JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 635-6430021-9193/79/01-0635/09$02.00/0

Vol. 137, No. 1

Plasmid Copy Number Control: Isolation and Characterizationof High-Copy-Number Mutants of Plasmid pE194

BERNARD WEISBLUM,'* MADGE YANG GRAHAM,t THOMAS GRYCZAN,2 AND DAVIDDUBNAU2

Department ofPharmacology, University of Wisconsin Medical Center, Madison, Wisconsin 53706,1 andDepartment ofMicrobiology, The Public Health Research Institute of The City ofNew York, Inc., New

York, New York 100162

Received for publication 14 October 1978

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance tomacrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For fullexpression, the resistance phenotype requires a period of induction by subinhibi-tory concentrations of erythromycin. A copy number in the range of 10 to 25copies per cell is maintained during cultivation at 320C. It is possible to transferpE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid ismaintained at a copy number of approximately 10 per cell at 370C, and resistanceis inducible. Tylosin, a macrolide antibiotic which resembles erythromycin struc-turally and to which erythromycin induces resistance, lacks inducing activity.Two types of plasmid mutants were obtained and characterized after selection onmedium containing 10 tig of tylosin per ml. One mutant class appeared to expressresistance constitutively and maintained a copy number indistinguishable fromthat of the parent plasmid. The other mutant type had a 5- to 10-fold-elevatedplasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistanceinducibly. Both classes of tylosin-resistant mutants were shown to be due toalterations in the plasmid and not to modifications of the host genome.

A plasmid, pE194 (molecular mass, 2.3 ± 0.1megadaltons), was isolated from cells of anerythromycin-resistant strain of Staphylococcusaureus described originally by Iordanescu (7, 8).The erythromycin resistance phenotype is indis-tinguishable from that originally discovered byChabbert (1), and the biochemical mechanismof this type of resistance has been determined tobe specific N6-dimethylation of adenine in 23SrRNA, as a consequence of which the affinitybetween erythromycin and the ribosome ismarkedly reduced (9, 10, 19). Plasmid pE194specifies co-resistance to macrolide, lincosamide,and streptogramin type B ("MLS") antibioticsand, upon transfer to Bacillus subtilis by DNA-mediated transformation, the inducible resistantphenotype found is indistinguishable from thatofthe original S. aureus strain from which pE194was isolated. In the present studies, the conse-quences of establishing this S. aureus plasmid inB. subtilis are described with special emphasison the regulation of plasmid DNA synthesis inthe new host background.

MATERIALS AND METHODSStrains. B. subtilis BD170 (trpC2 thr-5) was usedt Present address: Department of Microbiology and Im-

munology, Washington University, School of Medicine, St.Louis, MO 63110.

as the transformable recipient. S. aureus RN2442carrying pE194 was obtained from R. P. Novick.Gel electrophoresis. Agarose (0.8%; LE grade;

Seakem Marine Colloids Inc., Portland, Maine) wasused with tris(hydroxymethyl)aminomethane (Tris)-borate buffer (5). DNA samples were fractionated byelectrophoresis in agarose slabs (16 by 16 cm, 80 V, 50mA, 18 h). Gels were stained for 30 min with ethidiumbromide (EtBr; 1 ,ug/ml), destained in water for 30 to60 min, and photographed through combined 23A and8 Kodak filters on Polaroid 667 film, using long-wave-length UV light.To prepare crude lystates for electrophoretic anal-

ysis, overnight cultures (1 ml) ofB. subtilis were grownat 320C in VY medium (4), washed in TES (50 mMNaCl, 5 mM disodium ethylenediaminetetraacetate,30 mM Tris; pH 7.5), suspended in 1 ml of TEScontaining lysozyme (0.5 mg/ml), ribonuclease A (50Ag/ml), and ribonuclease Ti (0.5 ,ug/ml), and incu-bated at 370C for 30 min. An equal volume of predi-gested Pronase (1 mg/ml) in 1.6% Sarkosyl was added,and incubation continued for 2 h. Samples weresheared by mechanical agitation on a Vortex mixer for30 s before electrophoresis. For determination of ra-dioactivity in agarose gel slices, fluorescent bands wereexcised, dissolved in 0.2 ml of 30% H202-NH40H (19:1, vol/vol), and allowed to stand for 8 h at 500C. Fourmilliliters of Liquiscint counting fluid (National Diag-nostics Inc.) was added, and the samples were vigor-ously niixed before counting by liquid scintillation.Acrylamide gel electrophoresis was performed simi-larly, using 5% acrylamide-0.25% bisacrylamide gels.

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636 WEISBLUM ET AL.

