.I. Mol. Hiol. (1Wl) 219, 443-450

Bacterial DNA Supercoiling

and [ATP]/[ADP]

Changes Associated with a Transition to Anaerobic Growth Li-Shan Hsieh, Richard M. Burger and Karl Drlicat 355 First

Public Health Resea,rch Institute Avenue, New York, ,Y Ii 10016, V.S.A.

(Received 31 October 1990; accepted 21 February

1991)

Shifting Eseheriehia coli from aerobic to anaerobic growth caused changes in the ratio of ] ATP]/[ADP] and in negative supercoiling of chromosomal and plasmid DNA. Shortsly after lowering oxygen t,ension, both [ ATP]/[ADP] and supercoiling t,ransiently decreased. Under conditions of exponential anaerobic growth, both were higher than under aerobic conditions. These correlations may reflect an effect of [ATP]/[ADP] on DNA gyrase, since in oitro [ATP]/]ADP] influences the level of plasmid supercoiling attained when gyrase is either int,roducing or removing supercoils. When the supercoiling activity of gyrase was perturbed by a mutation in gyrB, a shift to anaerobic conditions resulted in plasmid supercoil relaxation similar to t)hat seen with wild-type. However, the low level of supercoiling in t’he mutant persisted during a time when supercoiling in wild-type recovered and then exceeded aerobic levels. Thus, changes in oxygen tension can alter DNA supercoiling through an effect on g.yrase, and correlations exist between changes in supercoiling and changes in the intracellular ratio of [ATP]/[ADP].

Keywords: DNA supercoiling;

1. Introduction DNA in bact,erial cells is under negative superhelical tension (Worcel & Burgi, 1972; Sinden et al., 1989; Bliska & Cozzarelli, 1987), a property thought to be regulated largely by the opposing activities of DNA gyrase (Gellert ef al., 1976a,b; Drlica & Snyder, 1978) and DNA topoisomerase I (Wang, 1971; Pruss et al., 1982; DiNardo et al., 1982). These correct, the changes in supercoiling enzymes expected to arise from changes in temperature (Goldstein di Drlica, 1984), from intercalation of dyes into DNA (Esposito & Sinden, 1987), and from physiological activities such as transcription (Pruss & Drlica, 1986; Liu & Wang, 1987; Wu et al., 1988). Regulation of supercoiling even extends to the level of topoisomerase gene expression: conditions that increase or decrease negative supercoiling also lead to corrective changes in the level of expression of gyrase and topoisomerase I (Menzel & Gellert, 1983, Tse-Dinh, 1985; Tse-Dinh & Beran, 1988). Cellular energetics is another factor that might influence supercoiling, since ATP is required for t Author to whom correspondence should he addressed.

gyrasr:

anaerobiosis:

ATP:

ADP

gyrase to introduce negative supercoiling into DNA and since gyrase removes negative supercoils in the absence of ATP (Gellert et al., 1976a, 1977; Sugino et al., 1977). Recently, it was shown in vitro that the ratio of [ ATP]/lADP] strongly influences the level of supercoiling reached in the presence of purified gyrase. regardless of whether gyrase is introducing or removing supercoils (Westerhoff et al., 1988). It is conceivable that intracellular DNA supercoiling also depends on [ATP]/[ADP]. While investigating the influence of anaerobic growth on DNA supercoiling we found that a transient decrease in supercoiling occurred shortly after lowering oxygen tension. Since the transition to anaerobiosis was known to cause transient changes in adenine nucleotide concentrations (Cole et al., 1967; Tkachenko et aE., 1988), we looked for correlations between [ATP]/[ADP] and supercoiling. Relaxation occurred at the same time that (ATP]/ (ADP] dropped. Eventually an elevated level of supercoiling was established which, under conditions of long-term exponential growth, was associated with an increased value for [ATP]/[ADP]. These two correlations fit with the idea that [ATP]/ (ADP] may be yet another factor involved in the control of DNA supercoiling in bacteria.

