comparison of plasmid dna topology among mesophilic and

JOURNAL OF BACrERIOLOGY, Mar. 1994, p. 1251-1259 Vol. 176, No. 50021-9193/94/$04.00+0Copyright X 1994, American Society for Microbiology

Comparison of Plasmid DNA Topology among Mesophilic andThermophilic Eubacteria and Archaebacteria

FRANCK CHARBONNIER AND PATRICK FORTERRE*Institut de Genetique et Microbiologie, Unite de Recherche 1354 Associee au Centre National de

la Recherche Scientifique, Universite de Paris-Sud, 91405 Orsay Cedex, France

Received 14 June 1993/Accepted 24 October 1993

Several plasmid DNAs have been isolated from mesophilic and thermophilic archaebacteria. Theirsuperhelical densities were estimated at their host strain's optimal growth temperature, and in somerepresentative strains, the presence of reverse gyrase activity (positive DNA supercoiling) was investigated. Weshow here that these plasmids can be grouped in two clusters with respect to their topological state. The groupI plasmids have a highly negatively supercoiled DNA and belong to the mesophilic archaebacteria and all typesof eubacteria. The group II plasmids have DNA which is close to the relaxed state and belong exclusively to thethermophilic archaebacteria. All archaebacteria containing a relaxed plasmid, with the exception of themoderately thermophilic methanogen Methanobcterium thermoautotrophicum Marburg, also exhibit reversegyrase activity. These findings show that extrachromosomal DNAs with very different topological states coexistin the archaebacterial domain.

Covalently closed DNA molecules so far isolated fromeukaryotes and eubacteria are negatively supercoiled to aboutthe same extent. Once purified of proteins, the native DNAmolecules exhibit a superhelical density in a range from- 0.040 to - 0.060 (4, 6, 48). Negatively supercoiled DNA hasa linking deficit, that is, the DNA has fewer duplex turns thanwould be found in a nicked or linear molecule of the samelength. The free energy of negative supercoiling permits allbiological processes such as DNA replication, transcription,transposition, and general or site-specific recombination totake place (50).

In mesophilic eubacteria, the level of supercoiling is deter-mined essentially by the opposing actions ofDNA topoisomer-ases, which either relax or produce negative superturns inDNA (18). In the eukaryotic nucleus, DNA is negativelysupercoiled around the histone core, whereas histone-freeDNA is relaxed by either DNA topoisomerase I or II (50).

In thermophilic organisms, the situation is not so clear. Themajor DNA topoisomerase activity detected in extremelythermophilic and hyperthermophilic archaebacteria is due toreverse gyrase (6, 29, 30). This ATP-dependent DNA topo-isomerase I is able to introduce positive superturns into DNA(23, 38, 40). Such DNA has an excess of links, which could helpto stabilize it at high temperatures. Reverse gyrase has alsobeen found in species of the order Thermotogales (5) and inCalderobacterium hydrogenophilum (3), which are thermophilicor extremely thermophilic eubacteria. These findings raisedthe question of the DNA topological state in all these differentorganisms.

It has been previously reported that DNA isolated from thearchaeophage SSV1 is positively supercoiled (39). However,since the cellular or viral SSV1 DNA was recovered after UVirradiation of Sulfolobus shibatae cells, it was not clear whetherthis unusual topological state takes place in cells under phys-iological conditions, since the stress due to UV irradiation caninfluence a variety of mechanisms that alter DNA topology. It

* Corresponding author. Mailing address: Institut de Gen6tique etMicrobiologie, URA 1354 CNRS, Batiment 409, Universite Paris-Sud,91405 Orsay Cedex, France. Phone: (33-1) 69 41 74 89. Fax: (33-1) 6941 78 08. Electronic mail address: [email protected].

was thus particularly interesting to investigate the topology ofplasmids isolated from strains grown in physiological condi-tions.We have recently found that the DNA of the plasmid pGT5,

isolated from the newly identified hyperthermophilic sulfur-metabolizing archaebacterium GE5, is relaxed at 95'C, theoptimal growth temperature of its host strain (9). This relaxedstate is completely different from that of the negatively super-coiled DNA of extrachromosomal elements that have beenpreviously studied in mesophilic eubacteria and eukaryotes.

In order to determine whether this unusual topologicalsituation is universal to the thermophilic organisms, we puri-fied several plasmid DNAs from mesophilic and thermophiliceubacteria as well as archaebacteria and determined theirsuperhelical densities at the physiological growth temperatureof the host strains.The results reported here strongly suggest the existence of

two groups of plasmids. The group I plasmids have a highlynegatively supercoiled DNA and were all isolated from eubac-teria and mesophilic archaebacteria. In contrast, group IIplasmids exhibit a relaxed DNA and belong exclusively tothermophilic archaebacterial group. An additional result isthat all thermophilic archaebacteria that possess a relaxedplasmid DNA show strong reverse gyrase-like activity, with theexception of a moderately thermophilic archaebacterium thatpossesses a relaxed plasmidic DNA but has no detectablereverse gyrase activity.

MATERIALS AND METHODS

Chemicals and enzymes. Alumina type A-5, cesium chloride,chloroquine, ethidium bromide, isoamyl alcohol, lysozyme,N-lauroyl sarcosine, and phenylmethylsulfonyl fluoride werepurchased from Sigma Chemical Co.; chloroform, dithiothrei-tol, ethanol, and ethylene glycol were from Prolabo; adenosinetriphosphate, BglII, bovine serum albumin, HindIII, leupeptin,pepstatin, and RNase were from Boehringer (Mannheim,Germany); phenyl-Sepharose and phosphocellulose were fromPharmacia; Indubiose A37NA was from IBF (Villeneuve laGarenne, France). All the other chemicals were from Merck(Darmstadt, Germany).

