JOURNAL OF BACTERIOLOGY, Apr. 1978, p. 318-3290021-9193/78/0134-0318$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 134, No. 1

Printed in U.S.A.

Characterization of Staphylococcus aureus PlasmidsIntroduced by Transformation into Bacillus subtilis

T. J. GRYCZAN, S. CONTENTE, AND D. DUBNAU*

Department ofMicrobiology, The Public Health Research Institute of The City ofNew York, Inc.,New York, New York 10016

Received for publication 21 November 1977

Covalently closed circular DNA from five Staphylococcus aureus plasmids hasbeen introduced into Bacillus subtilis. Four of these plasmids (pUBllO, pCM194,pSA2100, and pSA0501) have been selected for further study. These plasmidsreplicate as multicopy autonomous replicons in both Rec+ and Rec- B. subtilisstrains. They may be transduced between B. subtilis strains or transformed at a

frequency of 104 to 105 transformants per,g of DNA. The molecular weights ofthese plasmids were estimated, and restriction endonuclease cleavage site mapsare presented. Evidence is given that pSA2100, an in vivo recombinant ofpSA0501and pCM194 (S. Iordanescu, J. Bacteriol. 124:597-601, 1975), arose by a fusion ofthe latter plasmids, possibly by insertion of one element into another as a

translocatable element. Genetic information from three other S. aureus plasmids(pK545, pSH2, and pUB101) has also been introduced into B. subtilis, althoughno covalently closed circular plasmid DNA was recovered.

The development of a molecular cloning sys-tem in Bacillus subtilis is desirable from severalviewpoints. Such a system might be used toanalyze sporulation, germination, and transfor-mation and would provide an interesting com-parison with the Escherichia coli K-12 systemfor the study of gene expression. The ease ofintroduction ofDNA by means of the well-char-acterized transformation system of B. subtilis,the highly developed genetic map of this orga-nism, the extensive collection of available mu-tants, and the large body of available biochemi-cal data make B. subtilis an important candidatefor recombinant DNA studies. The nonpatho-genicity of B. subtilis and its absence from thenormal human body add to its attractiveness forsuch studies. Furthermore, the bacilli are impor-tant in the fermentation industry and in thecommercial production of antibiotics, enzymes,and insecticides. It is likely that the developmentof molecular cloning in B. subtilis will proveuseful in these areas of application.

Since recombinant DNA experiments with thebacilli have been hindered by the absence ofvectors with selectable markers, the recent re-port by Ehrlich (10) that several Staphylococcusaureus plasmids can replicate and express anti-biotic resistance in B. subtilis was of great inter-est. Aside from their use for molecular cloning,these plasmids are also potentially useful forstudies of plasmid biology, DNA uptake by com-petent cells, and genetic recombination in B.subtilis. For these reasons we undertook a study

of the molecular biology of the S. aureus plas-mids in B. subtilis.This paper describes the introduction by

transformation of a variety of S. aureus plasmidsinto B. subtilis and presents restriction endo-nuclease cleavage site maps of these plasmids.This information lays the basis for a variety ofstudies, including recombinant DNA experi-ments.

(This work was done in partial fulfillment ofthe requirements for the doctoral degree forT.J.G. and S.C. in the Department of Microbi-ology at the New York University School ofMedicine.)

MATERIALS AND METHODSBacterial strains and plasmids. The bacterial

strains used in this study and the plasmids containedtherein are listed in Table 1. All S. aureus strains wereobtained from R. P. Novick.

Isolation of plasmid DNA. Cultures of plasmid-positive cells of S. aureus grown on selective plates(GL agar with drug) (23) were used for the isolation ofplasmid DNA. Cleared lysates were prepared by amodification of the Clewell-Helinski technique (7) forS. aureus (22), using lysostaphin (Schwarz/Mann, Or-angeburg, N.Y.) followed by detergent (1.2% Brij, 0.5%sodium deoxycholate, 0.2 M ethylenediaminetetraace-tic acid, pH 8.4) lysis of protoplasts.

Plasmid DNA from B. subtilis was isolated by thesodium dodecyl sulfate-NaCI method (14). A sample(150 to 250 ml) of an overnight culture in VY (25 g ofDifco veal infusion, 5 g of Difco yeast extract, and1,000 ml of water) with selective antibiotic was har-vested by centrifugation, washed, and suspended in

318

VOL. 134, 1978 S. AUREUS PLASMID DNA IN B. SUBTILIS

TABLE 1. Bacterial strains andplasmids

Chromosomal P lasmidStrain Plasmid, phenotype marker(s) m.w.xlO6a References

RN2438 pUB112, Cmr 2.7 6, 18RN2425 pCM194, Cmr 2.0 17RN2443 pSAOS01, Smr 2.8 17RN2397 pUBllO, Kmr 3.0 18RN1956 pKS45, Kmr 14-15 29RN2430 pSH2, Kmr 9 32RN2444 pSA2100, Cmr Smr 4.7 17RN2399 pUB101, Penr Cadr Fusr 15 5BDSS trpC2 hisB2BD170 trpC2 thr-5BD224 trpC2 thr-5 recE4BD392 trpC2 thr-5 cysAl4

a Molecular weights are values from the references given, except for pUB110, pCM194, pSA0501, andpSA2100. The size determinations of these plasmids are presented in this report.