Transformation. Preparation of competent cellsfollowed by transformation, using covalently closedcircular plasmid DNA, was carried out as describedpreviously (3, 6).CsCI-EtBr centrifugation. To 6 ml of TES buffer

containing the DNA, 7.5 g of CsCl and 2 ml of EtBrsolution (1 mg/ml) were added, followed by centrifu-gation in a Ti 50 rotor at 42,000 rpm for 40 h. Thecovalently closed circular DNA was isolated and pu-rified by dye-buoyant CsCl density gradient centrifu-gation (12). Analytical density gradient centrifugationin neutral CsCl solution, followed by an optical scan ofthe photographic record, was performed as describedpreviously (17).

Restriction endonucleases. Enzymes were pur-chased from New England Biolabs or Bethesda Re-search Laboratories. The reaction conditions usedhave been described previously (6). The enzymesHaeIII, HhaI, and AluI used in some of the studieswere prepared by published procedures (22, 15, 14 and23, respectively).

Analy8i8 of methylated adenine. Analysis ofmethylated adenine in rRNA was performed by twomethods. For labeling with 14C, [methyl-14C]methio-nine was included in the growth medium. methyl-Y4C_labeled 23S RNA was isolated and then digested withTi ribonuclease, and the resultant digest was fraction-ated by two-dimensional electrophoresis (16). Thismethod permits examination of the total 23S rRNA toascertain the degree of altered methylation.

For labeling with [3H]adenine, cells were incubatedin a medium containing [8-3H]adenine, followed byisolation of 23S rRNA, depurination with acid andheat, fractionation of adenine plus methylated adenineas a group free from pyrimidines and guanine(s) bycolumn chromatography on Dowex 50, and fractiona-tion of the resultant adenine group into adenine, N6-monomethyl-adenine (m5A), and N6, N5-dimethyl ad-enine (m~2A), as described previously (10). This methodprovides a measurement of the relative number ofmethylated adenine residues in 23S rRNA.

Hybridization studie8. The concentrations ofpE194 sequences in B. subtilis and S. aureus DNApreparations were determined with a 32P-labeled com-plementary RNA probe prepared by transcription,using E. coli RNA polymerase, [a-32P]ATP, and theother three ribonucleotides in unlabeled form. Theincubation medium for preparation of the complemen-tary RNA probe contained (total volume, 0.1 ml): 40mM Tris-hydrochloride (pH 7.9); 4 mM MgCl2; 150mM KCI; 12 mM 2-mercaptoethanol; 1 mM MnCl2;MuM [32P]ATP (specific activity, 250 Ci/mmol); 0.5mMeach unlabeled GTP, CTP, and UTP; 1 Mug of pE194form I DNA, and 5 U of E. coli RNA polymerase.

After incubation for 60 min at 370C, 10 pl of 10%sodium dodecyl sulfate solution was added to thereaction mixture, and unincorporated substrates wereremoved by gel filtration over Sephadex G-50 equili-brated with water. The calculated specific activity ofthe 32P-labeled complementary RNA probe was 108cpm/,ug. The 32P-labeled complementary RNA washybridized to B. subtilis or S. aureus DNA prepara-tions affixed to nitrocellulose (Millipore Corp.) filters.The conditions used for preparation of the nitrocellu-lose filters and the hybridization reactions -were as

described by Denhardt (2). For preparation of thenitrocellulose filters, total cellular DNA (50 ,ug) wasdigested with endonuclease HaeIII to convert pE194form I DNA to open linear form. The volume of theincubation mixture was adjusted to 10 ml with water,boiled for 5 min, and chilled rapidly on ice, and thesalt concentration was adjusted to 0.9 M NaCl and 0.9M sodium citrate (6 x SSC). The mixture was ad-sorbed to a nitrocellulose filter (47 mm in diameter),dried, and baked as described previously (2). Fromeach filter, approximately 20 disks (5mm in diameter),each containing approximately 2 Mg of DNA, wereobtained with a paper punch.

RESULTS

Phenotypic expression of MLS antibioticresistance in B. subtilis. Covalently closedcircular pE194 DNA prepared from S. aureusRN2442 was used to transform sensitive B. sub-tilis BD170, followed by selection for resistanceon solid medium containing 5 ,tg oferythromycinper ml. One B. subtilis erythromycin-resistanttransformant selected for further study wastested with disks containing MLS antibiotics todetermine the extent of similarity between theerythromycin-resistant strains of S. aureus andB. subtilis. The results in Fig. 1 show the patternof resistance characteristic of induction on solidmedium described and analyzed in detail in ear-lier studies (19-21). The asymmetric inhibitionzone can be attributed to in situ induction by(initially) subinhibitory concentrations aroundthe disk which contains erythromycin.The biochemical alteration in resistant S. au-