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1252 CHARBONNIER AND FORTERRE

TABLE 1. Strain characteristics and sources

Organism Typea Optimal growth Plasmid Source Refer-temp ("C) used ence

Thermus sp. YS45 Eub T 65 pTYS45-1 R. A. D. Williams (London, U.K.)b 44Rhodothermus marinus R21 Eub T 65 pRM21 S. Ertnsson and A. Palsdottir 2

(Reykjavik, Iceland)Thermus thermophilus HB8 Eub ET 80 pTT8 R. K. Hartmann (Berlin, Germany) 41Halobacterium halobium GRB Arc Halo M 37 pGRB K. Ebert and W. Goebel (Munich, 20

Germany)Haloferax volcanii WR1l Arc Halo M 37 pHV11 M. Mevarech (Tel Aviv, Israel) 34Haloferax volcanii WR12 Arc Halo M 37 pHV12 M. Mevarech (Tel Aviv, Israel) 34Halobacterium volcanii DS2 Arc Halo M 37 pHV2 S. W. Cline and W. F. Doolittle 17

(Halifax, Canada)Methanococcus sp. C5 Arc Met M 30 pURB500 D. Tumbula (Athens, Ga.) 52Methanosarcina acetivorans C2A Arc Met M 35 pC2A K. R. Sowers (Los Angeles, Calif.) 46DSM 2834

Methanobacterium thermoautotrophicum Arc Met T 65 pME2001 L. Meile and T. Leisinger (Zurich, 33Marburg DSM 2133 Switzerland)

Sulfolobus shibatae DSM 5389 Arc Sm ET 78 pSSVl W. Zillig (Munich, Germany) 24Desulfurolobus ambivalens DSM 3772 Arc Sm ET 80 pSL10 A. Kletzin and W. Zillig (Munich, 54

Germany)Isolate GE5 Arc Sm HT 95 pGT5 G. Erauso and D. Prieur (Roscoff, 21

France)

aEub, eubacterium; Arc, archaebacterium; Halo, halophile; Met, methanogen; Sm, sulfur metabolizing; M, mesophilic; T, thermophilic; ET, extremely thermophilic;HT, hyperthermophilic.

b U.K., United Kingdom.

Strains. The characteristics and sources of strains are indi-cated in Table 1. Cell masses of Desulfurolobus ambivalens,Rhodothermus marinus R21, the methanogenic archaebacteria,and the species of the order Thermotogales were gifts. The restof the strains were cultivated in the laboratory. In all cases, theorganisms were cultured at the optimal growth temperatureunder the conditions described in the references cited in Table1. Cell masses were harvested within the late-logarithmicgrowth phase, and pellets were immediately frozen and storedat - 70°C.

Sources and purification of plasmids. pBR322 (from Esch-erichia coli HB101) was purchased from Pharmacia LKB(Milwaukee, Wis.), and M13mp19RF DNA (from E. coli F')was purchased from Boehringer. pTZ18 was isolated from E.coli JM109 grown at 37°C as described previously (32).pTYS45-1 from Thermus strain YS45 (44) was obtained fromR. A. D. Williams (London, United Kingdom), and pSL10-H6was obtained from A. Kletzin (Munich, Germany). Eubacterialand archaebacterial total DNAs were prepared according tothe method described elsewhere (10). Cells were lysed at roomtemperature by the addition of 1% N-lauroyl sarcosine and 1%sodium dodecyl sulfate. Proteolysis was effected by proteinaseK treatment (1 mg/ml) at 50°C for 3 h. After phenol andchloroform isoamyl alcohol (24:1) extractions, the DNA was

precipitated with 95% ethanol. After centrifugation at 15,000x g for 30 min at 4°C in a swingout rotor, the pellet was dried,suspended in 1 ml of Tris-HCl (pH 8.0)-2 mM EDTA andtreated with 20 ,ug of DNase-free RNase per ml at 37°C for 1h. Isolation of the covalently closed circular plasmidic DNAwas achieved by using a cesium chloride gradient as describedelsewhere (32). In the case of the eubacteria R marinus R21,Thermus thermophilus HB8, and the species of the orderThermotogales, cells were first lysed by resuspension in thepresence of 10 mg of lysozyme per ml and then incubated at37°C for 1 h (10).

Two-dimensional agarose gel electrophoresis. Electrophore-sis in the first dimension was performed in 0.7% agarose gels in90 mM Tris-borate (pH 8.0)-2 mM EDTA (TEB buffer)

containing chloroquine (42) for 16 h. After electrophoresis, thegel was soaked for 8 h at room temperature in TEB buffercontaining chloroquine. Electrophoresis in the second dimen-sion was performed in the same chloroquine-containing buffer,for 16 h. The corresponding conditions which were used foreach plasmid (i.e., the concentrations of chloroquine in thefirst and second dimension, the applied electrophoretic field,and the measured critical temperature of the gel during thefirst migration) are listed in Table 2. All migrations in bothdimensions were carried out for 16 h, except in the case ofpURB500 (Fig. 1, lane E), which migrated in the first dimen-sion for 24 h. In order to visualize large amounts of topoiso-

TABLE 2. Electrophoretic migration conditions of plasmidic DNAs

Special electrophoretic conditions

First Second

Plasmid Size electrophoresis electrophoresisPlad(bp) Chloro- Electric Gel Chloro- Electric

quine field temp quine field(,Lg/ml) (V/cm-) (OC) (,ug/ml) (V/cm-)

pTZ18 2,880 2.5 2 23 5 1.3pBR322 4,362 2.5 2.5 26 5 1.6M13mpl9 7,500 2.5 2.8 32 5 1.7pTYS45-1 5,900 5 2.5 29 10 1.7pRM21 2,927 5 1.9 26 10 1.3pTT8 9,700 2.5 3.3 27 10 1.5pGRB 1,781 5 1.8 22 10 1.3pHVll 3,000 2.5 2 23 5 1.3pHV12 5,000 5 2.7 28 10 1.3pHV2 6,354 5 2.7 34 10 2.2pURB500 8,700 5 2.6 33 10 1.3pC2A 5,100 5 2.7 28 10 1.3pME2001 4,500 2.5 2.5 26 5 1.6pSSVl 15,400 0 2.5 26 5 1.6pSL10 7,700 2.5 2.8 12 5 1.7pGT5 3,450 2.5 2 23 5 1.3