0.1 volume of 25% sucrose-0.1 M NaCl-0.05 Mtris(hydroxymethyl)aminomethane, pH 7.5. Lysozyme(Worthington Biochemicals Corp., Freehold, N.J.) (0.5mg/ml) was added, and the mixture was incubated at37°C for 15 min. To 20 ml of suspension, 4.8 ml of 5 MNaCl, 1.2 ml of 0.5 M ethylenediaminetetraacetic acid(pH 8.5), and 26 ml of 2% sodium dodecyl sulfate-0.7M NaCl were added, in that order, and the suspensionwas inverted once (gently) and placed on ice for 18 hat 4°C. The lysate was centrifuged at 15,000 x g for 45min, and the supernatant was collected and made 0.3M in sodium acetate. Two volumes of 95% ethanolwere added, and the mixture was incubated at -20°Cfor at least 2 h. The precipitate was collected bycentrifugation at 5,000 x g for 30 min, dissolved in5 ml of TES [30 mM tris(hydroxymethyl)-aminomethane, pH 7.5, 50 mM NaCl, 5 mM ethylene-diaminetetraacetic acid], and digested with pancreaticribonuclease (50 jug/ml) and Ti ribonuclease (1 U/nd)for 30 min at 37°C, followed by incubation with pre-

digested Pronase (500,ug/ml) until nearly clear. Puri-fication of covalently closed circular (CCC) plasmidDNA was performed by cesium chloride-ethidium bro-mide dye buoyant-density centrifugation (26). CCCDNA (lower band) was dripped from the gradients,extracted three times with equal volumes of isopro-panol to remove the ethidium bromide, dialyzed for 3to 5 h against 3 to 4 liters (three changes) of buffer [10

mM tris(hydroxymethyl)aminomethane, pH 7.5, 4mM NaCl, 0.1 mM ethylenediaminetetraacetic acid],and stored at 4°C until use. DNA concentrations were

determined by using a Gilford UV spectrophotometer,and the purity and form of isolated plasmid DNA wereexamined by electrophoretic analysis on agarose. Gen-erally, 100 to 150 jig of purified pUBllO, pSA0501,pSA2100, or pCM194 CCC DNA was obtained from100 ml of culture.Plasmid transformation and transduction and

preparation of bacterial DNA and competentcells. Transformation was carried out using frozencompetent cells prepared as described (8). IncubationofDNA and cells was for 30 min at 37°C using 5 ug ofDNA per ml. The transformation medium used was as

described (8) except that 1 mM ethyleneglycol-bis(fi-aminoethyl ether)-N,N'-tetraacetic acid was added(Contente and Dubnau, manuscript in preparation).

Phage AR9 (2) transducing lysates were prepared as

described (19). Selection for antibiotic resistance was

carried out by the overlay method in tryptose bloodagar base agar after a 1.5-h delay at 37°C to allow forexpression. Selective concentrations of antibiotic were:

chloramphenicol (Cm), 5 yg/ml; kanamycin (Km), 5,ug/ml; streptomycin (Sm), 35,Ig/ml; and fusidic acid(Fus), 5 ,ug/ml. Chromosomal DNA was isolated as

described previously (8).Restriction endonuclease. Reaction mixtures

contained 0.2 to 1.0 ILg of plasmid DNA in a finalreaction volume of 20 to 50 1L. Digestions were run for30 to 120 min at 37°C with 0.5 to 2 U of enzyme perreaction mixture. The composition of the reactionmixtures is shown in Table 2. Reactions were termi-nated by heating at 65°C for 10 min. Before sampleswere loaded on agarose gels, 5 ,lI of tracking dye (60%sucrose [wt/vol], 0.1 M ethylenediaminetetraaceticacid, 0.15% bromophenol blue) was added to eachsample. In many cases, double digestions were carriedout simultaneously in the same buffer. Following is a

list of those double digestions that were carried out inEcoRI buffer: EcoRI-HindIII, EcoRI-XbaI, EcoRI-HaeIII, XbaI-HaeIII, EcoRI-HindII. Following is a

list of those digestions carried out in HindIII buffer:HindIII-XbaI, HindIII-HaeIII, HindIII-HindII.HaeIII-HindII codigestions were carried out in HindIIbuffer, and XbaI-HindII codigestions were in XbaIbuffer. In some cases, digestions were carried outsequentially with two enzymes. After incubation withthe first enzyme, the sample was heated for 5 min at65°C, and buffer components were added to bring thecomposition of the buffer as close as possible to thatdesired for the second enzyme. Following is a list ofenzyme pairs used in this sequential fashion, with theenzymes in each pair presented in the order used:HpaII-HaeIII, HpaII-HindIII, HpaII-BamHI, HpaII-HindII, HpaII-XbaI, BamHI-XbaI, BamHI-EcoRI,BglII-BamHI, BglII-EcoRI. All restriction endonucle-ases were obtained from New England BioLabs, Bev-erly, Mass., with the exception of HindII, which was

purchased from Miles Laboratories, Inc., Elkhart, Ind.Agarose gel electrophoresis. Plasmid DNA and

plasmid DNA fragments were resolved on vertical 0.8to 1.5% agarose gels (30, 33). Agarose (LE) was pur-chased from Seakem Marine Colloid Corp., Portland,

319

320 GRYCZAN, CONTENTE, AND DUBNAU

TABLE 2. Restriction endonuclease reaction mixturesConstituents (final concentrations)

2-Mercapto- Bovine serumEnzyme Tris NaCl KC1 MgCl2 ethanol albumin

(mM, pH) (mM) (mM) (mM) (mM) (iig/ml)

5677

20 106 10

66666.666

66la

666lala6666666

Ref.

