JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 416-421Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 2Printed in U.S.A.

Isolation of Covalently Closed Circular Deoxyribonucleic Acidfrom Streptomyces coelicolor A3(2)

HILDGUND SCHREMPF, HERMANN BUJARD, DAVID A. HOPWOOD, AND WERNER GOEBEL*

Gesellschaft fair Molekularbiologische Forschung mbH, 3301 Stockheim/Braunschweig, Germany*; Institutfair Molekulare Genetik der Universitdt Heidelberg, Heidelberg, Germany; and John Innes Institute,

Norwich, England

Received for publication 20 September 1974

Covalently closed circular deoxyribonucleic acid (DNA) was isolated from twostrains of Streptomyces coelicolor A3(2), representing two of the known fertilitytypes. In each of the two strains circular DNA of about 20 x 106 daltons could bedetected, amounting to about 1.5% of the total cellular DNA. The possible func-tion of this DNA is discussed.

Extrachromosomal deoxyribonucleic acid(DNA) elements, which are either plasmids ordefective phage genomes, have been isolatedfrom a wide variety of bacterial species. How-ever, as far as we are aware, no supercoiled DNAhas been identified until now in streptomycetes.Genetic studies on Streptomyces coelicolorA3(2) have indicated that initial fertility (IF)strains harbor a plasmid designated as SCP1(16, 17). This plasmid, as well as acting as a sexfactor leading to mobilization of chromosomalmarkers (7), confers on strains that carry it theproperty of producing a diffusible substancethat inhibits strains lacking the plasmid. Thelatter are designated ultrafertility (UF) strains.The SCP1 plasmid is transferred efficientlyfrom IF to UF strains when they are growntogether (16).

Although SCP1 of S. coelicolor A3(2) is genet-ically the best characterized plasmid among thestreptomycetes, circumstantial evidence is ac-cumulating for the control of several phenotypicproperties by extrachromosomal elements incertain other streptomycetes; these include theproduction of melanin (5), aerial mycelium(12), and antibiotics (1, 12). Thus, plasmidsmay turn out to be important genetic determi-nants in this group of bacteria, as in theenterobacteria, pseudomonads, and staphylo-cocci.

In this report the isolation of supercoiledDNA from both IF and UF strains of S.coelicolor A3(2) is described.

MATERIALS AND METHODSBacterial strains. Strains 12 and 1098 of S.

coelicolor A3(2), each carrying the chromosomalmarker pheA1, were used for the isolation of DNA; thestrains are of IF and UF fertility, respectively, 1098having been derived from 12 after ultraviolet irradia-

tion (17). Strain 1190, a UF strain bearing thechromosomal markers hisAl uraAl strAl, was used asindicator of the diffusible inhibitor produced by IFstrains. Samples of mycelium from each culture usedfor DNA isolation were tested for inhibitor produc-tion, and so for presence of the SCP1 plasmid, bystreaking on lawns of spores of strain 1190 on plates of"complete" agar medium (6). IF colonies gave rise tozones of nonsporulating and nonpigmented growth of1190, whereas UF colonies did not (16). Spore suspen-sions for use as inocula for the production of liquid-grown mycelia were prepared from cultures grown onslants (6).

Reagents. [Methyl-3H]thymidine (specific activ-ity, 24 Ci/mmol) was obtained from the Radiochemi-cal Centre, Amersham, England. Lysozyme and pro-teinase K were purchased from Merck, Darmstadt,Germany. Ethidium bromide was obtained from Cal-biochem, Los Angeles, Calif.Media for the growth of mycelium. Minimal salts

medium contained (per liter): glucose, 20 g; glycine,2.6 g; monosodium glutamate, 2.2 g; K2HPO4.3H2O,0.5 g; MgSO4.7H2O, 0.5 g; FeSO4.7H20, 0.025 g;CuSO4.5H20, 0.012 g; Mn S04 7H20, 0.016 g;ZnSO4.7H20, 0.030 g; CaCl2 .2H20, 0.05 g; andL-phenylalanine, 100 mg. The medium was adjustedwith KOH to pH 7.2. Sucrose-Casamino Acids-gly-cine medium contained 34% sucrose, 2.4% CasaminoAcids (Difco), 1% MgCl2, 0.5% glucose, and 0.5% gly-cine.

Lysis of the strains and isolation of supercoiledDNA. Two methods of growth and lysis were usedduring this investigation.