-2. Materials

and Methods et al.. 1981) is a derivative

Strain ,JTTI (Sternglanz

ot

Escharichiar:oli KI2 having the genetic markers pyrF strAZ95. Strain KI)ll:! is a gyrB22h’ of JTTI. KDI 12 was constructed by transducing the yyrHZ% mutation from strain SD7 (DiR’ardo rf al.. 1982) into ,JTTI. KDI 12 is wilti-tvpr for @A. (‘ells were grown at 37°C‘ in LB liquid medium (Miller. 1972) containing 1 ‘&) (w/v) glucose, 0.1 M-sodium phosphate buffer (pH 6.8) and 200 pg streptoinyc~irl/rrII. Glturcl growth was routinely measured as an increase in turbidit? calorimeter; control expertusing a Klrtt-Summerson ment~s in which protein synthesis was measured by incorporation of radioactive leucinr produced the same result,s as t,urbiditv measurc~mrnts. Anaerobiosis was achieved by bubbling bacterial cultures with an anaerobic gas mixture (IGoblend, I’nion (‘arbide C’orporation: X5’:; nitrogen, loo, hydrogen. So0 carbon dioxide) that had been scrubbed t)?; passage through a powdered zinc suspension in POPM-phenazinr methosulfatr (pH 4). Aerobic growth was obtained b> bubbling water-saturated air vigorouslythrough bacterial (*uItures. Cells were shifted to anaerobic> conditions at N Klrtt, reading of 30. which corresponds to roughly 3 x 10” cells/ml. llnder t,he anaerobic growth condit’ions emplo.yrd, strain *ITT1 was expected to generate ATI’ I)> fermentation. The anaerobic8 growth rate was about 0.4 times the aerobic rate, but growth rate ~WTSCis expected to have little effect on DEA supervoiling ((Goldstein 8r Drlica, 1984). During the transition from aerobic, to anaerobic growt,h. no distinct lag in growth rak was ohsrrvrd for strain .JTTl qa12:i

----_

-__

tions of c~hloi~oquinf~ were f~~arrlinr~cl to 1nsur(’ ttlat IllL,g:;It,ivc suprrc*oiling was being monitort~cl. ~l~c.troJ)tior’~sis w-as c*arritlfl oilt at rooni t~emprr‘aturt~ af 2 to 3 \‘!‘c,tn in X0 rriu-‘l’ris-I)hos1)t~at,(,. using (W/V) agarose t (’ 8 ~wEDTA (pH 7.2) (Maniatis P[ nl.. 1982) as a I,llfh.

nirA(Jnr)

tlwivativc

(b) (‘ornpfrr&on

of

DNA

8upercoiliny

C”hromosomal DICA supercoiling under various condtions was compared by titration of negative supercoils in nucleoids extracted from E. coli cells. For these measurtaments cells were radioactively labeled by growt,h in r3H]thymidine (10 &i/ml) for one generation and then lysed by a modification of the method of Stonington Kr Pettijohn (1971) as described (Drlioa $ Snyder. 1978). (lells grown anaerobically were harvested anaerobicall\ and were lysed under anaerobic conditions (these c*ondltions required a S-fold increase in lysozyme ooncentration). 1,ysates were loaded directly onto sucrose densit,!: gradients along with “Clabeled bacteriophage T4R used as a sedimentation standard. Sucrose gradients cont~ained varying concentrations of ethidium bromide to titrate the negative supercoils. Centrifugation conditions. gradient fractionation, and radioactive counting were as described (Drlica & Snyder, 1978). Sucleoid sedimentat,ion corfficients were determined from the distribution of radioactivity in the gradients relative to the bacteriophage standard (s = 1025 (Cummings. 1964)). Plasmid supercoiling under various conditions was compared by gel electrophoresis in the presence of uhloroyuine. For these measurements plasmid DI\iA from strain ,JTTl, transformed with pUC9 (Vieier & Messing. 1982), was extracted by an alkaline/sodium dodecyl sulfate miniprep method (Ish-Horwicz 8~ Burke, 1981) as modified b> Treisman (1985) but omitting the polyethylene glyrol step. During elertrophoresis in appropriate concent,rations of chloroquine. plasmid topoisomers of different linking numbers were resolved. From the relative electrophoretic mobility of the topoisomer population it was possible to deduce relative superhelix densities. Several caoncentra-