reus cells responsible for the resistance pheno-type has been identified as a specific N6-di-methylation of adenine in 23S rRNA (9, 10).Similar results were found when 23S RNA fromB. subtilis transformants was tested. First, anexamination of the fractionated Ti ribonucleasedigest of in vivo methyl-14C-labeled 23S rRNArevealed the presence of a single componentwhich was absent from the sensitive strain (Fig.2). A similar finding for resistant cells of S.aureus was reported previously (9). To deter-mine whether adenine was dimethylated in theresistant transformant, B. subtilis cells weregrown with [8-3H]adenine, and the relativeamounts of mgA and adenine were deterninedafter purification of 23S rRNA. Results of theanalysis presented in Fig. 3 show the absence ofm2'A and m5A in the sensitive and uninduced B.subtilis strains, whereas in the induced strainapproximately 0.2% of the radioactivity presentin the total sample comigrated as a distinct peaktogether with m9A. On the basis of the foregoingdeterminations, we conclude that the geneticinfornation encoded by pE194 is expressed sim-ilarly in both the S. aureus strain from which

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CONTROL OF PLASMID REPLICATION 637

the plasmid was isolated and the new B. subtilistransformant.Physical state of pE194 in B. subtilis and

S. aureus. Both S. aureus(pE194) and B. sub-

.' :1 :-:.'.. S '*1:. i. i,

tilis(pE194) were found to contain covalentlyclosed circular DNA as judged by EtBr-CsClcentrifugation, and digestion of this purifiedplasmid fraction with several restriction endo-nucleases failed to show any differences. At thelevel of resolution afforded by these techniques,pE194 can be demonstrated in both the parentS. aureus and recipient B. subtilis strains.

B. subti I i s 170

FIG. 1. Erythromycin-inducible resistance in S.aureus and B. subtilis into whichplasmidpE194 hadbeen introduced. Induction on solid medium in situwas demonstrated with the use oferythromycin disksapposed to disks containing other MLS antibiotics.The testpanel contained (in addition to erythromycin[15 pg per disk]) clindamycin, carbomycin, tylosin,and vernamycin B alpha (20 pgper disk each) as testMLS antibiotics. The relative locations of the diskson the plate are: (top row in each plate, from left toright) carbomycin, erythromycin, and clindamycin;(bottom row in each plate, from left to right) tylosin,erythromycin, and vernamycin B alpha. The trun-cated zones of inhibition seen in b and d are due toinduction of resistance by erythromycin in situ to theMLS antibiotic contained in the respective test disks.

B.subtilis 170 (pE]94)

FIG. 2. Altered methylation of 23S rRNA fromerythromycin-resistant B. subtilis transformed withpE194. Growing B. subtilis cells were incubated with[methyl- "Cimethionine. 23S rRNA was preparedand digested with Ti ribonuclease, and the resultantdigest was fractionated by two-dimensional separa-tion on cellulose acetate and diethylaminoethyl-cel-lulose by the method of Sanger et al. (16); autora-diography was then performed.

as

D 06

al1 1 0

V-1

1 42:3 1r_

I0110 20 30 40

b

i B.subtil is

. 170(pE194)I \ uninduced

10 .20 3. 4

1 0 20 30 40

c

10 20 30 40

FRACTION NUMBER

FIG. 3. m6A in 23S rRNA from erythromycin-resistant B. subtilispE194 transformants. 23S rRNA in intactcells was labeled by growth in [3H]adenine. mSA was demonstrated and estimated quantitatively afterpurification of adenine plus methylated adenine and separation of adenine from m 9A by paper chromatog-raphy with isopropanol-concentrated ammonia-water (85:1.3:15, vol/vol/vol) as the solvent. The number ofcounts in mgA relative to those in adenine provides a measure of the number ofmgA residues per 23S rRNA.This amounts to approximately 0.2%, or two residues per 23S rRNA.

VOL. 137, 1979

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638 WEISBLUM ET AL.

Constitutively resistant mutants have beenfound useful in previous studies of inducibleerythromycin resistance (21). The rationale forthe isolation procedure for such mutants is asfollows. Whereas erythromycin can act as aninducer at subinhibitory concentrations, otherMLS antibiotics normally cannot (Fig. 1). Tosurvive, one class of such mutants must havem2A-containing 23S rRNA at the time of platingon selective medium. Among survivors of induc-ible S. aureus cells plated in this manner, suchconstitutive forms can be obtained with highefficiency.To compare the range of phenotypes obtain-

able, 20 independent mutants of S. aureus-(pE194) and 20 mutants of B. subtilis(pE194)were selected on the basis of ability to grow at370C on solid medium containing 10 ,ug of thenoninducing macrolide tylosin per ml. In bothseries, patterns of resistance similar to thosedescribed earlier for S. aureus were found (21).Examination of DNA in crude lysates extractedrevealed two mutant types. Nineteen of the B.subtilis mutants contained an intense fluores-cent band with mobility indistinguishable fromthat of the less-intense pE194 band from thewild-type strain. Similar mutants have been ob-tained by using clindamycin or carbomycin forselection (B. Weisblum, unpublished data). Theplasmid profile of the remaining B. subtilis mu-tant was indistinguishable from that of the in-ducible parent. Figure 4 shows typical agarosegel electrophoresis patterns of B. subtilis strainscarrying pE194 and one each of the two varietiesof tylosin resistance plasmids. In similar gelsprepared S. aureus(pE194) and its tylosin-resist-

