. D. Davis. 1965. Inhibition of nucleic acid synthesis by novobiocin. Biochem. Biophys. Res.

Commun. 18:796-800. 34a.Snyder, M., and K. Drlica. 1979. DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid. J. Mol. Biol 131:287-302. 35. Sparling, P. F., J. Modolell, Y. Takeda, and B. D. Davis. 1968. Ribosomes from Escherichia coli merodiploids heterozygous for resistance to streptomycin and to spectinomycin. J. Mol. Biol. 37:407-421. 36. Staudenbauer, W. L. 1975. Novobiocin-a specific inhibitor of semiconservative DNA replication in permeabilized Escherichia coli cells. J. Mol. Biol. 96:201-205. 37. Staudenbauer, W. L. 1976. Replication of Escherichia coli DNA in vitro: inhibition by oxolinic acid. Eur. J. Biochem. 62:491-497. 38. Staudenbauer, W. L. 1976. Replication of small plasmids in extracts of Escherichia coli: requirement for both DNA polymerases I and III. Mol. Gen. Genet. 149:151158. 39. Sugino, A., N. P. Higgins, P. O. Brown, C. L. Peebles, and N. R. Cozzarelli. 1978. Energy coupling in DNA gyrase and the mechanism of action of novobiocin. Proc. Natl. Acad. Sci. U.S.A. 75:4838-4842. 40. Sugino, A., C. L. Peebles, K. N. Kreuzer, and N. R. Cozzarelli. 1977. Mechanism of action of nalidixic acid: purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-

435

VOL. 140, 1979

DNA GYRASE SUBUNIT A MUTANTS

closing enzyme. Proc. Natl. Acad. Sci. U.S.A. 74:47674771. 41. Sumida-Yasumoto, C., and J. Hurwitz. 1977. Synthesis of OX174 viral DNA in vitro depends on OX replicative form DNA. Proc. Natl. Acad. Sci. U.S.A. 74:4195-4199. 42. Sumida-Yasumoto, C., A. Yudelevich, and J. Hurwitz. 1976. DNA synthesis in vitro dependent upon 4X174 replicative form I DNA. Proc. Natl. Acad. Sci.

U.S.A. 73:1887-1891. 43. Taketo, A., and H. Watanabe. 1967. Effect of nalidixic acid on the growth of bacterial viruses. J. Biochem. 61: 520-523. 44. Wickner, S., M. Wright, L. Berkower, and J. Hurwitz. 1974. Isolation of dna gene products of Escherichia coli, p. 195-215. In R. B. Wickner (ed.), DNA replication. Marcel Dekker, New York.