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SUPERCOILING OF MESOPHILIC AND THERMOPHILIC PLASMIDS 1253

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FIG. 1. Comparative topoprofiles of archaebacterial and eubacte-rial plasmid DNAs, analyzed by two-dimensional agarose gel electro-phoresis where chloroquine was present in both dimensions. (A)halophilic archaebacterial plasmids. Lanes B and C, plasmids pHV11and pGRB, respectively. (B) Methanogenic archaebacterial plasmids.Lanes E and G, plasmids pURB500 and pME2001, respectively. (C)Plasmids from thermophilic sulfur-metabolizing archaebacteria. LanesH, I, and J, plasmids pGT5, pSL1O, and pSSV1, respectively. (D)Thermophilic eubacterial plasmids. Lanes K, L, and M, plasmidspTYS45-1, pRM21, and pTT8, respectively. The mesophilic eubacte-rial plasmids used as controls were pTZ18 (A, A', A"), M13mp19RF(D, D', and D"), and pBR322 (F, F', F", and F"'). Arrows 1 and 2 showthe direction of the run in the first and second dimensions, respec-

tively. Plasmid open circular form II (*) and linear form III (O) are

indicated. Radioactive detection of the plasmid is shown by dashed-line squares. Arrows with the value and the topological sign indicatethe major topoisomer of each plasmid as detected by densitometricanalyses. We named topoisomers with respect to first or secondmigration, depending on the configuration of the arch. If the arches ofthe plasmid and its control show a top, this band is assigned the value0, corresponding to the relaxed topoisomer in the first migration;topoisomers are then numbered and designated first-dimension topo-isomers (open numbers). However, if the two arches show only a leftbranch, the band at the leftmost position is assigned the value 0,corresponding to the relaxed topoisomer in the second migration; inthis case, topoisomers are numbered and designated second-dimensiontopoisomers (solid numbers). In all cases, this rule permits insertion ofa marker into the arches, whatever their configuration, and determi-nation of the relative sign of the major topoisomers of each plasmidand its control. The special electrophoretic conditions used for eachplasmid are described in Materials and Methods and in Table 2.

mers of the plasmid pSL1O (lane I), the first migration wasperformed at 12°C with the same electrophoretic parametersas used for the eubacterial controls (lanes D' and F"). Afterelectrophoresis, the gel was stained with ethidium bromide andchloroquine was eliminated with MgSO4 (1 mM). Polaroidphotographs were taken under 254-nm UV illumination. Den-sitometric analyses were performed as described elsewhere (9).

Radioactive detection of plasmids. Stringent conditions wereused to visualize specific detections (32). The plasmid pSL10probe was prepared by incubating 25 ng of the constructedplasmid pSL10-H6 with HindIll (15 enzymatic units) in bufferB (Boehringer) for 1 h at 37°C. To prepare the probe for pTT8detection, 25 ng of the plasmid was linearized by 10 enzymaticunits of BglII in buffer M (Boehringer) for 1 h at 37°C. ThepSL10 and pTT8 probes were 32p labelled with a randompriming kit (Megaprime DNA labelling system; Amersham,Amersham, United Kingdom) and hybridized for 16 h at 650C.

Determination of the plasmid superhelical density at phys-iological temperatures. To calculate the superhelical densityvalue, the major topoisomer of the distribution in two-dimen-sional gel electrophoresis was first determined for each plas-mid and its control (see the legend of Fig. 1). Densitometricanalyses, as previously described (9), permitted determinationof its value with, in most cases, a relative precision of +0.5,corresponding to the limit of accuracy of the band-countingmethod (28). For example, the major values for the second-dimension topoisomers of the plasmids pTZ18 (Fig. 1, lane A)and pHV1 1 (lane B) are, respectively, - 1 and -5.

Furthermore, we determined the superhelical density of theplasmid at the electrophoresis temperature, with eubacterialplasmids of known superhelical densities as internal markers.The superhelical density of the plasmid pTZ18 had beencalculated to be -0.052 at 250C (9), according to the band-counting method (28), and was used to calculate the superheli-cal densities of both the other eubacterial controls, pBR322and M13mpl9, as -0.053 at 25°C.

Using these values, we determined the major topoisomer ofeach control at the gel temperature, in the absence of anymigration. The observed differences between the estimatedvalues for the major topoisomers in the two-dimensional gelwere used to calculate the modification of the winding angleintroduced by chloroquine during the first dimension of theelectrophoresis. For example, in Fig. IA, lane B, we calculateda value of - 15 at 230C for the major topoisomer of pTZ18 inabsence of migration. After two-dimensional electrophoresis,the pTZ18 major topoisomer is -1 at 23°C; the observeddifference therefore is 14 negative superturns for 2,880 bp. Inthe case of pHV11, the deduced difference is thus 14.6 negativesuperturns for 3,000 bp. Since its major topoisomer afterelectrophoresis is -5, the difference led us to calculate a valueof - 19.6 for the major topoisomer of pHV1 1 at 23°C in theabsence of electrophoresis.The effect of temperature on DNA supercoiling also has to

be taken into account. Each superhelical density, determinedat the gel temperature, was corrected according to the equa-tion relating the rotation angle of the DNA double helix withtemperature. We have previously determined this equation tobe - 0.0100/°C/bp in a range of temperatures from 25 to 950C(9). For the above-cited example of pHV11, this equation givesa value of 1.20 negative superturns lost between 37 and 23°C.This led to the calculated values of - 18.4 for the majortopoisomer of pHV1 1 at 37°C and - 0.065 for the correspond-ing superhelical density at 37°C.