-.: .. -; s.:1~~~~~-

FIG. 4. Restriction endonuclease digestion pat-terns ofpE194 DNA isolated from S. aureus (a, c, e,

g) and B. subtilis (b, d, f, h). (a and b) UndigestedDNA. Results ofdigestion withXbaI (c and d), HaeIII(e and f), and HpaII (g and h). Lane i contains a

HindIII digest of bacteriophage lambda.

ant derivatives, only a faint plasmid band wasvisible, similar to that seen in band c of Fig. 4. Afurther test was performed by digesting withrestriction endonucleases, covalently closed cir-cular pE194 DNA prepared from S. aureus andtotal DNA from B. subtilis tyl-6 (Fig. 5). Pat-terns obtained from digestion of the plasmidmaterial present stood out strongly against abackground of chromosomal DNA fluorescence,indicative of a high copy number in the B.subtilis tylosin-resistant strain used. From theseobservations, we infer that the intense band seenin the agarose gels corresponds to a high copynumber of pE194 in 19 of 20 of the tylosin-resistant mutants of B. subtilis. For furtherstudy, one typical S. aureus mutant (tyl-1) andone typical B. subtilis mutant showing an ap-parently increased copy number (tyl-6) werechosen as representative strains for detailedcomparative investigation.To determine whether the tylosin resistance

phenotype and the altered copy number weredependent on changes in the host cell or in theplasmid, covalently closed circularDNA purifiedby CsCl-EtBr centrifugation from S. aureus-(pE194 tyl-1) and from B. subtilis(pE194 tyl-6)was used to transform B. subtilis BD170 toerythromycin resistance. Of 200 erythromycin-resistant colonies derived from each transfor-mation, all were found to be tylosin resistant

no enz Hae III Hha I

a b c d e f

Alu I

g h

FIG. 5. Comparative analysis ofpE194 from S. au-reus and B. subtilis. Total cellular DNA from B.subtilis(pE194 tyl-6) (lanes b, d, f, and h) and, forcomparison, pE194prepared as covalently closed cir-cular DNA from S. aureus RN2442 (lanes a, c, e, andg) were digested with restriction endonucleasesHaeIII, HhaI, and AluI, respectively, followed byelectrophoretic fractionation of the resultant digestin 5%polyacrylamide gel. The highpE194 copy num-ber in B. subtilis(pE194 tyl-6) can be inferredfrom thefluorescence intensity of plasmid bands relative tochromosomal DNA.

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CONTROL OF PLASMID REPLICATION 639

when the tyl-1 and tyl-6 donors were used. Noneof those derived from pE194 was tylosin resist-ant. Lysates obtained from one transformantderived from each donor were examined by aga-rose gel electrophoresis. Only the transformantderived from pE194 tyl-6 showed an intensefluorescent band corresponding to the mobilityof pE194. Plasmid bands from all three trans-formants had indistinguishable mobilities. Thus,expression of tylosin resistance in both the tyl-1and tyl-6 mutants is attributable to an alterationof the plasmid rather than of the host DNA.The plasmid-mediated tylosin resistance of thestrains carrying pE194 tyl-6 is correlated withincreased copy number. The high copy numberof pE194 in the B. subtilis tyl-6 mutant couldalso be visualized directly if total DNA extractedfrom the tyl-6 strain was digested with restric-tion endonucleases HaeIII, HhaI, and AluI. Ourresults are shown in Fig. 6. The fragments ob-tained by digestion of total tyl-6 DNA containeda high multiplicity of fragments with mobilities

B.subtilis 170

B.subtilis 170 (pEl94)

170 (pE194 tyl-6 )

1.731 1.703 1.692 gm/ccM.luteus B.subtilisStandard

FIG. 6. Analysis of total DNA extracted from B.subtilis BD 170(pE194) and (pE194 tyl-6) by densitygradient centrifugation in CsCl solution. Total DNAextracted from the three named strains was spun toequilibrium in CsCl solution. Buoyant densities oftheconstituents and of the Micrococcus luteus internalreference standard are annotated directly on thefigure.