(i) A 10-ml amount of minimal salts medium,supplemented with 10 UCi of [Methyl-3H]thymidineper ml (24 Ci/mmol), was inoculated with spores andshaken at 30 C for 50 h. The mycelium was harvestedby centrifugation at 15,000 rpm for 15 min at 4 C andwashed twice with TES buffer (0.03 M tris(hydrox-ymethyl)aminomethane [Tris], 0.005 M ethylene-diaminetetraacetic acid [EDTA], and 0.05 M NaCl,pH 8.0). The mycelium was then suspended in 1ml of 0.01 M Tris-hydrochloride buffer (pH 7.3)containing 25% sucrose. To this suspension 0.3 ml of

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0.25 M EDTA, pH 8.0, and 2 mg of lysozyme wereadded. 32P-labeled supercoiled ColEl DNA was addedas an internal control for nuclease activity during thelysis procedure. After incubation of the mixture for 30min at 37 C, sodium dodecyl sulfate (SDS) to aconcentration of 0.5% and 200 mg of proteinase K perml were added, and the mixture was kept for a further30 min at 37 C. Then the concentration of SDS wasraised to 1% and the suspension was incubated foranother 15 min at 37 C. Following the method ofVapnek and Rupp (15), sodium chloride was added toa final concentration of 1 M. The suspension was keptin ice for at least 2 h and then cleared by centrifuga-tion at 16,000 rpm and 4 C for 15 min in a BeckmanJ21B centrifuge.

(ii) Alternatively, the mycelium was harvestedfrom a 10-ml culture grown for 70 h in the sucrose-Casamino Acids-glycine medium in the presence of 10UCi of [3H]thymidine per ml. The washed cells weresuspended in 1 ml of 34% sucrose in TE buffer (0.01 MTris-hydrochloride, 0.001 M EDTA, pH 8.0), and 0.2ml of 0.2 M EDTA (pH 8.0) and 0.2 ml of a lysozymesolution (25 mg/ml in 0.01 M Tris-hydrochloride, pH8.0) were added and incubated for 5 min at 30 C. Themixture was quickly cooled in ice, and 0.2 ml ofEDTA(0.25 M) and SDS (final concentration, 1%) wereadded. Sodium chloride was then added as before,and the lysate was cleared in the same way.Dye-buoyant centrifugation. A 2-ml amount of

the cleared supernatant fluid, 0.5 ml of 0.25 M EDTA(pH 8.0), 1 ml of TES (pH 8.0), 0.5 ml of ethidiumbromide (1 mg/ml), and 3.65 g of CsCl were mixed in anitrocellulose tube for the Ti5O type rotor. Dye-buoy-ant centrifugation was carried out at 2 C and 42,000rpm for 36 h using a Beckman L2-65B centrifuge.Fractions of 15 drops were collected from the bottomof the tube in small vials. An 0.02-ml amount of eachfraction was spotted on filter-paper squares andassayed for radioactivity.

Sucrose gradient centrifugation. Fractions fromcesium chloride gradients containing closed circularDNA were pooled and dialyzed against 0.1 x SSC(0.015 M NaCl, 0.0015 M sodium citrate, pH 7.0) and0.0025 M EDTA for 50 min. Samples of 0.1 to 0.3 mlwere layered on linear 5 to 20% sucrose gradients inTES buffer or on alkaline sucrose gradients whichcontained, in addition, 0.2 M NaOH and 0.7 M NaCl.After centrifugation at 20 C and 45,000 rpm in theSW50.1 rotor of the Beckman L2-65B centrifuge,10-drop fractions were collected on filter-papersquares.Counting of radioisotopes. The filter-paper

squares were dried and washed with 10% trichlora-cetic acid, twice with ethanol, and finally with ether.The dried filters were put in vials containing scintilla-tion fluid [5 g of 2.5-diphenyloxazole and 50 mg of1,4-bis-[2]-(5-phenyloxazolyl)benzene in 1 liter oftoluene] and counted in an Intertechnique liquidscintillation counter SL30.

Electron microscopy. Samples (100 Mliters) con-taining circular DNA were dialyzed against 0.15 Mammonium acetate (pH 7.2) and prepared for electronmicroscopy by the droplet method of Lang and Mitani(10). DNA of bacteriophage PM2 was used as the

internal length standard. Electron microscopy wasperformed with a Siemens I electron microscope, andphotographs were analyzed as described earlier (2).

RESULTSThe mycelium of S. coelicolor A3(2) can be

effectively lysed by lysozyme and SDS. In thefirst experiments, mycelium, grown for 50 h inminimal salts medium, was treated with lyso-zyme for 30 min at 37 C and subsequently lysedin the presence of proteinase K first with 0.5%and then with 1% SDS. When supercoiled32P-labeled ColEl DNA (molecular weight, 4.2x 10") was present in the mixture during thelysis procedure, nearly all of the ColEl DNAcould be reisolated as covalently closed mole-cules.