100 28100 35200 25100 12

313

100 24100 11100 30100 27100 4,31

15

27100 20100 27,36100 27

a Dithiothreitol was used in place of 2-mercaptoethanol.

Me. The Tris-borate buffer of Greene et al. (12) was

used throughout. Gels were usually run at a constantvoltage of 50 V (4.1 V/cm) at room temperature untilthe tracking dye just ran off the gel (3 to 4 h). In someexperiments, better fragment resolution was obtainedby running the gels at 15 V for 18 h. Gels were stainedin ethidium bromide (1 ,ug/ml) for 30 min at room

temperature and destained in water for 30 to 60 min.Fluorescence was visualized with a long-wave UV lightTransilluminator (C-5, Ultraviolet Products Inc., SanGabriel, Calif.) and photographed on Polaroid 665 filmthrough combined Kodak gel filters 23A and 8.

Estimation of plasmid DNA fragment molecularweights was performed using X-HindIII fragments as

standards (1, 21). The published values for these frag-ments in megadaltons were averaged (14.4, 6.13, 4.09,2.82, 1.5, 1.32, 0.367) and used to determine unknownfragment sizes graphically, assuming a logarithmic re-

lationship between molecular weight and electropho-retic mobility. In some experiments, A-EcoRI frag-ments were employed as standards (16, 34).

RESULTS

Transformation of S. aureus plasmidsinto B. subtilis. Table 3 shows the transfor-mation of various S. aureus plasmids into B.subtilis. Plasmid transformants were grown inVY plus the selective drug, lysed, and analyzedfor the presence of CCC DNA on cesium chlo-ride-ethidium bromide gradients. In the case offive plasmids (pUBllO, pUB112, pCM194,pSA0501, and pSA2100), bands denser thanchromosomal DNA were observed on these gra-dients, indicating the presence of CCC DNA. Inthe case of three other plasmids (pUB101,pK545, and pSH2), no plasmid band was ob-served, although transformation of B. subtilisfor the Fusr and Kmr characters of these plas-mids was observed repeatedly. This may be anindication of low plasmid copy number in the B.

subtilis host, plasmid integration into the hostchromosome, or loss during isolation.When plasmid DNA isolated from the B. sub-

tilis transformants was used to transform B.subtilis recipients, higher transformation fre-quencies were obtained than with plasmid DNAisolated from S. aureus, except in the case ofpUB112 (Table 3). These results may indicatethat a restriction-modification system is opera-tive under these conditions, as suggested byEhrlich based on similar findings (10).

In the case of pSA2100, pCM194, pSA0501,pUB110, and pUB112, plasmid transformationoccurred readily into strain BD224, a recombi-nation-deficient recipient, at about the same fre-quency as into BD170, a Rec+ strain that isisogenic with BD224 (Table 4). Chromosomaltransformation into BD224 was not detectable(Table 4) (9). CCC DNA was successfully re-covered from the rec strain (data not shown).pUB10, pCM194, and pSA2100 DNA isolated

from B. subtilis transformants showed charac-teristic frequencies of transformation into B.subtilis competent recipients. Comparisons weremade at 5 ,ig of plasmid DNA per ml, which isin the linear range of response to DNA concen-tration (Contente and Dubnau, unpublisheddata). The average number of transformants permilliliter of BD170 competent culture was 7.49x 105 (pSA2100), 4.36 x 104 (pUB110), and 3.79X 104 (pCM194). These values represent theaverage of five separate determinations each.

Figure 1 shows agarose gel patterns ofpUB110, pUB112, pCM194, pSA0501, andpSA2100 plasmid DNA isolated by cesium chlo-ride-ethidium bromide centrifugation from S.aureus and from B. subtilis. In each case, elec-trophoretic migration was identical for the plas-

66

106

AluIBanHIBglI IEcoRIHaeIIIHindI IHindI I IHpaIHpaIIKpnIPatISalISetISmaIXbaIXhoI

6, 7.6 506, 7.4 50

10, 7.4100, 7.5 50

6, 7.4 610, 7.9 607, 7.4 60

10, 7.410, 7.46, 7.5 506, 7.4 506, 7.9 10014, 7.5 906, 7.4 66, 7.9 1506, 7.4 150

J. BACTERIOL.

S. AUREUS PLASMID DNA IN B. SUBTILIS

mid DNA recovered from either host. Theseplasmid DNA preparations were screened forthe presence of cleavage sites for a variety ofrestriction endonucleases. Typical results maybe seen in Fig. 1 and are summarized in Table 5.