indistinguishable from those obtained by diges-tion of pE194 (form I DNA) purified from S.aureus, included for comparison. From visualinspection, we infer that pE194 is present at ahigh copy number and that no discernible rear-rangements occurred which would produce analtered restriction pattern.Estimation ofpE194 copy number. pE194,

with a buoyant density of 1.692g/cm3, is easilyresolvable from B. subtilis chromosomal DNA,which has a buoyant density of 1.703 g/cm3 inCsCl. DNA extracted, respectively, from B. sub-tilis(pE194) and B. subtilis(pE194 tyl-6) grownat 370C was banded to equilibrium by densitygradient centrifugation in CsCl solution (Fig. 6).The DNA sample from the tyl-6 mutant showeda main band at 1.703 g/cm3, which was close to1.702 reported for B. subtilis DNA (17), and adistinct satellite banding at 1.692 g/cm3, whichwas undetectable in the photometric tracings ofthe DNA sample from the plasmid-free strainand was possibly seen as a minor shoulder in B.subtilis(pE194). We infer that tyl-6 contains anelevated pE194 copy number, amounting to ap-proximately 10% of the total cellular DNA, or2.5 x 108 daltons. For a plasmid with a molecularweight of approximately 2.4 x 106, this impliesthe presence of about 100 copies of pE194 tyl-6per chromosome.The satellite buoyant density, 1.692 g/cm3,

corresponds to that of pE194 extracted directlyfrom erythromycin-resistant S. aureus, as wellas to the density reported for total bulk chro-mosomal DNA from S. aureus (17). Since pE194does not band as a distinct satellite in DNAsamples extracted from S. aureus, further quan-titative studies were performed by hybridizing32P-labeled complementary RNA probes pre-pared from form I pE194 (obtained by dye-buoy-ant CsCl density gradient centrifugation) to totalDNA from various sources. To estimate the copynumber of pE194 in the parental and the tyl-6strains, as well as in S. aureus strains carryingpE194, equal amounts of total cellular DNAfrom each source were denatured and fixed tonitrocellulose (Millipore Corp.) membrane fil-ters. The extent of hybridization was then deter-mined as a function of increasing concentrationsof pE194 complementary RNA probe in thehybridizationmixture (Fig. 7). For the B. subtilisstrains, the tyl-6 mutant contains 10 to 100 timesas much pE194 complementary sequences asdoes B. subtilis(pE194). Hybridization to DNAfrom S. aureus(pE194) and DNA from S. au-reus(pE194 tyl-1) shows that the tyl-1 copy num-ber is the same in both strains. This methodappears to underestimate consistently the levelof pE194 in B. subtilis, for reasons which are notyet clear.

b

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640 WEISBLUM ET AL.

Lysates of B. subtilis(pE194), B. subtilis-(pE194 tyl-1), and B. subtilis(pE194 tyl-6) whichhad been uniformly labeled with [methyl-3H]-thymidine at 320C were subjected to CsCl-EtBrcentrifugation. Comparison of radioactivitypresent as covalently closed circular DNA andin the main chromosomal DNA band yieldedthe following estimates of copy number: 10,pE194; 10, pE194 tyl-1; and 65, pE194 tyl-6.Similarly, [methyl-3H]thymidine-labeled lysatesfrom cells grown at 320C were sheared, and totalDNA was examined by agarose gel electropho-resis. Plasmid (covalently closed circular andopen circular) bands and chromosome bandswere excised and radioactivity was determined.Copy numbers determined in this way were: 17,pE194; 18, pE194 tyl-1; and 84, pE194 tyl-6. It is

a B.subtilis tyl-6 b S.aureus

10 tylp i

coG 7 09pE194)quencesinB.subtilisand S. aureu sensitive

102thromycin andtosensitive

10 20 50 100 200 500 10 20 50 100 200 500

pE194 3P-cRNA PROBE ADDED (THOUSAND COUNTS PER MINlUTE)

FIG. 7. Comparison of pE194 complementary se-quences in B. subtilis and S. aureus resistant toerythromycin and tylosin. Hybridization of pE194complementary RNA (cRNA) to membrane filterscarrying 2 pg of total cellular DNA purified from thestrains indicated was measured as a function ofincreasing concentration ofprobe added. The extentof hybridization to DNA from both the pE194-carry-ing strains and the tyl-1 mutants ofB. subtilis and S.aureus fall within one order of magnitude of eachother, whereas the extent of hybridization to DNAfrom tyl-6 (measured only in B. subtilis) was at leastone order ofmagnitude higher.

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apparent from these studies that the tyl-6 mu-tant is altered in copy control and that the tyl-1mutant appears indistinguishable in this regardfrom the parent plasmid.