Table 3 shows the calculated superhelical densities at phys-iological temperatures for each plasmid we have analyzed. Thereported values are the averages of several different plasmidic

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TABLE 3. Superhelical densities of several mesophilic andthermophilic plasmidic DNAs

Optimal SuperhelicalHost organisms and growth Plasmid density at optimalthermotolerance type temp growth temp(OC)

EubacteriaMesophile strains 37 pTZ18 -0.051a

37 pBR322 -0.050 ± 0.00237 M13mpl9 -0.049 ± 0.002

Thermophile 65 pTYS45-1 -0.057 ± 0.00165 pRM21 -0.063 ± 0.001

Extreme thermophile 80 pTT8 - 0.059 ± 0.002

ArchaebacteriaHalophile 37 pGRB -0.068 ± 0.001

37 pHV11 -0.068 ± 0.00237 pHV12 -0.060 ± 0.00337 pHV2 -0.060 ± 0.004

Mesophilic methanogen 30 pURB500 -0.058 ± 0.00335 pC2A -0.048 ± 0.002

Thermophilic methanogen 65 pME2001 -0.013 ± 0.003Extreme thermophile 78 pSSV1 +O.OlSb

80 pSL10 +0.007 ± 0.004Hyperthermophile 95 pGT5 -0.003 ± 0.003

a Independent data calculated by the method described by Keller (28).b Estimated value (see the explanation in the text).

DNA extractions. In the case of pHV11, the average is - 0.068+ 0.002 at 370C.Preparation of crude cell extracts. Crude cell extracts were

prepared as described elsewhere (9), with some modifications.After addition of alumina (wt/wt) in a sterile crucible, frozencells (about 2 to 3 g) were crushed for 15 min at 40C. Brokencells were then suspended at 40C in 10 ml of buffer A {50 mMNa2HPO4-NaH2PO4 [pH 7.0], 1 mM [each] dithiothreitol,EDTA, EGTA [ethylene glycol-bis(,B-aminoethyl ether)-N,N,N',N'-tetracetic acid], 0.4 mM phenylmethylsulfonyl flu-oride, and 1 p,g [each] of leupeptin and pepstatin A per ml}containing 1.5 M NaCl and 1 M (NH4)2SO4. Alumina waseliminated by centrifugation at 2,000 x g for 15 min at 40C.The supernatant (crude cell extract) was carefully removed,immediately frozen in liquid nitrogen and stored at - 70°C.Protein concentration was measured by the method describedby Bradford (8).

Preparation of partially purified extracts. In the cases ofMethanobacterium thermoautotrophicum Marburg and T ther-mophilus HB8, the procedure for partial purification wasadapted from the method described by Bouthier de la Tour etal. (5, 6). About 5 mg of proteins from crude cell extracts wereloaded onto a phenyl-Sepharose column (0.5 by 2 cm) equili-brated with buffer A containing 1.5 M NaCl and 1 M ammo-nium sulfate at a flow rate of 1.5 ml/h. The column was washedwith 5 ml of the equilibrating buffer (step 1). The proteins werestep eluted with 5 ml of buffer A containing 0.3 M NaCl (step2), 0.3 M NaCl and 30% ethylene glycol (step 3), 0.3 M NaCland 60% ethylene glycol (step 4), and then 0.3 M NaCl and80% ethylene glycol (step 5). Collected fractions of 500 ,lI(steps 1 to 3) or 250 ,ul (steps 4 and 5) were tested fortopoisomerase activity. Protein concentrations were measuredas described by Bradford (8).The fractions of the 60% ethylene glycol step were pooled

and dialyzed against buffer A containing 0.1 M NaCl and 15%ethylene glycol. Fractions were loaded onto a phosphocellulosecolumn equilibrated with the dialysis buffer at the same flowrate as mentioned above. The column was washed with 5 ml of

the same buffer (step 1'), and the proteins were finally stepeluted with the same buffer containing 1 M NaCl (step 2').Fractions of 300 pul (steps 1' and 2') were tested for topo-isomerase activity, and protein concentrations were deter-mined as described elsewhere (8).

Topoisomerase assays. Topoisomerase activity was assayedby incubating 500 ng of pTZ18 DNA with crude cell extracts orsemipurified fractions at the optimal salt and temperatureconditions for each organism. The standard reaction mixture(20 ,ld) contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 30jig of bovine serum albumin per ml, 12% ethylene glycol, and0.5 mM (each) dithiothreitol and EDTA. Two microliters of adilution of the crude cell extract or the fraction to be tested wasadded, and the mixture was incubated for 20 min at the optimalgrowth temperature of each considered strain. After additionof 2 ,ul of 10 mg/ml proteinase K, incubation was performed at50°C for 15 min. The products of the incubations wereanalyzed by two-dimensional gel electrophoresis in order todistinguish between negatively and positively supercoiled to-poisomers.

RESULTS

Estimation of plasmid topology at physiological tempera-tures. In order to elucidate and compare the DNA topology ofseveral plasmids, all purification steps of the different plasmidsmust first be identical. The carrier strains were all cultured atthe optimal growth temperature (Table 1) and harvestedwithin the late logarithmic phase. Secondly, all plasmids werepurified by the total DNA extraction method (see Materialsand Methods), which involves the fewest DNA manipulations,in order to recover all the DNA molecules (10). Hence, eachplasmid was analyzed along with the eubacterial plasmid ofcorresponding size as a control by two-dimensional electro-phoresis (7). To visualize the topoisomers of the plasmid andits control in the same gel, chloroquine (42) was added in thefirst dimension of the electrophoresis. The running conditionswere optimized for each plasmid to get the clearest resolutionof the topoisomers (Table 2). Higher concentrations of chlo-roquine (two- or fourfold that in the first dimension) wereadded in the second electrophoresis in order to separate thetopoisomers that were either negatively or positively super-coiled in the first dimension.