The single HindIII site observed on pCM194confirms the report of Ehrlich (10). In all cases.no difference was seen in the number of BamHI,EcoRI, XbaI, and HindIII sites when plasmidDNA was isolated from S. aureus or B. subtilis,

TABLE 3. Transformation ofB. subtilis byplasmid DNA

Plasmid SourceSelected Trans-

Recipient markera formants/ml

pUBilO S. aureusB. subtilis

pSA2100 S. aureusB. subtilis

pCM194 S. aureusB. subtilis

pUB112 S. aureusB. subtilis

pSAOS01 S. aureuspUB101 S. aureuspK545 S. aureus

pSH2 S. aureus

BD392BD392

BD170BD170

BD392BD392

BD392BD392

BD170

BD170

BD170

BD170

Km 4 x 1032 X 104

Cm 1.2 x 1045.8 x 105

Cm 1.4 x 1039.3 x 103

Cm 8.7 x 1027.4 x 102

Sm 2 X 104Fus 1.5 X 102Km 5 X 102Km 5 x 102

a Number of resistant clones per milliliter obtained from competent cells incubated in the absence of plasmidDNA was: Km, <10; Cm, <10; Sm, 60; Fus, 20.

TABLE 4. Plasmid transformation into a recE4 strain

Selected Transfornants/mlaDNA marker BD170 (Rec') BD224 (recE4)

pUBllO Kmr 1.81 x 105 9.8 x 104pSA2100 Cmr 1.26 x 106 2.94 x 105pCM194 Cmr 2.54 x 104 1.2 . 104B. subtilis Smr Thr' 3.2 x 106 <10

a Transformations were carried out using 5 ILg of DNA isolated from B. subtilis per ml. Control experimentsusing [3H]DNA to measure uptake showed that the recE4 cells were about half as competent as the Rec+ (datanot shown).

A B C D

FIG. 1. Comparison ofplasmid DNA isolated from B. subtilis and S. aureus on 0.8% agarose gels. Fromleft to right the lanes show: (A) pUB110 (S. aureus), pUB110 (B. subtilis), pUB110 (S. aureus) EcoRI digested,pUB110 (B. subtilis) EcoRI digested, pUB110 (S. aureus) BamHI digested, pUB110 (B. subtilis) BamHIdigested, pUB10 (S. aureus) XbaI digested, pUBZ10 (B. subtilis) XbaI digested, pUB112 (S. aureus), pUBJ12(B. subtilis); (B) pCM194 (S. aureus), pCM194 (B. subtilis), pCM194 (S. aureus) HindIII digested, pCM194 (B.subtilis) HindIII digested; (C) pSAO501 (S. aureus), pSA0501 (B. subtilis); (D) pSA2100 (S. aureus), pSA2100(B. subtilis).

321VOL. 134, 1978

322 GRYCZAN, CONTENTE, AND DUBNAU

TABLE 5. Restriction endonuclease cleavage sitesEnzyme (number of cuts)

Plasmid AiuI BacmiIa BgZII EcoRIa HaeIII HindII HindIIIa HpaI HpaII KpnI PstI Sall SmaI SstI XTaI* XhoaI

pUBl10 5 1 1 1 4 2 0 0 4 0 0 0 0 0 1 0pSA0501 6 0 0 1 0 1 1 0 2 0 0 0 0 0 1 0pSA2100 12 0 0 1 1 1 2 0 3 0 0 0 0 0 1 0pCM194 6 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0

a These enzymes were tested on plasmids isolated from S. aureus and from B. subtilis. In all cases the numberof sites and the fragment size distributions were identical. The remaining enzymes were tested only on plasmidDNA isolated from B. subtilis.

TABLE 6. Transduction of S. aureus plasmidsbetween B. subtilis straines

Transductants/mlPlasmid Plasmid marker His+

pCM194 2.68 x 103, Cmr 4 x 102pSA2100 5.9 x 102, Cmr 6.7 x 102pUBllO 8.8 x 103, Kmr 5 x 102

a Bacteriophage AR9 was grown on BD170(pCM194), BD170 (pSA2100), and BD170 (pUBllO)and used to transduce BD55 trpC2 hisB2. Selectionwas for His+ or for drug resistance.

nor were the resulting fragment sizes different.Thus, these plasmids can replicate in B. subtilisand, to a first approximation, neither lose norgain genetic material.Transduction of plasmids in B. subtili8.

The generalized transducing phage AR9 is ca-pable of transducing pCM194, pSA2100, andpUBllO between B. subtilis strains (Table 6).pSA0501 was not tested. The frequency of trans-duction ofpCM194 and pUBllO was higher thanthat of the hisB2 chromosomal marker. Thisdifference in transduction efficiency has beenobserved with several auxotrophic markers, re-cipient strains, and transducing lysates. Thetransductants selected for the plasmid markershave been shown to contain plasmids that areelectrophoretically indistinguishable from theplasmids harbored by the donor strains.

Stability of pUBlIO and pSA2100 char-acters in B. subtili To estimate the stabilityof pUBllO and pSA2100 in B. subtilis, plasmid-bearing strains were grown overnight in VYbroth, plated on TBAB, grown overnight again,and replicated to TBAB containing antibiotic(Km, Cm, or Sm). Of more than 2,000 coloniesof BD170 (pUBllO) replicated to Km, no sensi-tive colonies were detected.With BD170(pSA2100), however, a 22 to 84%

loss of both plasmid characters was observed inseveral experiments. The Cmr character was lostmore frequently than Smr in one experiment:88%, compared to 77%. When growth occurredin the presence of either Cm (5 ,ug/ml) or Sm(15 ,ug/ml), no loss of either resistance characterwas detected.