For ATI’ and ,\l)l’ det~,rniinations c.rlls wart’ gro\vLl under the aerobic or anaerobic, c.onditions used for supe’caoiling measurements. A.t various t)imrs. 3 ml portions were removed and quickly transf(irrtxd to tubes c.ontainin,g 0.17 ml of 60”$, (v/v) perchloric~ acaid on icr. In thtb (‘ase of anaerobic experiments. (*are was t)akrn to maintain all anaerobic> environment, unt)il the sample a as trr*at,chdwit,h perchloric, acid. Aft)rr 1 h t hts ac,id-trtaattd ct~lls w(~rt~ neutralized with 5 .M-KOH and tjhrn c~t~ntrifugc4 at 10,000 g for 10 min to remove vrll debris. Samplrs werIb diluted and assayed for AT1 and Al)l’ using a Iuc4f&,ascl assay as drsc*ribrd bv (‘hal)mari r4 trl. ( I!17 I ). Reag(~ilts for the Iuciferasr reac+iorr wtare procluc+s of tiorhringt,,, (XT]’ t)iolurninc,s~rnc,(, (‘LS). I~iolrlr~~inrsc~t:~ic~rbj its tl~~tc~c+etl using a I+c*kman LSXWO liquid sc*int,illation sl)e(‘t ronlrtc~r set to caount both coincident itIl(I nc,ri-c,oillc.itlt.tlt l)hotons. Since ATI’ and Al)]’ were iiesayt~tl on itlrntic3l I)ortic,lls. their ratio c~ultl be detc~rnrint~d Lvithorrt t1irec.t c~al(*ulatio~l of intra(.rllular c~orrc~rlltl~atiotls.

3. Results We examined thra influrrrct> of oxygen t.ension on chromosomal DNA suprrcviling hy vxt rac%irrg nucleoitls f’rorn straiII .J’lTl growing t~spont~ntially under aerobic2 or anaerobic~ chontlitions and then t,itratirig thv negatjive supt~rcoils hy srtiiment at ion int,o a wries of su(arose gradients c~mt ainillg various concent rations of cthidium hrotnidv. lntt~cdat~ion of ethidium t)romide into I)NA rcrnoves rrrgcativv supercGls and tkreases the nuclroid sdirntantat ion rate; iL minimum srdiment~atiori ratr is :~ttaincd when t,he negative suprrcoils are fully titrated. Higher dye concent,rations introduce positive supercoils and increase thcl nucaleoid sedimentation ratcb. At, low I)S\;A concentration the t:thidiurn l~rornitle concentration generating the sedimentation minimum is relat’ed to nt~g,rat~iv~~ suprrhelical density. LAs shown it) Figure 1(a) nu&oids isolat~~cl from cells growing anaerobically required 25o/,, higher dye cmoncentrat,ion li)r c~onipletc~ supercGl rr~laxation. This caorrrspollds to WI incarrase in super(*oiling of’ ahut I7 ‘) ,) (calculatctl using the Scatchard given 1)~ Hinton &. Bode equation and constants (1975) with t.he assumption t,trat at low l)Nr\ WIIcentration the total arid free dytx c,orrc,t.rltratiorls arc nearly idcntic~al). The d&a in Figure, I (a) also show t,hat the nuclroid sedirnrnt,at ion rate is lowtAr during anaerobic growth: the reason f’or this is c.nrrc*ntl> under study. Exposure to anaerobic c3mtlitions for 20 minutes caused supercaoiling to inc*reasc to 1he same’ level ohserved a f ter estensivt, anarrohic~ growth (Fig. I(h)). However. when we rxaniirred chromosomal supert~oiling at, short.er t imrs after lowering oxygen tension. we ohservrti that it first decreased

IO

30 Time after shift (mid

Figure 2. Chromosomal supercoiling during the transition to anaerobic growth, Exponentially growing cultures of strain JTTI were shifted to anaerobic conditions for various times. Nucleoids were extracted anaerobically and analyzed by sucrose gradient centrifugation in the prrsent’? of rthidium bromide (EBr) as described for Fig. 1. Filled circles indicate the ethidium bromide concent,ration at the sedimentation minima from plots similar to those shown in Fig. 1. These values are proportional to superhelix density. The point at the extreme right of t,he Figure was obtained from a long-term anaerobic culture growing exponent’ially as described for Fig. l(a).