Fig. 1A and B (lane E) shows that all plasmids from themesophilic archaebacteria were relaxed by chloroquine in thefirst electrophoretic dimension and are more negatively super-coiled than their respective controls at the gel running tem-perature. This is the case for the halophilic plasmids pHV11(lane B) and pGRB (lane C) and the methanogenic plasmidpURB500 (lane E). Similarly, Fig. 1D shows that all theplasmids from thermophilic and extreme thermophilic eubac-teria were relaxed by chloroquine and are also more negativelysupercoiled than their controls at the running temperature.This is so for the eubacterial plasmids pTYS45-1 (lane K),pRM21 (lane L), and pTT8 (lane M).

In contrast, Fig. 1B (lane G) and C shows that all theplasmids from thermophilic, extremely thermophilic and hy-perthermophilic archaebacteria were positively supercoiled bythe addition of chloroquine during the first migration. This isthe case for the archaebacterial plasmids pME2001 (lane G),pGT5 (lane H), pSL10 (lane I), and pSSV1 (lane J). In the caseof pGT5, this has already been shown via a similar extractionmethod (9) and is presented anew in order to compare theplasmid's topologies under the same conditions. The resultsindicate that 2.5 pug of chloroquine per ml, in the firstdimension, was sufficient to produce almost 50% very com-

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FIG. 2. Superhelical densities of several mesophilic and thermo-philic plasmids as a function of the optimal growth temperature oftheir host strain. Superhelical densities of plasmids from archaebacte-ria (0) and eubacteria (0) are indicated. The ellipses show the twogroups of plasmids (I and II) that we have defined.

pacted and positively supercoiled forms of pSL10 DNA, andmoreover, all topoisomers of pSSV1 were visualized withoutaddition of chloroquine, indicating the very different state ofthese plasmids from that of their negatively supercoiled con-trols.To calculate the superhelical densities of the various plas-

mids, we determined the major topoisomer for each by densi-tometric analysis. In the cases of pSL10 and pTT8, we useddifferent exposure times, since all topoisomers were not visibleon the same autoradiogram. In the case of pURB500, we failedto completely resolve the topoisomers of both this plasmid andthe plasmid control on the same gel. Nevertheless, the value ofthe major topoisomer from the control plasmid was obtainedby densitometry with an accuracy of ± 1. In the case of pSSV1,we failed to observe discrete topoisomers under our condi-tions, because of the molecule size (15.4 kb); nevertheless, thebroad heterogeneity of topoisomer distribution, the absence ofspots (indicating completely negatively or positively super-coiled DNAs), and the presence of many relaxed topoisomersat the top of the arch, led us to consider a value of around 0 forthe major topoisomer in these conditions.

Defined as the number of superhelical turns in a circularduplex DNA per 10.5 bp (49), the superhelical density permitsdirect comparison of DNA molecules differing in size. More-over, if this value is determined at the growth temperature ofthe host strain, it allows estimation of the topological state ofthe plasmids under in vivo physiological conditions. In this way,we used the equation relating the DNA winding angle to thetemperature (-0.010°/°C/bp), which we had previously vali-dated for a range of temperatures from 25 to 95°C (9). Thesame equation was also recently reported by Duguet in a rangeof temperatures from 21 to 83°C (19). The superhelical densityof each plasmid was thus determined (Table 3). The reportedvalues are averages for several different plasmid DNA extrac-tions.We have plotted the superhelical density of each plasmid as

a function of the optimal growth temperature of the respectivestrains. Figure 2 shows that we can define two major groups,denoted by the ellipses. The first group consists of plasmidsfrom mesophilic archaebacteria and from all eubacteria, evenif the latter are thermophilic or extremely thermophilic. In this

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FIG. 3. Topological conversions of pTZ18 DNA after incubationwith non- or semipurified extracts from five thermophilic organisms.The assays were performed in the absence ( - ) or presence (+) of 1.25mM ATP. The incubation products were analyzed by two-dimensionalgel electrophoresis. A first migration without chloroquine was per-formed at 1.8 V/cm-l. A second migration was performed in thepresence of 2.5 pg of chloroquine per ml at 1 V/cm- l. Arrows 1 and2 show the first and second electrophoresis runs, respectively. In eachlane, left and right branches of the arch correspond to the negativelyand positively supercoiled topoisomers, respectively. (A) negativelysupercoiled pTZ18 DNA control. (B, C, and D) pTZ18 DNA incu-bated at 150 mM NaCl with crude cell extracts from strain GE5 (B), D.ambivalens (C), and S. shibatae (D) for 20 min at 95, 80, and 78°C,respectively. (E and F) pTZ18 DNA incubated in the presence of 30mM NaCl with the most active fraction step eluted from the phospho-cellulose column (see Materials and Methods) from M. thermoautotro-phicum Marburg (E) and T. thermophilus HB8 (F) for 20 min at 65 and80°C, respectively. Open circular form II (*) and linear form III (K)pTZ18 DNA are shown. -_, restriction fragments from the weakcontaminating genomic DNA of the pTZ18 DNA.