Inducibility of erythromycin resistancein B. subtilis(pE194 tyl-6). The causal rela-tionship between high plasmid copy numberseen in B. subtilis tyl-6 and the resultant tylo-sin resistance phenotype is not apparent directlyfrom the work described above. Especially per-tinent is the question whether the informationfor rRNA methylation and the resistance phe-notype is still inducible in B. subtilis(pE194 tyl-6) despite the high copy number. An experimentdesigned to provide a definitive answer (outlinedin Table 2) was performed as follows. PlasmidDNA isolated from the two tylosin resistanceplasmids pE194 tyl-1 and pE194 tyl-6, as well asfrom the wild-type plasmid pE194, was used totransform the sensitive recipient strain of B.subtilis. All three covalently closed circular plas-mid DNA preparations had been isolated fromB. subtilis by CsCl-EtBr centrifugation. Eryth-romycin-resistant and tylosin-resistant trans-formants were selected after plating on agar withand without inducing levels (0.1 ytg/ml) of eryth-

TABLE 1. Estimation ofpE194 copy number in B.subtilis

Plasmid Total Chromo- pE194DNA" circular somal Ratio copyfrom: DNA DNA Rto num-

(cpm) (cpm) ber

pE194 361 22,700 0.016 17pE194 tyl-1 443 26,050 0.017 18pE194 tyl-6 1,752 21,700 0.081 84

a B. subtilis strains carrying each of the three plas-mids were used, and lysates were prepared and ana-lyzed as described in the text. DNA bands were stainedwith EtBr and excised from the gel. Radioactivityassociated with the various bands was counted. Tocalculate copy number, the molecular weights ofpE194 and the B. subtilis chromosome were taken as2.4 x i06 and 2.5 x 10i, respectively.

TABLE 2. Test of inducibility ofpE194 tyl-6 (schematic)aINDUCIBILITY OF B.subtilis TRANSFORMED WITH pEI94+, (tyl. ), AND (tyl-l) DNA

Plate at:

1 hr 2 hr 3 hr 4 hr

Ery Tyl5 pg/ml 5 jug/ml

a DNAase, Deoxyribonuclease; Ery, erythromycin; Tyl, tylosin.

+pEl94 DNA5 mug/ml

O min

B.subtilis5 x 108/mi

+DNAase+Ery 0.1 jug/ml

20 min

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CONTROL OF PLASMID REPLICATION 641

romycin. Plates were incubated for various timesat 370C to allow expression with or withoutinduction, followed by a challenge with the se-lecting antibiotic by overlay. Figure 8 shows theresults. Few tylosin-resistant transformantswere detected using pE194 DNA (Fig. 8) withoutinduction, whereas the number of erythromycin-resistant transformants gradually increased toabout 4 x 104/ml. The high initial level (2 x 104transformants per ml) presumably representsinduction on the plates even in the presence ofa "selecting" concentration of erythromycin.With induction before challenge, equivalentnumbers of both tylosin- and erythromycin-re-sistant transformants were obtained, and thenumbers rose rapidly to about 2 x 105/ml. WhenpE194 tyl-6 DNA was used (Fig. 8b), both eryth-romycin-resistant and tylosin-resistant trans-formants were obtained without induction. Thelatter increased 100-fold during the first 90 minof delay. If challenge is preceded by a period ofinduction, the number of both erythromycin-resistant and tylosin-resistant transformants in-creased markedly, and again a pronounced ini-tial rise in the yield of tylosin-resistant coloniesis evident. We conclude that in the pE194 tyl-6

107a) pE194 donor b) pE11

io06rEry ,induced

10 rEry .uninduced

T-yl induced

mutant resistance to both erythromycin and ty-losin is inducible. It is likely that the high copynumber of this plasmid allows sufficient "es-cape" synthesis ofrRNA methylating activity toconfer tylosin resistance without induction. Theappearance of tylosin-resistant transformantsprobably reflects this escape synthesis togetherwith the rapid replication of the plasmid to ahigh copy number.pE194 tyl-1 DNA was used in a similar exper-

iment (Fig. 8c). Prior induction produced only asmall increase in the number of tylosin- or eryth-romycin-resistant colonies. The kinetics of ap-pearance and the number of these transformantsare very similar to those seen for tylosin- anderythromycin-resistant transformants with in-duction using pE194 DNA. This pattern wouldbe expected for a low-copy-number plasmidwhich specifies constitutive resistance to MLSantibiotics. We cannot rule out the possibilitythat pE194 tyl-1 may be partially constitutive.Whatever the detailed explanation for the ki-netic behavior shown in Fig. 8, the pE194 parentplasmid and the pE194 tyl-6 mutant derivativeconfer inducible MLS antibiotic resistance,whereas the tyl-1 derivative confers constitutive

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

TIME AT WHICH ERYTHROMYCIN OR TYLOSIN W4AS ADDED FOR SELECTION (HOURS)