Restriction endonuclease cleavage sitemapping of pUBllO. Table 7 presents thefragment sizes obtained by digestion of pUBilODNA (isolated from B. subtilis) with EcoRI,BamHI, BglII, XbaI, HpaII, Hindll, HaeIII,and AluI. The fragment sizes were estimated bygraphical interpolation using data obtained fromstandard fragments subjected to electrophoresison the same gels as the unknown fragments (seeMaterials and Methods). The average value fortotal pUBllO molecular weight obtained fromthe four single-site restriction enzymes, based on37 determinations, was 3.00 ± 0.03 x 106. Thevalues from the other restriction endonucleasedigestions listed in Table 7 were not averaged,since the graphical estimation of their fragmentmolecular weights involved extrapolation. Thesum of fragment sizes from these other diges-tions however, is close to 3.0 Mdal, suggestingthat, at least for HpaII, HindIl, and HaeIII, nofragments were lost. Double digestion experi-ments were performed to construct a map ofsome of these restriction sites (Table 8). In allcases, the sum of the fragment sizes from a givendigestion was close to 3 Mdal, indicating that allmajor fragments were detected and each frag-ment was represented once, with the exceptionof XbaI-HpaII fragment C, which is clearly a"double."The double digestion experiments with the

four single-site enzymes permitted an unambig-uous ordering ofthese sites. The BamHI, EcoRI,and XbaI sites are within 0.5 Mdal of one an-other. The relative fragment sizes suggest thatthe XbaI site lies between the EcoRI andBamHI sites. The BglII site is separated fromthese three, and the sizes of the Bglll-EcoRIand the BglJI-BamHI fragments place this siteon the map between BamHI and EcoRI.The HpaII double digestion experiments show

that the BamHI, XbaI, and EcoRI sites arelocated in HpaII fragment A. This is consistentwith their close proximity to one another. TheBglII site is in HpaII fragment B, since, in a gelcontaining BglII-HpaII digestion fragments to-gether with a channel containing HpaII frag-ments, A and C were intact, B was no longerpresent, and a new fragment with a molecularweight of 0.35 x 101 appeared. HpaII fragment

J. BACTERIOL.

S. AUREUS PLASMID DNA IN B. SUBTILIS

C can occur in two positions relative to frag-ments A and B. The correct position is inferredfrom Table 9, based on the fit between predictedand observed BglII-BamHI and BglII-EcoRIdigestion fragment sizes. The clockwise-counter-clockwise orientation ofHpaII fragment B is lesscertain. However, the BglII-EcoRI and BglII-BamHI digestion products were more consistentwith Fig. 2 than with the altemative orientationof HpaII fragment B. For instance, the alterna-tive orientation would predict BglII-EcoRIdigestion fragments of molecular weights of 1.25x 101 and 1.62 x 10O rather than the observed1.40 x 106 and 1.55 x 10' fragments. Similarly,the BglH-BamHI fragments would be 1.75 and1.12 Mdal rather than 2.00 and 1.00 Mdal. Thepredicted values, based on the preferred orienta-tion shown in Fig. 2, are 1.45 and 1.45 Mdal forBglII-EcoRI and 1.95 and 0.92 Mdal for BglII-

323

BamHI, in good agreement with the observedvalues in Table 8 (1.55, 1.40 and 2.00, 1.00). TheHpaII-D fragment was not mapped in this study.Detection of this fragment required the use of1.5% agarose gels in short runs.The XbaI, BamHI, and EcoRI sites are lo-

cated in HindII fragment A. Cleavage of thisfragment with each of these enzymes yieldedtwo new fragments. The sizes of these fragmentsplace the two HindII sites on either side of andnear the BglII site. BglII-HindII digestions con-firmed that HindII fragment B contains thesingle BglII site. Electrophoresis of the Hindll-HpaII digestion products showed that HpaIIfragment B contains a HindII site, while HpaIIfragments A and C were recovered apparentlyintact. The second HindII site must be locatednear one of the HpaII sites, since only four ofthe five fragments expected on a 0.8% agarose

TABLE 7. Fragments produced by restriction endonuclease digestion ofpUBI0

Number of Sum ofdetermi- Fragment size (Mdal) fragment

Enzyme nations A B C D sizes(Mdal)

Bami-I 8 3.05 3.05BglII 4 3.04 3.04EcoRI 18 2.97 2.97XbaI 7 3.02 3.02HpaII 10 1.99 0.49 0.45 0.10 3.03HindII 1 2.63 0.28 2.91HaeIII 4 2.39 0.32 0.22 0.09 3.02AZuI 2 1.34 0.60 0.3 a 0.2 2.44

a This fragment is overabundant and is inferred to be a "double."