In later experiments the increased sensitivityto lysozyme of Streptomyces mycelium grown inthe presence of high concentrations of sucrose(7) and glycine (13) was exploited. The optimalconcentrations of Casamino Acids, sucrose, andglycine were determined, the medium finallychosen being that described in Materials andMethods. Although cultures of S. coelicolorgrow very slowly in this medium, mycelium,harvested after 70 to 80 h, could be lysed veryquickly with SDS after treatment with lyso-zyme for only 5 min at 30 C.Using a modification of the method of Vapnek

and Rupp (15), the lysate produced by either ofthe two procedures was cleared by low-speedcentrifugation. The supernatant, which con-tained 25 to 40% of the total cellular DNA, wascentrifuged to equilibrium in a cesium chloride-ethidium bromide gradient. The IF strain andthe corresponding UF strain of S. coelicolorA3(2) both contain a small amount of DNAbanding at a position corresponding to a higherbuoyant density than most of the extractedcellular DNA (Fig. 1A and 2A). The DNA ofthese denser bands was further analyzed bysucrose gradient centrifugation.Both strains contain one species of DNA

sedimenting at 42 to 43S under neutral condi-tions (Fig. 1B and 2B). Under alkaline condi-tions again only one DNA species sedimentingat 86 to 90S could be detected in each strain(Fig. 1C and 2C). Based on the data of Claytonand Vinograd (3), these sedimentation valuesindicate that this DNA represents supercoiledDNA with a molecular weight of about 20 x 106.No larger covalently closed circular DNA couldbe detected when shorter centrifugation timeswere used or when the cleared lysates wereassayed directly on neutral and alkaline sucrosegradients.The slowly sedimenting peak in Fig. 1B and C

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4. 8 12 16 20 214 28 32 36

Fraction Number

FIG. 1. (A) Cesium chloride-ethidium bromidecentrifugation of a cleared lysate of a 10-ml culture ofS. coelicolor A3(2) strain 12 (IF type) grown inminimal salts medium in the presence of 10 gCi of[3H]thymidine per ml. The lysate was prepared asdescribed. After centrifugation in a cesium chloridegradient in the presence of ethidium bromide, frac-tions of 15 drops were collected from the bottom of thetube. Samples (0.02 ml) of each fraction were spottedon filter paper disks, which were assayed for radioac-tivity. (B) Neutral sucrose gradient analysis of thesupercoiled DNA. The fractions of the denser DNAsatellite band were pooled and dialyzed. An 0.2-mlamount was layered on a neutral 5 to 20% sucrose

does not represent supercoiled DNA. The quan-tity of these small fragments of DNA varies indifferent experiments and is sometimes presentalso in preparations from UF strains. This DNAprobably represents small pieces of chromo-sonal DNA.

Electron microscopic analysis revealed thatthe isolated DNA molecules are indeed circular(Fig. 3). The molecules isolated from the IFstrain have a contour length of 10.3 + 0.3 umand those of the UF strain 9.8 + 0.2 ,um,corresponding to molecular weights of 21.4 + 0.6X 106 and 20.4 4 0.4 x 106, respectively (Table1). Although these data indicate that the circu-lar DNA from IF and UF strains differedslightly in size, further investigations would benecessary to confirm a difference.The amount of supercoiled extrachromosomal

DNA isolated was about the same whetherthe cells were lysed quickly after growth in thesucrose-Casamino Acids-glycine medium orwhether the lysis procedure was performed overa longer period of time, as was necessary whenthe cultures were grown in the minimal saltsmedium.The amount of extrachromosomal DNA rep-

resents about 1.0 to 1.5% of the total DNA inboth strains. Taking the estimate of genomesize for S. coelicolor of 5.2 x 109 daltons (14),this amount would correspond to three to fourcopies of supercoiled DNA per chromosome andthis would represent a minimal value as a loss ofsupercoiled DNA during the lysis procedurecannot be excluded. However, the estimate ofgenome size is so far based on a single report,the details of which have not yet been pub-lished.