FIG. 8. Expression oferythromycin and tylosin resistance after transformation ofB. subtilis with (a) pE194,(b) pE194 tyl-6, and (c) pE194 tyl-1 DNA. B. subtilis BDJ 70 competent cells were incubated for 20 min at 37°Cwith 5 pg of covalently closed circular DNA per ml. Deoxyribonuclease (50 ,ug/ml) was added to terminateuptake, and the transformed cells were plated at 37°C on medium either with or without an inducingconcentration (0.1 pg) of erythromycin per milliliter. At the indicated times, theplates were overlaid with agarcontaining selecting concentrations of erythromycin or tylosin. The number of transformants per milliliter oftransformed culture is shown for erythromycin selection with and without induction and for tylosin selectionwith and without induction as indicated di;Fectly in the labels.

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642 WEISBLUM ET AL.

resistance. Thus, our data clearly indicate thatat least two distinct genetic mechanisms of ty-losin resistance can be established by mutationsof pE194. The first, exemplified by pE194 tyl-6,results in a high copy number and may resultfrom an alteration in initiation of replication.We propose to refer to these mutations as "cop"(e.g., pE194 cop-6) in accordance with a pub-lished recommendation (11). The second mech-anism, exemplified by pE194 tyl-1, results inconstitutive expression of the resistance pheno-type and may result from altered control oftranscription.

DISCUSSIONBy use of the selection methods described

above an unlimited number of mutants withaltered copy number control (cop) can be ob-tained. We expect that these mutants will beuseful in studies of the control of DNA replica-tion. Progress in understanding the complexityof transcriptional control will likewise benefitfrom examination of constitutive mutants.The relative numbers of cop and constitutive

mutants selected with tylosin in B.subtilis(pE194) are a function of the selectiontemperature (A. G. Shivakumar, J. Hahn, andD. Dubnau, unpublished data). The steady-statecopy number ofpE194 can be lowered by growthat temperatures greater than 32°C. The proba-bility of plasmid loss by segregation is a functionof both growth temperature and the copy num-ber; high-copy mutants have a lower probabilityof plasmid loss at elevated temperatures, andtheir isolation is thus favored by selection ontylosin plates at 370C compared with that at32°C. pE194 may be more stably maintained at370C under these conditions in S. aureus thanin B. subtilis. This model is supported by thefinding of tylosin-resistant B. subtilis mutantswith constitutive R-determinant gene expressionwhen selection is performed at 30°C, a temper-ature which favors stable maintenance of pE194in B. subtilis. The complementary experiment,selection of cop mutants of S. aureus by culti-vation at higher temperatures (e.g., 40 to 42°0),would provide additional support for this inter-pretation.To date, pE194 is the smallest plasmid de-

scribed which specifies inducible resistance. Shi-vakumar and Dubnau (unpublished data) havestudied expression of pE194 in the B. subtilisminicell system (13). pE194 segregates readilyinto B. subtilis minicells, where it specifies be-tween six and eight major peptides. One of theseis clearly induced by exposure of the minicellsto low concentrations (0.05 ,ug/ml) of erythro-mycin. This peptide is also expressed inducibly

J. BACTERIOL.

in minicells containing pE194 cop-6, but consti-tutively in the tyl-1 mutant.

Part of the impetus for constructing B. sub-tilis(pE194) was to determine the genetic com-plexity of the erythromycin resistance mecha-nism in terms of the relative contributions ofplasmid and chromosome to the resistance phe-notype. From the present studies we infer thatthe determinants are specified by the plasmidexclusively in the sense that chromosomal con-tributions, if any, are present in all gram-positiveorganisms including naive strains such as B.subtilis. Moreover, these studies provide ameans for development of general methods foramplification of plasmids which specify induc-ible functions, the expression of which can beselected and possibly utilized in recombinantsystems.

ACKNOWLEDGMENTSWe thank R. P. Novick for the gift of strains and for

valuable discussions, and Annabel Howard and Susan Crosbiefor expert secretarial assistance. S. B. Holder provided experttechnical assistance. D. Womble and R. H. Rownd assisted inthe analytical ultracentrifugation studies, E. Lund and J. E.Dahlberg assisted in the oligonucleotide mapping studies, andR. Burgess provided RNA polymerase for probe preparation.S. Silver made many helpful suggestions for improvement ofthe manuscript.

This work was supported by Public Health Service grantAI-10311 to D.D., from the National Institute of Allergy andInfectious Diseases, and NSF grant PCM77-19390, as well asby research grants from The Upjohn Co. and Eli Lilly and Co.to B.W.

LITERATURE CITED1. Chabbert, Y. 1956. Antagonisme in vitro entre

1'erythromycine et la spiramycine. Ann. Inst. Pasteur(Paris) 90:787-790.