TABLE 8. Fragments produced by digestion ofpUBO1 with two restriction endonucleasesNumber of Sum ofdetermi- Fragment size (Mdal) fragment

Enzyme pair nations A B C D E sizes(Mdal)

BamHI-EcoRI 3 2.45 0.52 2.97Xbal-EcoRI 2 2.70 0.30 3.00BamHI-XbaI 1 2.80 0.20 3.00BglII-EcoRI 2 1.55 1.40 2.95BgZII-BaHII 2 2.00 1.00 3.00EcoRI-HpaII 2 1.10 0.71 0.50 0.45 ( )a 2.76BamHII-HpaII 2 1.73 0.S0 0.45 0.32 a)a 3.00BgtII-HpaII 3 2.07 0.47 (0.20)bc 0.11 3.15XbaI-HpaII 2 1.42 0.50 (0.44)b )a 2.80HindII-HpaII 1 1.92 0.41 0.36 0.35 )a 3.04HaeIII-HpaIId 2 1.69 0.40 0.30 0.26 ( )a 2.65HaeIII-HindII 1 2.34 0.36 ( )a 2.70HaeIII-EooRI 2 1.35 0.88 0.40 0.31 ( )a 2.94HaeIII-BanmHI 2 1.40 0.76 0.40 0.29 ( )a 2.85HaeIII-XbaI 2 1.20 0.98 0.40 0.29 ( )a 2.87BglII-HaeIII 3 2.43 0.19 0.14 0.09 ( )a 2.85HindII-EcoRI 1 1.5S 1.17 0.25 2.97HindII-Ba'r&I 1 1.70 0.94 0.25 2.89HindII-XbaI 1 1.50 1.22 0.25 2.97BgtII-HindII 1 2.85 0.25 ( )a 3.10

a Indicates that fragments were not detected, but were predicted from the number of single digestionfragments (Table 6).

I This fragment was of high intensity and appeared to be a "double."c This value is based on a single determination in 1.5% agarose. The migration of the corresponding band on

0.8% agarose was not measured, since it had entered the holding gel.d These two determinations were on 0.8% agarose. On 1.5% agarose, a HaeIII-HpaII digest revealed six of the

eight expected bands: 1.73, 0.40, 0.30, 0.15, 0.11, and 0.09 Mdal.

VOL. 134, 1978

324 GRYCZAN, CONTENTE, AND DUBNAU

TABLE 9. Position ofpUBIIO-HpaII fragment C

Observed Expected fragmentsaEnzyme pair fragments Orientation iD Orientation 2D

BgZII-BamHI 2.00 2.00 2.451.00 0.95 0.50

BgZII-EcoRI 1.55 1.50 1.951.40 1.45 1.00

a These are inferred by summing the shortest intervening fragments obtained from the HpaII, BglII, BamHI,EcoRI, and XbaI double digestion data of Table 7.

b In orientation 1, HpaII fragment C is adjacent to the end of HpaII fragment A, that is nearest the BamHIsite. In orientation 2, it is adjacent to the end of fragment A that is nearest the EcoRI site, as shown:

EcoRI BanHI

I I

HpaI II I

A HpaIII

c HpaII

BgV[ I

-aHpaI I

Orientation 1

HpaII c HpaII

EcoRI BarnHI

I I I

A HpaIIOrientation 2

gel were detected in the HpaII-HindII digests.The sixth fragment (HpaII-D) would not bedetected on this gel. The fragment sizes obtainedfrom the XbaI, EcoRI, BamHI, and BgIII dou-ble digestions with HindII determined that thissecond HindII site is very near the HpaII sitethat joins HpaII fragments A and B, but may beon either side of this site.Three of the four HaeIII sites were positioned

in a similar manner. EcoRI, XbaI, and BamHIcleavages occur in HaeIII fragment A, resultingin the appearance in each case of two new frag-ments. The sizes of these fragments, togetherwith the known order of the EcoRI, XbaI, andBamHI sites, position the two HaeIII sites thatdefine fragment A, suggesting that one is closeto the HpaII site that joins HpaII fragments Band C, and the other is in HpaII fragment A.The BglII site is located in HaeIII fragment B.Inspection of the gels from the HaeIII-HpaIIdouble digestions revealed that HpaII fragmentsA and B each contained a HaeIII site, andfragments C and D appeared unaffected byHaeIII. Only six of the expected eight HpaII-HaeIII fragments were detected. HaeIII frag-ment A contained at least one HpaII site. Thisis consistent with the conclusions drawn above.The location of HaeIII fragment D was notdetermined. Finally, the HindII-HaeIII doubledigestion showed that Hindll fragment A con-tains a HaeIII site, while HaeIII fragment B

BglI I

I I

B HpaI I

contains a HindII site. The pUB110 mappinginformation is summarized in Fig. 2. Twelverestriction endonuclease sites were located. Theprecise position of two of the HaeIII sites wasnot determinable from our data. Another HaeIIIsite and a HpaII site have not been mapped atall.