group, the plasmid superhelical densities vary from - 0.068 to- 0.048. All these group I plasmids are very negatively super-coiled at the host's physiological growth temperature. Thesecond group comprises plasmids from thermophilic, ex-tremely thermophilic, and hyperthermophilic archaebacteria.No eubacterial plasmid falls into this group. The plasmidsuperhelical densities vary from -0.013 to +0.015. All thesegroup II plasmids are in a relaxed state at the physiologicalgrowth temperature of the host strain. They exhibit only a fewnegatively or positively supercoiled topoisomers at their in vivoequivalent temperatures.These results show that two types of plasmids can exist in

vivo. One has negatively supercoiled DNA; the other hasrelaxed DNA.Topoisomerase activity in archaebacteria and eubacteria. In

order to understand the difference between strains that possessrelaxed group II plasmids and negatively supercoiled group Iplasmids, we investigated the existence of a reverse gyrase-likeactivity in several strains of archaebacteria and eubacteria.Figure 3 shows the products of incubation of negativelysupercoiled pTZ18 DNA with non- or semipurified fractionsfrom thermophilic organisms. For each organism, optimal saltand temperature conditions for topoisomerase activity weredetermined. The conversion of pTZ18 DNA substrate (Fig. 3,panel A) to a distribution of topoisomers (panels B to F) wasanalyzed by standard two-dimensional agarose gel electro-phoresis. In all the gels, the left branch of the arch is made upof negatively supercoiled topoisomers, while the right branchrepresents positively supercoiled DNA.

In the cases of the hyperthermophilic and extremely ther-

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mophilic archaebacterial strains GE5, D. ambivalens, andSulfolobus shibatae (Fig. 3, panels B, C, and D, respectively),the negatively supercoiled pTZ18 DNA became completelypositively supercoiled after incubation with the crude cellextracts at the respective temperatures of 95, 80, and 78°C. Thereaction is strictly ATP dependent (Fig. 3; compare results inthe presence or absence of ATP) for the standard reactionmixture containing 150 mM NaCl, 1.25 mM ATP, and 1, 12.5,or 15 ng of crude extract proteins for GE5, D. ambivalens, orS. shibatae, respectively. This ATP-dependent activity, able toproduce a large quantity of positively supercoiled topoisomers,is characteristic of reverse gyrase activity. In the case of GE5,the reverse gyrase activity has already been detected in anotherpreparation of crude cell extracts (9) and we present anew thisresult here in order to compare the detected reverse gyraseactivities of several strains under the same conditions. In thecase of D. ambivalens (Fig. 3, panel B), a low ATP-indepen-dent relaxation activity was detected and incubation in thepresence or absence of ATP showed the presence of nucleaseactivity, as indicated by the appearance of linear form III anda larger quantity of the open circular form II of plasmidpTZ18.No ATP-dependent activity was observed by incubating

pTZ18 DNA in 150 mM NaCI with crude cell extracts from thethermophilic archaebacterium M. thermoautotrophicum Mar-burg and the extremely thermophilic eubacterium T ther-mophilus HB8, at 65 and 80°C, respectively. On the contrary, aprominent ATP-independent relaxation activity and a highlevel of nucleolytic activity were detected, irrespective of theNaCl or KCl concentration in the reaction mixture (30, 50, 300,or 600 mM). As suggested by the results obtained by Bouthierde la Tour et al. for species of the order Thermotogales (5, 6),reverse gyrase activity could be masked by these other twoactivities in the thermophilic organisms. Thus, we tried furtherpurification steps. Partial purification of DNA topoisomeraseactivities from M. thermoautotrophicum Marburg and T ther-mophilus were first achieved by phenyl-Sepharose chromatog-raphy (fraction FI). The crude extract proteins were boundunder high salt conditions and step eluted with increasingethylene glycol concentrations under low-salt conditions. Inboth cases, we detected a single peak of topoisomerase activitywhich was eluted at 60% ethylene glycol. The most activefractions were incubated at low (30 mM NaCI) or medium (150mM NaCl) ionic strength in the standard reaction mixture. Inall cases, the ATP-independent relaxing activity was the onlynoticeable detected topoisomerase activity. Further purifica-tion was performed by chromatography on phosphocellulose(fraction FII). Proteins of the pooled 60% ethylene glycolfractions were loaded onto the column in a low-salt buffer (0.1M NaCI) and step eluted at high salt concentration (1 MNaCI). The most active fraction (80 and 100 ng of proteins)was incubated in the reaction mixture containing 30 mM NaCland pTZ18 DNA at 65 and 80°C, forM thermoautotrophicumMarburg (Fig. 3E) and T. thermophilus HB8 (Fig. 3F), respec-tively. In neither of these cases did we observe reverse gyrase-like activity, but we did again detect a major ATP-independentrelaxation activity. In the case of M thermoautotrophicumMarburg, the ATP-independent activity seems to be enhancedcompared with the low level of nuclease activity (Fig. 3E),while this contaminating activity is again coupled with theATP-independent activity in T. thermophilus HB8 (Fig. 3F).These results are summarized in Table 4. The extreme

thermophilic S. shibatae and D. ambivalens, and the hyperther-mophilic GE5 all possess readily detectable reverse gyraseactivity able to produce positively supercoiled topoisomers athigh temperatures in the presence of ATP. In the case of these

TABLE 4. Presence of reverse gyrase activity in several mesophilicand thermophilic organisms

Presence of DNAOptimal Last characteristica

Organism(s) growth purificationtemp stpurfcto Positive ATP-(OC) step super- dependent

coiling activity

EubacteriaE. coli HB101 37 Crude extract - -T. thermophilus HB8 80 FIIb - -

ArchaebacteriaHalobacterium sp. GRB 37 Crude extract - -

M. thermoautotrophicum 65 FII - -

MarburgS. shibatae 78 Crude extract + +D. ambivalens 80 Crude extract + +Isolate GE5 95 Crude extract + +

a +, presence; -, absence.b FII, phosphocellulose fraction.

archaebacteria, this activity is detected by incubating a verysmall amount of crude extract proteins in the test reaction.Although Bouthier de la Tour et al. have shown that this

activity was present in many thermophilic organisms (5, 6), wefailed to observe reverse gyrase activity in the extreme ther-mophilic eubacterium T thermophilus HB8. This result hasalready been obtained by Collin et al. (11) after incubatingcrude extracts of several other strains of Thermus. We alsofailed to observe reverse gyrase activity in the case of thethermophilic archaebacterium M. thermoautotrophicum Mar-burg. These last two organisms exhibit a major ATP-indepen-dent relaxation activity, as is the case for the mesophiliceubacteria and archaebacteria (Table 4).