2. Denhardt, D. T. 1966. A membrane-filter technique forthe detection of complementary DNA. Biochem. Bio-phys. Res. Commun. 23:641-646.

3. Dubnau, D., and R. Davidoff-Abelson. 1971. Fate oftransforming DNA following uptake by competent Ba-cillus subtilis. I. Formation and properties of the donor-recipient complex. J. Mol. Biol. 56:209-221.

4. Dubnau, D., R. Davidoff-Abelson, B. Scher, and C.Cirigliano. 1973. Fate of transforming deoxyribonu-cleic acid after uptake by competent Bacillus subtilis:phenotypic characterization of radiation-sensitive re-combination-deficient mutants. J. Bacteriol. 114:273-286.

5. Greene, P. J., M. C. Betlach, H. M. Goodman, and H.W. Boyer. 1974. The EcoRI restriction endonuclease.Methods Mol. Biol. 7:87-111.

6. Gryczan, T. J., S. Contente, and D. Dubnau. 1978.Characterization of Staphylococcus aureus plasmidsintroduced by transformation into Bacillus subtilis. J.Bacteriol. 134:318-329.

7. Iordanescu, S. 1976. Three distinct plasmids originatingin the same Staphylococcus aureus strain. Arch. Roum.Pathol. Exp. Microbiol. 35:111-118.

8. Iordanescu, S. 1977. Relationships between cotransdu-cible plasmids in Staphylococcus aureus. J. Bacteriol.129:71-75.

9. Lai, C.-J., J. E. Dahlberg, and B. Weisblum. 1973.Structure of an inducibly methylatable nucleotide se-

on February 14, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CONTROL OF PLASMID REPLICATION 643

quence in 23S ribosomal ribonucleic acid from eryth-romycin-resistant Staphylococcus aureus. Biochemis-try 12:457-460.

10. Lai, C.-J., and B. Weisblum. 1971. Altered methylationof ribosomal RNA in an erythromycin-resistant strainof Staphylococcus aureus. Proc. Natl. Acad. Sci. U.S.A.68:856-860.

11. Nordstrom, K., W. Geobel, B. C. KHlne, and R. H.Rownd. 1977. Phenotypic and genetic symbols for plas-mid copy number mutants. Plasmid 1:117.

12. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dye-buoyant density method for the detection and isolationof closed circular duplex DNA: the closed circular DNAin HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521.

13. Reeve, J. N., N. H. Mendelson, S. L. Coyne, L. L.Hallock, and R. M. Cole. 1973. Minicells of Bacillussubtilis. J. Bacteriol. 114:860-873.

14. Roberts, R. J., P. A. Myers, A. Morrison, and K.Murray. A specific endonuclease from Arthrobacterluteus. J. Mol. Biol. 102:157-165.

15. Roberts, R. J., P. A. Myers, A. Morrison, and K.Murray. 1976. A specific endonuclease from Haemo-philus haemolyticus. J. Mol. Biol. 103:199-208.

16. Sanger, F., G. G. Brownlee, and B. G. Barrell. 1965.A two-dimensional fractionation procedure for radio-active nucleotides. J. Mol. Biol. 13:373-398.

17. Szybalski, W. 1968. Use of cesium sulfate for equilibriumdensity gradient centrifugation. Methods Enzymol.12B:330-360.

18. Tanaka, T., and B. Weisblum. 1975. Systematic differ-ence in the methylation of ribosomal ribonucleic acidfrom gram-positive and gram-negative bacteria. J. Bac-teriol. 123:771-774.

19. Weisblum, B. 1975. Altered methylation of ribosomalribonucleic acid in erythromycin-resistant Staphylococ-cus aureus, p. 199-206. In D. Schlesinger (ed.), Micro-biology-1974. American Society for Microbiology,Washington, D.C.

20. Weisblum, B., and V. Demohn. 1969. Erythromycin-inducible resistance in Staphylococcus aureus: surveyof antibiotic classes involved. J. Bacteriol. 98:447-452.

21. Weisblum, B., C. Siddhikol, C. J. Lai, and V. Demohn.1971. Erythromycin-inducible resistance in Staphylo-coccus aureus: requirements for induction. J. Bacteriol.106:835-847.

22. Yang, R. C., A. Van de Voorde, and W. Fiers. 1976.Cleavage map of the simian-virus-40 genome by therestriction endonuclease III of Haemophilus aegyptius.Eur. J. Biochem. 61:101-117.

23. Yang, R. C., A. Van de Voorde, and W. Fiers. 1976.Specific cleavage and physical mapping of simian-virus-40 DNA by the restriction endonuclease of Arthrobac-ter luteus. Eur. J. Biochem. 61:119-138.

VOL. 137, 1979

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