Restriction endonuclease cleavage sitemapping of pSA2100. Table 10 presents acatalog of fragments obtained from the digestionof pSA2100 DNA with EcoRI, XbaL HindIll,HpaII, HindII, and HaeIII. The aver-age of total fragment sizes obtained from theenzyme digestions shown in Table 10 is 4.69 +0.21 Mdal. Double digestion experiments arepresented in Table 11. The double digestionswith single-site enzymes allowed the unambigu-ous positioning of these sites relative to oneanother, with the results shown in Fig. 3. Thetwo HindIII sites could also be placed based ondouble digestion data. For instance, the XbaIand EcoRI sites are in HindIII fragment B,while the Hindll and HaeIII sites are in HindIIIfragment A. This locates one HindIII site be-tween the HindII and EcoRI sites and the otherbetween the XbaI and HaeIII sites. The threeHpaII sites could also be located by doubledigestion experiments. The HindII and HaeIIIsites are in HpaII fragment A. The XbaI siteand one HindIII site are in HpaII fragment B,and the EcoRI and the other HindIII site are in

J. BACTERIOL.

II

S. AUREUS PLASMID DNA IN B. SUBTILIS

BamHI

EcoRIT

HaemHpa I

_-~kMndIrHae HndFIG. 2. Restriction endonuclease cleavage site map ofpUB110. The order of the HindII and HaeIII sites

shown in parentheses is uncertain, as is their precise position on the map. The exact position of anotherHaeIII site is also not determined, as indicated. In addition, one other HaeIII and another HpaII site havenot been mapped. The HpaII fragment B, which contains the BglII site, may exist in the reverse orientation.The relative distances shown are from average fragment sizes determined on agarose gels.

TABLE 10. Fragments produced by restrictionendonuclease digestion ofpSA2100

Sum ofNumber of fragmentdetermi- Fragment size (Mdal) sizes

Enzyme nations A B C (Mdal)

EcoRI 3 4.40 4.40XbaI 2 4.75 4.75HindII 4 4.75 4.75HaeIII 1 4.50 4.50HindIII 2 3.10 1.63 4.73HpaII 5 2.13 1.47 1.22 4.82

HpaII-C. These data, together with the averagefragment sizes measured from gels, determinethe map shown in Fig. 3.Restriction endonuclease cleavage site

mapping ofpCM194. The restriction fragmentcatalog of this plasmid is given in Table 12. Theaverage molecular weight of this plasmid, basedon fragment sizes produced by the singlesite enzymes, was 2.00 + 0.10 x 106. The threesingle sites were easily mapped from the doubledigestion experiments, with the results shown inFig. 4A.Restriction endonuclease cleavage site

mapping of pSA0501. Table 13 presents therestriction fragment catalog for pSA0501. Theaverage molecular weight of this plasmid, basedon the fragment sizes generated by the five singleenzyme digestions shown in Table 13, was 2.80+ 0.20 x 106. The four single cleavage pointscould be unambiguously ordered based on thedouble digestion experiments. Double digestionsalso revealed that the EcoRI and HindIII sitesare in Hpa II-B and the XbaI and HindII sitesare in HpaII-A. This places one HpaII site be-tween XbaI and EcoRI and the other betweenHindII and HindIII. The resulting map is shownin Fig. 4B.

DISCUSSIONSeveral S. aureus plasmids which confer re-

sistance to antibiotics are readily transferredinto B. subtilis strains by transformation. Reiso-lation of plasmid DNA and analysis on agarosegels and by restriction endonuclease cleavagedemonstrates that no gross alteration of theseplasmids occurs during uptake and replicationin B. subtilis. The ease of transformation into arecE4 strain demonstrates that no Rec-mediated

325VOL. 134, 1978

326 GRYCZAN, CONTENTE, AND DUBNAU

TABLE 11. Fragments produced by digestion ofpSA2100 with two restriction endonucleasesa

Number ofdetermi-

Enzyme pair nations

EcoRI -XbaIEcoRI-HindI IIHindI I I-XbaIEcoRI-HpaI IXbaI-HpaI IHind II-HpaIIEcoRI-HaeIIIXbaI-HaelIIHindIII-HaeIIIEcoRI-HindI IXbaI-HindI IHin dI I I -Hin dl IHpaII-HindIIHaeII I-HindIIHpaII-HaeIII

Sum offragment

Fragment size (Mdal) sizesA B C D E (Mdal)

2 3.652 3.012 3.283 2.183 2.103 2.062 3.002 2.782 1. 6602 3.802 2.641 2.234 1.532 4.201 1.42

1.081.231.151.461.371.061.682.071.531.042.201.571. 27C0.561.32

( )b0.541.041.220.95

0.300.220.55 0.19

0.900.91

1.23 0.66

4.73

4.974.984.914.814.684.854.854.844.844.704.984.764.63

a The first three double digests were analyzed on 0.8% agarose gels and the rest on 1.4%.b This fragment was not detected but was predicted from the number of single digestion fragments (Table

10).c This fragment is overabundant and is inferred to be a "double."

EcoRIHindIHa

HpalFIG. 3. Restriction endonuclease ckeavage site map of pSA2100. The relative distances shown are from

average fragment sizes determined on agarose gels. The darker segments identify thejunction regions betweenthe pSA0501 andpCM194 moieties (see Fig. 4).

process is required for this transformation eventand supports the finding that these plasmidsreplicate autonomously in B. subtilis.