DISCUSSIONS. coelicolor A3(2) is the only representative

of the streptomycetes whose genetics has beenextensively studied (7). These genetic investiga-tions have indicated that IF strains of S. coeli-color possess a transmissible plasmid (SCP1)which carries information for the production of

gradient and centrifuged at 45,000 rpm for 45 min at20 C in an SW50.1 rotor using a Beckman L2-65centrifuge. Fractions of 10 drops were collected fromthe bottom of the tubes on filter paper disks andassayed for radioactivity. (C) Alkaline sucrose gradi-ent analysis of the supercoiled DNA. A dialyzedsample of the satellite DNA band was layered on analkaline 5 to 20% gradient and centrifuged for 45 minunder the same conditions as described in (B). Svalues given are related to supercoiled 32P-labeledColEl DNA, which was used as an internal markerand which sediments at 23S under neutral and 48Sunder alkaline conditions. Symbols: 0, 3H-labeledDNA of S. coelicolor; x, 32P-labeled ColEl DNA.

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16000

12000

SUPERCOILED DNA FROM S. CEREVISIAE

A

8000 +

4000

800-t

600

400L

200 .

6004

400f.

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4 8 12 16 20 24 28 32 36

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4 8 I12 1'6 20 24 28 32 36

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iN 1t-} !v I ,+IO____ ~ J*+*~~~~'I

L 8 12 16 20 24 28 32 36Fraction Number

FIG: 2. (A) Cesium chloride-ethidium bromidecentrifugation of a cleared lysate of S. coelicolorA3(2)strain 1098 (UF type). (B) Neutral sucrose gradientanalysis of the supercoiled DNA of strain 1098. (C)Alkaline sucrose gradient analysis of the supercoiledDNA of strain 1098. The centrifugation conditionsand the symbols are the same as in Fig. 1.

a diffusible substance that inhibits the growthand development of UF strains. These latterstrains are interpreted as having lost the SCP1plasmid. Plasmid loss, rather than mutation ordeletion of plasmid genes without loss of theplasmid itself, is indicated by the rather fre-quent concomitant loss of a group of characters

419

including inhibitor production, resistance to theinhibitor, and certain fertility properties. More-over, the derived UF strains are readily rein-fected with SCP1 by mixed growth with an IFstrain, thereby regaining the full IF phenotype.

Covalently closed circular DNA molecules inthe supercoiled configuration characteristic forall plasmids that have been isolated fromEnterobacteriaceae, pseudomonads, strepto-cocci, and staphylococci could also be isolatedby a modified procedure from S. coelicolorA3(2). The circular DNA molecules isolatedfrom an IF and UF strain have contour lengthsof 10.3 4 0.3 and 9.8 i 0.2 Am, respectively,corresponding to molecular weights of 21.4 + 0.6x 106 and 20.4 i 0.4 x 106. However, it wasfound that both the IF strain and the isogenicUF strain carry approximately the sameamount of circular DNA of almost the samesize. The amount is equivalent to about three tofour copies per chromosome, assuming that thesize of the chromosomal DNA of S. coelicolor is5.2 x 109 daltons (14). These findings make itunlikely that the circular DNA so far identifiedrepresents the SCP1 plasmid. No other plas-mids were found in this study, although themethod used should have allowed the isolationof plasmid molecules up to the size of 100 x 106daltons (unpublished data).

Several possibilities can be considered for thefunction of the circular DNA. (i) It may repre-sent the genomes of spontaneously inducedlysogenic or defective phages. A defective lyso-genic phage, 4C31, has in fact been demon-strated in A3(2) strains by Lomovskaya et al.(11), but its DNA has not been characterized.Although we failed to detect free phages in thesupernatant of our cultures, the possibilitycannot be excluded that the DNA circles repre-sent genomes of phages that remain inside thecell. (ii) The DNA may represent plasmidsdetermining functions common to both IF andUF strains and different from the functionscoded for by the SCP1 plasmid. (iii) It couldconceivably represent SCP1 plasmids with thesame physical properties in both strains butwith altered genetic determinants in the UFstrain. Although, as mentioned above, the ge-netic data favor an interpretation of plasmidloss for the origin of the UF from the IF strain, amutational origin cannot be completely ex-cluded. In this context it will be interesting todetermine if the extrachromosomal DNA iso-lated from UF strains is indeed about 106daltons smaller than that of IF strains. (iv) Inspite of its apparent homogeneity and thesimilarity in the amount of supercoiled DNA inboth strains, the extrachromosomal DNA of the

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Cnor Mol wt No. ofDNA lentoh(urmI (mega- molecules

daltons) measured

Strain 12 (IF type) 10.3 ±0.3 21.4 ±0.6 20Strain 1098 (UF type) 9.8 ± 0.2 20.4 ± 0.4 38PM2 (Pseudomonas 3.22 ± 0.06 6.70 ± 0.13phage as internallength standard)

laq1tIV, Plunvilu III UL'VUl 11- aiiu %-J nulallao. 1 1llointerpretation is consistent with the finding thatchromosomal marker transfer occurs, at a lowfrequency, in UF x UF crosses (7). If thesupercoiled DNA molecules represent or includethe SCP1 plasmid of S. coelicolor A3(2), itsphysical size and hence its genetic information,would be considerably smaller than that of thefertility factor F of Escherichia coli (4).