CEerl (pg/ml)

Figure 1. Effect of anaerobic growth on chromosomal l)NA supercoiling. (a) Titration of negative supercoils in nucleoids from cells growing exponentially under aerobic or anaerobic conditions. 3H-labeled nucleoids were extracted from E. coli strain ?JTTl, and their sediment.ation rate relative t,o bacteriophage T4B (s = 1025) was determined by eentrifugation in sucrose gradients (Lontaining thr indirated c%oncentrations of ethidium hromide (EHr). (0) Cells grown aerobically; (0) cells grown anaerobicaally. Tn both cases the cells were grown overnight under &her aerobic or anaerobic conditions. diluted. and grown to mid-log phase prior to nucleoid extraction. The broken vertical line is to aid in comparison of the c‘urves. This experiment was repeated 3 times with identical results. (b) Chromosomal DKA supercoiling

before flxcreding aerobic levels (Fig. 8). This trans&it relaxation of about 15yb occurred during t,hfa first ten minutes after lowering oxygen tension. We fr>untf a similar effect of anaerobic growth on plasmid DSA supercoiling. Strain .JTTl . t ransformed with J’lasmid puc’9, was grown under aerobic or anaerobic conditions. I)uring esponential growth. pI:C9 was extracted from t.hr cells. and plasmid supercoiling was assessed by gel rlrVtjrophorrsis in the presence of chloroyuinc~, HII intercalat’ing dye that allows the tjopoisornfhrs to 1)~

following a shift to anaerobic conditions. ‘H-labeled nucleoids were extracted and analyzed as described for (a) after 20 min incubation under anaerobic1 condit,ions.

20

40 Time (mm)

60

Figure 4. [ATP]/[ADI’] during a transition to anaerobic growth. Strain .JTTl. transformed with pIY(“!l. was shifted from aerobic to anaerobic* conditions. and at various times portions were removed anaerobically for determination of [ATP]/[ADPJ as described in Materials and Methods.

(I

D

t

d

e

f

h

I

i

(c 1

Figure 3. Effert of aerobic and anaerobic growth on plasmid supercoiling. (a) Supercoiling of pUC9 extracted from cells growing exponentially. Strain ,JTTl t.ransformed with pUC9 was grown overnight under aerobic or anaerobic conditions, diluted, and then grown aerobically or anaerobically to mid-log phase. Cultures were treated for I min with rifampicin (160 pg/ml) to eliminate possible t.ranscriptional effects on plasmid supercoiling (for pUC9 ident,ical results were obtained when rifampicin was omitted). Plasmid DKA was then quickly isolated and subjected to gel electrophoresis in the presence of chloroquine (22.5 pg/ml). Direction of migration is from top t,o bottom; topoisomers migrating more rapidly have more negative supercoils. Lane 5, anaerobic growth; lane b, aerobic growth. (b) Supercoiling of pUC9 during the transition from aerobic to anaerobic growth. St,rain ,JTTl, transformed with pUC9 and growing exponentially. was shifted from aerobic to anaerobic conditions. At various times portions were treated with rifampicin for I min and plasmids were prepared as described for (a). Plasmid supercoiling was compared by electrophoresis in the prrsence of chloroquine (12.5 @g/ml); under these condit.ions more negatively supercoiled topoisomers migrate more rapidly (migration is from top to bottom). Length of time

displayetl. I’lasmid superc*oiling was more rqativc~ hy 1.5 bands or t;?{, when pL!(Yl was obtaim~cl from cells growing anaerobically (Fig. 3(a)). As with chromosomal supercoiling, a shitt from aerobic to anaerobic c*onditions caused plasmid supercoiling to transiently decrease, in this case by about 2.5 t,opoisomer hands or lci’!;, (Fig. S(I))). Addition of rifampicin immediately before rtlrnovai of oxygen increased relaxation to about four hands (data! not, shown). Apparently, expression of new protein masks part of the relaxation. The opposite shift. from anaerobic to aerobic conditions. elicited a simple relaxation of DNA (Fig. S(e)).

(b) Nupwcoiling Shifting conditions

rind / A!PPj/fA

ljP/

strain JTTl from aerobic to anaerobic resulted in a 67”o decrease in ) ATI’]/

under anaerobic conditions: 0 min (lane a), 2 min (lanr b). 5min (lane (a), 8 min (lane d), 12 min (lane P)> 15 rnin (lane f). 20 min (lane g), 30 min (lane h) and -10 min (lane i). (c) Supercoiling in pI:C9 during the transition from anaerobic to aerobic growth. Strain .JTT\. t.ransformed with pLJC9, was grown under anarrohica caonditions. Cells were shifted to aerobic growth hy vigorous aeration, and at various times portions were t,reatrd with rifampicin for I min and quickly chilled. Plasmid was extracted and the topoisomers were separated by gel electrophoresis in the presence of chloroyuine (22 pg/ml). Times of aerobic growth are as follows: 0 rnin (lane a). 2 min (lane b), 5 min (lane c), 10 min (lane d). 15 min (lane e). 20 min (lane f), 25 min (lane g), 30 min (lane h). 40 min (lane i) and 60 min (lane j).