DISCUSSIONWe have shown previously that the plasmid pGT5 from the

hyperthermophilic archaebacterium GE5 is relaxed at physio-logical temperatures (9). In order to investigate the universal-ity of such an unusual topological situation, we isolated severalmesophilic and thermophilic plasmids constituting a represen-tative collection. We analyzed their topological state by deter-mining their superhelical density at the optimal growth tem-perature of their host strain.Our results suggest that a plasmid can exhibit two different

topological states, depending on the strain harboring the DNA.The first group contains plasmids that have a highly negativelysupercoiled DNA. All the host strains of these group Iplasmids are either eubacteria or mesophilic archaebacteria.Their superhelical densities vary from - 0.068 to - 0.048; thisis in the range of the values previously described for eubacteria(4, 45, 48). The second group consists of plasmids that haveDNA which is close to the relaxed state. Their superhelicaldensities vary from -0.013 to +0.015. Up to now, we havefound that these group II plasmids exist exclusively in thermo-philic, extremely thermophilic, and hyperthermophilic archae-bacteria. In our investigations, no plasmid has been found tohave a superhelical density value between - 0.048 and - 0.013.As there is a lack of intermediate superhelical density

plasmids, the two groups seem to be distinct. The question liesin whether we can assume that the difference between thevalues of their superhelical densities is significant. We do thinkthat this is the case. We made several independent extractionsfor each plasmid and never observed significant variations of

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I EUKARYOTES|

Calderobacterium hydrogenophilum [75]0

Thermotoga [80]0Fervidobacterium [751] /

Thermus thermophilus HB8 [801()Thermus YS45 [65](i)Rhodothermus marinus R21 [65] 0

I EUBACTERIA I

Methanosarcina acetivorans C2A [351]0

Halobacterium volcanfi DS2 [3710Halobacterium sp. GRB [3710

Haloferaxvolcanii rstrainWRIl [37](0(strain WR12 [37] 0

Methanobacterium thermoautotrophicum strain W [65]MMethanothermus fervidus [85] |strain Marburg [651]®

Methanococcus sp. C5 [30] 03_ Isolate GE5 [951 ( 0

ARCHAEBACTERIA

Desulfurolobus ambivalens [80] ()

Sulfolobus shibatae [78] ®)0

E. coli strain JM109 [3710strain HB101 [37]1strain F' [371]

FIG. 4. Schematic comparison of the appearance of thermophily, the presence of reverse gyrase activity, and a topologically relaxed plasmidDNA among archaebacteria and eubacteria. The unrooted phylogenetic tree is from D. A. Cowan (16). Dashed lines indicate independently knownphylogenetic positions and do not indicate the phylogenetic distances. The optimal growth temperature of each strain is indicated in brackets, withextreme temperatures in boldface type. The plasmid DNA topology (in open circles) is indicated for negatively supercoiled plasmid DNA (-) andrelaxed plasmid DNA (0). The presence of reverse gyrase activity in the strain is shown by shaded circles. The presence of reverse gyrase-likeactivities in M. thermoautotrophicum (strain W), M. fervidus, and strains of Thermotoga and Fervidobacterium has been investigated by Bouthierde la Tour et al. (5, 6). Reverse gyrase activity in C hydrogenophilum has been detected by Andera et al (3).

superhelical density values. Also, the hyperthermophilic ar-chaebacterial strain GE5 was cultured at several growth tem-peratures from 80 to 95°C and plasmid pGT5 was purified andanalyzed. The superhelical density values vary from -0.0125to - 0.006 (9). The mesophilic and halophilic strain Haloferaxvolcanii WRl1 was also cultured between 30 and 50°C, andplasmid pHV11 was analyzed. The superhelical density valuesvary from - 0.075 to - 0.055 (35).

In E. coli, plasmid supercoiling can be affected by the level oftranscription and/or by modification of environmental param-eters, such as the salt concentration in the growth medium ora shift from aerobic to anaerobic conditions (31, 51, 53).Nevertheless, the supercoiling fluctuations observed in thesecases are smaller than the supercoiling difference between thetwo plasmid groups defined here. For example, Figueroa andBossi reported a decrease in superhelical density of - 0.016 +0.002 following transcriptional activation of a reporter plasmid(22) and others concluded that the level of DNA supercoilingin vivo is primarily determined by topoisomerase, not bytranscription (14, 15). Similarly, the absolute values in themodification of superhelical density of pUC9 plasmid, follow-ing shifts between two different salt concentrations or betweenthe anaerobic and aerobic states, range from 0.006 to 0.016 (26,27).We never observed different populations of DNA molecules

for the same DNA but saw a major Gaussian distributionexcept for the eubacterial plasmid pTT8 from T. thermophilus.In that case, very strong radioactive detection permitted us toobserve, albeit in a very small amount (less than 5%), a secondpopulation composed of positively supercoiled plasmids. Onecan speculate that this unusual topological state reveals apopulation of plasmids that have been transiently positivelysupercoiled in the course of transcription or replication.

In conclusion, the two states of plasmid supercoiling de-tected in this work probably reflect important differences in thetopological state of chromosomal DNA. Interestingly, it hasbeen shown recently that the site-specific integrase encoded bythe virus SSV1 from S. shibatae recombines linear and relaxedsubstrates efficiently, whereas negatively or positively super-coiled DNAs are poor substrates (37). This suggests thatintracellular DNA from hyperthermophilic archaebacteriacould indeed be relaxed in vivo. The finding of relaxedplasmids in vivo might seem surprising, although recent studieshave shown that mitochondrial DNA from the eukaryoteCrithidia fasciculata is relaxed in the giant in vivo kinetoplastnetwork (43).