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SUPERCOILED DNA FROM S. CEREVISIAE

The availability of strains of S. coelicolorA3(2), bearing derivatives of SCP1 in whichchromosomal segments have been inserted (9),and strains of a different species, Streptomyceslividans, into which the SCP1 plasmid has beentransferred from S. coelicolor A3(2), (8) shouldhelp to distinguish between these possibilities.

ACKNOWLEDGMENTSWe gratefully acknowledge the collaboration of Helen M.

Wright in the development of the growth medium for rapidlysis and of J. Siss in the electron microscope studies.

This work was supported by a grant from the DeutscheForschungsgemeinschaft.

LITERATURE CITED1. Boronin, A. M., and L. G. Sadovnikova. 1972. Elimination

by acridine dyes of oxytetracline resistance in Ac-tinomyces rimosus. Genetika 8:174-176.

2. Bujard, H. 1970. Electron microscopy of single strandedDNA. J. Mol. Biol. 49:125-137.

3. Clayton, D. A., and J. Vinograd. 1967. Circular dimer andcatenate forms of mitochondrial DNA in human leuca-emic leucocytes. Nature (London) 216:652-657.

4. Freifelder, D. R., and D. Freifelder. 1968. Studies on E.coli sex factors. II. Some physical properties of F' lacand F DNA. J. Mol Biol. 32:25-35.

5. Gregory, K. F., and J. C. C. Huang. 1964. Tyrosinaseinheritance in Streptomyces scabies. II. Induction oftyrosinase deficiency by acridine dyes. J. Bacteriol.87:1287-1294.

6. Hopwood, D. A. 1967. Genetic analysis and genomestructure in Streptomyces coelicolor. Bacteriol. Rev.31:373-403.

7. Hopwood, D. A., K. F. Chater, J. E. Dowding, and A.Vivian. 1973. Advances in Streptomyces coelicolor

genetics. Bacteriol. Rev. 37:371-405.8. Hopwood, D. A., and H. M. Wright. 1973. Transfer of a

plasmid between Streptomyces species. J. Gen. Micro-biol. 77:187-195.

9. Hopwood, D. A., and H. M. Wright. 1973. A plasmid ofStreptomyces coelicolor carrying a chromosomal locusand its interspecific transfer. J. Gen. Microbiol.79:331-342.

10. Lang, D., and M. Mitani. 1970. Simplified quantitativeelectron microscopy of biopolymers. Biopolymers9:373-379.

11. Lomovskaya, N. D., N. M. Mkrtumian, N. L. Gostim-skaya, and V. N. Danilenko. 1972. Characterization oftemperate actinophage kC31 isolated from Strep-tomyces coelicolor A3(2). J. Virol. 9:258-262.

12. Okanishi, M., T. Ohta, and H. Umezawa. 1970. Possiblecontrol of formation of aerial mycelium and antibioticproduction in Streptomyces by episomic factors. J.Antibiot. 23:45-47.

13. Okanishi, M., K. Suzuki, and H. Umezawa. 1974. Forma-tion and reversion of streptomycete protoplasts: cul-tural condition and morphological study. J. Gen.Microbiol. 80:389-400.

14. Sermonti, G., and A. M. Puglia. 1974. Progressive fertili-zation in Streptomyces coelicolor. In Proceedings, 2ndInternational Symposium on Genetics of IndustrialMicroorganisms, Sheffield. Academic Press Inc., Lon-don.

15. Vapnek, D., and W. D. Rupp. 1971. Identification of in-dividual sex factor DNA strands and their replication

during conjugation in thermosensitive DNA mutants ofEscherichia coli. J. Mol. Biol. 60:413-424.

16. Vivian, A. 1971. Genetic control of fertility in Strep-tomyces coelicolor A3(2): plasmid involvement in theinterconversion of UF and IF strains. J. Gen. Microbiol.69:353-364.

17. Vivian, A., and D. A. Hopwood. 1970. Genetic control offertility in Streptomyces coelicolor A3(2): the IF-fer-tility type. J. Gen. Microbiol. 64:101-117.

421VOL. 121, 1975

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