Control of DNA

Supercoiling

447

[ADP] within five minutes (Fig. 4). As pointed out above, plasmid DNA supercoiling decreased by as much as four bands (25%). After 10 to 15 minutes of anaerobic incubation both [ATP]/[ADP] and supercoiling began to increase. Eventually both exceeded that associated with aerobic growth, although supercoiling increased more rapidly (compare Figs 2 and 4). Under conditions of exponential growth, identical to those described for Figure l(a), [ATP]/[ADP] was 25 and 3.25 for aerobic and anaerobic growth, respectively (plasmid supercoiling increased by I-5 bands]. Both for the rapid drop and for the increase seen with anaerobic exponential growth the relationship between changes in [ATPJ/[ADP] and changes in supercoiling was comparable to that observed in vitro with purified gyrase in the presence of spermidine (note that the relationship is not expected to be linear (Westerhoff et al.. 1988)). (c) ilna~robiosis

and gyrase-DNA

interactions

We next used the gyrase inhibitor oxolinic acid to a,sk whether an increase in gyrase-chromosome interactions was associated with the increase in supercoiling observed during long-term anaerobic growth. Oxolinic acid traps a reaction intermediate in which the DNA has been cleaved by gyrase, and analysis of the fragments reveals information about the interaction of gyrase with DNA (for a review, see Drlica & France, 1988). Cultures, growing exponentially under aerobic or anaerobic conditions as described for Figure 1(a), were labeled with [‘“Cl- or respectively, and incubated with ] 3H]thymidine, 5 pg oxolinic/ml acid for ten minutes. Cells were lysed and DNA size was compared by sedimentation analysis of mixed lysates in neutral sucrose gradients (Drlica et al., 1990). Comparison of number-average molecular weights (Snyder & Mica? 1979) indicated that chromosomal DNA from cells growing anaerobically was one-third the size of DNA from cells growing aerobically (data not shown). At 5 pg/ml the effective drug concentration was saturating: identical results were obtained with twice the oxolinic acid concentration, and under both aerobic and anaerobic conditions oxolinic acid inhibit’ed DNA synthesis by the same amount’ (great,er than 9496 within 5 min) using a variety of concentrations from iipg/ml up to lOO~g/ml (data not shown). Thus, it appears that gyrase-DNA interactions involving DNA cleavage are more frequent under anaerobic conditions, supporting the hypothesis that gyrase is involved in the increase in supercoiling associated with anaerobiosis.

(d) Phmid

supercoiling

in a gyrR

mutant

Perturbation of gyrase by mutation provides another way t’o examine involvement of the enzyme

in changes in supercoiling.

Among the mutations

available are several that arose as suppressors to topA mutations (Russ et nl., 1982; DiNardo et al..

40 Ttme (mm)

60

SO

Cc)

Figure 5. Effect of a gyrB mutation on plasmid supercoiling

during

the

transition

to

anaerobic

growth.

(a) Supercoiling of pUC9 extracted from a gyrB226 mutant. E. coli strain KD112. transformed with p1’(‘9 and growing exponentially, was treated as described for Fig. 3(b). Hasmid pUC9 from strain ,JTTl, growing aerobically, was electrophoresed in lane a as a reference. Length of time under anaerobic conditions: 0 min (lane b), 2 min (lane c)? 5 min (lane d), 10 mitr (lane e). 80 min (lane f), 30 min (lane g). 40 min (lane h), 50 nun (lane i). 69 min (lane j), 75 min (Jane k). 90 min (lane I). (b) Super-coiling of pUC9 extracted from wild-type (*ells. Strain ,JTTl, transformed with pUC9 and growing expo nentially, was shifted from aerobic to anaerobic? condtions, and at various times portions were processed as described for Fig. 3(b). Length of anaerobic incubation time is as indicated for (a). (c) Comparison of supercoiling change in pUC9 extracted from wild-type and a gyrB226 mutant. Midpoirns of plasmid topoisomer distributions shown in (a) and (h) were determined by visual inspection. Changes in average linking number were expressed relative to that observed for pUC9 from strain ,JTTl grown under aerobic conditions ((a), lane a: (b). lane b). These linking number changes are plotted for various times of incubation under anaerobic conditions (note that linking number becomes more positive when negative super-coils relax). Symbols: strain .JTTl (Wild-tyJ)e. 0): KDll2 (g~rR226. 0).