In three cases, S. aureus plasmid characterswere transferred to B. subtilis by transforma-

tion, but no plasmid DNA could be isolated(Table 3). These observations were repeatedfour times. These plasmids (pUB101 [FUsr],pK545 [Kmr], and pSH2 [Kmr]) may have inte-grated in part or in toto, or the plasmid may

J. BACT1ERIOL.

S. AUREUS PLASMID DNA IN B. SUBTILIS

have been undetected for some other reason,such as low copy number.pUB10, pSA2100, pCM194, and pSA0501 can

be isolated from fully grown cultures of B. sub-tilis with a yield of about 1 ,ug of CCC DNA perml of culture. This permits us to calculate thatthese plasmids exist with copy numbers ofroughly 20 to 50. pUB110 is quite stable in B.subtilis even in the absence of Km. It has beenshown that it replicates in B. subtilis with acopy number of about 50, cannot be amplifiedwith Cm, but can replicate in various tempera-ture-sensitive mutants when incubated at thenonpermissive temperature for chromosomalDNA synthesis (Shivakumar and Dubnau, man-uscript in preparation). pSA2100, however, isunstable unless grown in the presence of eitherCm or Sm. This instability may be due to thepresence of the seg mutation in the Sm moiety(17) and might be circumvented by introductionof the Seg+ allele.

Iordanescu (17) isolated pSA2100 as an SmrCmr in vivo recombinant between pCM194-Cmrand pSA0501_Smr. The molecular weight ofpSA2100 (4.69 + 0.21 x 106) suggests that, withinthe limits of the methods, it may have derived

TABLE 12. Fragments produced by single anddouble digestion ofpCM194 with restriction

endonucleasesFragment size Sum of

Number of (Mdal) fragmentdetermi- sizes

Enzymes nations A B (Mdal)

HindIII 3 1.97 1.97HpaII 2 2.06 2.06HaeIII 1 2.00 2.00HpaII-HindIII 4 0.96a 1.92HpaII-HaeIII 2 1.35 0.64 1.99HindIII-HaeIII 2 1.68 0.37 2.05

a This fragment is inferred to be a "double."

A Oell

from a fusion of the two smaller plasmids (2.00+ 0.10 x 106 and 2.80 ± 0.20 x 106). Our restric-tion site mapping entirely supports the fusionmodel (Fig. 3 and 4), since all sites in pSA0501and pCM194 are represented in pSA2100 withcomplete conservation of the map order. Thejunctions between the Cmr and Smr moieties ofthe composite plasmid are indicated in Fig. 3and 4; they occur between the HindIII andHaeIII sites ofpCM194 and between the HindIIand XbaI sites ofpSA0501. Analysis ofthe struc-ture of these junctions will require heteroduplexanalysis to determine whether complete conser-vation of genetic material occurred during fusionand whether homologous segments exist onpCM194 and pSA2100. Electron microscopymight also reveal whether repeated sequencesrepresented by stem-loop structures occur onthese plasmids, since insertion of one into an-other as a translocatable element is an attractivemodel for the in vivo formation of pSA2100.pUB110, pSA2100, pSA0501, and pCM194 ap-

pear to have many of the properties required ofgood cloning vectors. First, they are easily trans-ferred by transformation and transduction fromone strain to another. Second, they can be iso-lated from B. subtilis in high yield and withcomparative ease by conventional means. Theplasmids replicate in B. subtilis as multicopyautonomous replicons, and pUB110 at least issusceptible to further amplification. A largenumber of restriction endonuclease sites havebeen mapped, many of which appear once on agiven plasmid and are known to produce over-lapping ends. Finally, the frequencies of trans-formation of pUB110, pCM194, and pSA2100(104 to 105 transformants per ,tg of DNA) arehigh and should permit the efficient selection ofrecombinant molecules. The decisive property,namely, the ability to support the replication

B

FIG. 4. Restriction endonuclease cleavage site maps ofpCM194 (A) andpSA0501 (B). The relative distancesshown are from average fragment sizes determined on agarose gels. The darker segments identify the regionswhere fusion occurred to produce pSA2100.

VOL. 134, 1978 327

328 GRYCZAN, CONTENTE, AND DUBNAU

TABLE 13. Fragments produced by single and double digestion ofpSA0501 with restriction endonucleases

Enzymes

EcoRIHindIIIXbaIHindIIHpaIIEcoRI -HindI I IXbaI-HindI IIEcoRI-XbaIHindI II-HpaIIEcoRI -HpaI IXbaI-HpaIIHindII-HpaI IHindI II-HindIIHindI I-EcoRIHindII-XbaI

a This fragment was undetected.

Number ofdetermi-nations

Fragment size (Mdal)A B C

3 2.812 2.792 2.823 2.945 1.612 2.653 1.673 1.753 1.593 1.613 1.504 1.261 2.152 1.962 2.50

1.22( )1.141.101.051.021.270.911.101.110.76

Sum offragmentsizes(Mdal)

2.812.792.822.942.83

2.812.85

0.19 2.830.22 2.850. 16 2.930.87 3.04

3.253.073.26

and expression of foreign DNA inserted in oneor another of the available restriction sites, re-mains to be tested.

ACKNOWLEDGMENTS

We thank E. Dubnau, L. Mindich, R. P. Novick, and I.Smith for valuable discussions, and A. Howard for expertsecretarial assistance.

This work was supported by a Public Health Service re-

search grant, AI-10311, awarded to D. Dubnau by the NationalInstitute of Allergy and Infectious Diseases.

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