In order to understand what intracellular factor(s) areresponsible for the relaxed-DNA state of plasmids from ther-mophilic archaebacteria, we have investigated the presence ofreverse gyrase in strains harboring the two types of plasmids.Figure 4 groups our results together from a phylogenetic pointof view.

All organisms that do not possess reverse gyrase activityexhibit negatively supercoiled DNA, with the exception of M.thermoautotrophicum Marburg (see the discussion below).These organisms consist of mesophilic archaebacteria and alleubacteria, even extreme thermophiles such as Thermus sp.Alternatively, all organisms that possess reverse gyrase activityharbor relaxed plasmid DNA; up to now, these latter organ-isms have been found exclusively to comprise thermophilicarchaebacteria. As Bouthier de la Tour et al. (5, 6) have shownthat extremely thermophilic eubacteria of the order Thermo-togales also possess reverse gyrase, it would have been veryinteresting to analyze the topology of extrachromosomalDNAs in these strains. Unfortunately, our extraction methodfailed to detect any plasmid in such bacteria of the order

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Thermotogales as Fervidobacterium sp. and several Thermotogaspecies.We previously proposed that the relaxed state of the plasmid

pGT5 might be the result of a balancing out of the reversegyrase activity on protein-free DNA and the negative super-coiling of the DNA around histone-like proteins (9). Thediscovery of a relaxed plasmid (pME2001) in a strain that doesnot possess reverse gyrase activity argues against this model.We failed to detect positive supercoiling activity in M. therno-autotrophicum Marburg, even after using two purification stepsand different salt conditions. This result is in agreement withthe fact that reverse gyrase has never been detected inorganisms that have an optimal growth temperature below70°C, and the optimal growth temperature of M. thermoau-totrophicum Marburg is 65°C. In particular, Bouthier de laTour et al. did not find any reverse gyrase activity in the relatedorganism M. thermoautotrophicum strain W (6).

However, one cannot completely exclude the possibility thatreverse gyrase is present in M. thermoautotrophicum Marburgbut cannot be detected under our experimental conditions.Although the gene encoding Sulfolobus acidocaldarius reversegyrase has been recently cloned (13), it cannot be used to checkthe presence of reverse gyrase in M. thermoautotrophicum,since it does not give a hybridization signal with DNA fromother archaebacteria known to possess reverse gyrase (12). Inany event, the patterns of DNA topoisomerase activitiesappear strikingly different in M. thermoautotrophicum Marburgand in archaebacteria with demonstrated reverse gyrase. In-deed, Marburg exhibits strong and readily detectable ATP-independent DNA relaxation activity, which is absent orscarcely noticeable in archaebacteria containing reverse gyrase(29). Thus, M. thermoautotrophicum Marburg might be animportant link between two subdomains in the archaebacterialworld, one possessing reverse gyrase activity and relaxedplasmids and the other without reverse gyrase and harboringnegatively supercoiled plasmids (Fig. 4).Another problem with our previous model to explain the

relaxed state of archaebacterial plasmid is the recent findingthat the histone-like protein HMf, discovered first in Methano-thermus fervidus, induces nucleosome-like structures in whichthe DNA is positively supercoiled (36, 47). Accordingly, thismodel is probably too simple or not valid for all thermophilicarchaebacteria.A protein homologous to HMf has been detected recently in

M. thermoautotrophicum AH (47). The relaxed state ofpME2001 in Marburg could also be related to the presence ofthis unusual histone-like protein. However, HMf-like proteinshave not been detected up to now in nonmethanogenic ther-mophilic archaebacteria and a gene encoding a putative HMf-like protein has been detected in the mesophile Methanococcusvoltae (1).

Since DNA gyrase is essential to maintain the negativesuperhelicity of eubacterial plasmids, still another hypothesis isthat the topological state of plasmids in archaebacteria iscorrelated with the presence or absence of DNA gyrase.Indeed, a gene encoding a type II DNA topoisomerase-whose sequence is closely related to those of eubacterial DNAgyrases-has been isolated in a strain of Haloferax (25),whereas a type II DNA topoisomerase without gyrase activityhas been detected in Sulfolobus sp. (29). This is in agreementwith the finding of negatively supercoiled plasmids in halobac-teria and relaxed plasmids in members of the order Sul-folobales.

In conclusion, complete understanding of the biologicalmechanisms that take place in the organisms having a relaxedplasmidic DNA needs to be reached. One consideration,

preeminent for future investigations, is our need to completelyelucidate the pattern of DNA topoisomerase content in allbranches of the archaebacterial domain as well as the organi-zation and structure of the "thermophilic" chromosome and itsassociated proteins.

ACKNOWLEDGMENTSWe thank S. Erntsson and A. Palsdottir for the generous gift of R

marinus, R. K. Hartmann for T. thermophilus, K. Ebert, W. Goebel,S. W. Cline, and W. F. Doolittle for Halobacterium species, M.Mevarech for Haloferax species, D. Tumbula for Methanococcusspecies, K. R. Sowers for M. acetivorans, L. Meile and T. Leisinger forM. thermoautotrophicum, A. Kletzin and W. Zillig for D. ambivalensand S. shibatae, R. Huber for Fervidobacterium and Thermotogaspecies, G. Erauso and D. Prieur for strain GE5, and R. A. D. Williamsfor the plasmid pTYS45-1. We thank D. Vicky Gavrias for helpfuldiscussion and critical reading of the manuscript. We are grateful to D.Armel Guyonvarch for his help in bibliographic research assisted bycomputer.

This work was supported by Association pour la Recherche sur leCancer (A.R.C.), and the Ministere de la Recherche et de la Tech-nologie. F. Charbonnier was supported by a Ph.D. grant from A.R.C.

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