I!%?: Richardson et d.. 1984; Ra.ji et rrl., 19X5). (‘otnpensatory mutations of this type lower thth supercoiling activit,y of gyrase iv1 z&v (Gellert et cfl.. 1983). One of these, yyrB226 (IXNardo rt al.. 1982), when present in an otherwise wild-type background (strain KD112), lowered ohromosomal DNA suprrcoiling by about 17 “1; (data not shown). It lowered plasmid supercoiling by about IGo/,, (Fig. 5(a)). lanes a and b). We examined the effect of this mutation on t.hcbt,imcl-course of plasmid snperc+oiting changtl following a shift from aerobic: to anaerobic cortditions. Plasmid plTC9 relaxed by about two topoisomer bands or about 15% (Fig. 5(a)). This was similar to the relaxation seen in the isogenic wit& t,ype strain JTTl (Fig. 5(b)), but in t,he mut,ant this low level of supercoiling persisted for at least 60 minutes, during which time supercoiling in ,TTTl recovered and .exceeded aerobic levels (Fig. S(V)). Thus, gyrase plays a role in t’hr recovt~ry of sulJrating t)he correlations that ti)tlow rcduc~tion of oxjrgc’tt tension. It is important to emphasize t.hat tht~rc~tnav I)(, many factors involvrd in thca (~otitrol of sul,f~rcGling and t’hat their relative importattcrb may vary with thrs ?xperirncbntitt c-onditiotts. For c~xantplt~ j ;\Tl’l/ IA ])I’] may 1)~ an imltortant fac%or imrrrt~~liatt~l~ aft,er os>‘gert lension is Io\vt~rcd. bitt lat cxt’ 0t titar facstors dominate t’o raise supf~r(‘oiling bcfort, 1CiTl’ll IADP] tevc>ts in(~reasc~((*omparr Figs 2 antI 1). ;\ shift, in dominant t’ac%ors is also suggestrtt 1)~.(*antmutant and uiltl-typtl parison of‘ t hr qyrH226 (Fig. 5(c)). The two strains b~~hav~~in a sitnitar wan immediatrly after oxygr~n tlepri~ntiott. 1)11t the) dif&r in their ability to re(‘over supercoils. ‘I’hfi f’aetors t.hat dominate during the rtac*ovrr\ IjClitSt’ have not bt%*rtidt~titified: one might bt, t.he ittc*rc~as~~tl rtxpressioti of gyrase t~xpC~($r~tlto bc ;1SSO(~i;lt(yI \vit II I)NA rrlaxation (Menzet & (:ellrrt. I!#~).

The idpa t.hat. anat~robic growth tcatls I o ill1 increase in supercoiling arose’ from the oljservat ion that somr mutations mapping at or ttt’at’ thr gyrase genes conferred sensitivity to anaeroltiosis (Yamamoto & Droffrtc~r. I%%). i1S if iItlarrot)ic* growth required ctrvatcd It~vc& of ttcgati\,r> srtpt’rcoiling that c~~ultl not be attained in t tt(l trtrrt ants. Previous efforts t.0 docsutnettt an ittc*rrastJ itt sttl)c’t’ coiling have been incomplete. For t~xampl~. (.hromosoma, l)NA supereoiling c.artnot 1)~ inferrt-ct front nucleoid sedimentation studies perfot~mett iIt a sing+ c,ortcentratiotl of int~erc~itlating (Iye (Yamamot 0 1(: Droffner. 1985). and n-hen a tlif%rrttc*c in super. coiling w-its ol~srrvetl in plasmid rut~ract,etl from cvtls grown overnight under ufarobic or attaerot)ic, c,otttlitions (f)orman f~f nl.. 19X8). it was rtttc.lt>;tr whrt htlt this differenc*r arose’ from t ttfl dccrrasc* itt supercoiling assoc~iatt~d \+it h ac~robic~ stat iottar\ phasc~ (f>orman ~‘f (II.. 1988). f rom an itt(*r