Vol. 141, No. 1JOURAL OF BACTERIOLOGY, Jan. 1980, p. 246-2530021-9193/80/01-0246/08$02.00/0

Characterization of Chimeric Plasmid Cloning Vehicles inBacillus subtilis

THOMAS GRYCZAN, A. G. SHIVAKUMAR, AND DAVID DUBNAU*Department ofMicrobiology, The Public Health Research Institute of The City ofNew York, Inc., New

York, New York 10016

Restriction endonuclease cleavage maps of seven chimeric plasmids that maybe used for molecular cloning in Bacillus subtilis are presented. These plasmidsall carry multiple antibiotic resistance markers and were constructed by in vitromolecular cloning techniques. Several of the antibiotic resistance markers wereshown to undergo insertional inactivation at specific restriction endonucleasesites. Kanamycin inactivation occurred at the BglII site of pUBilO derivatives,erytbromycin inactivation occurred at the HpaI and Bcll sites of pE194 deriva-tives, and streptomycin inactivation occurred at the HindIll site of pSA0501derivatives. A stable mini-derivative ofpBD12 was isolated and characterized. Byusing these plasmids, we identified proteins involved in plasmid-coded kanamycinand erythromycin resistance. The properties and uses of these chimeric plasmidsin the further development of recombinant deoxyribonucleic acid technology inB. subtilis are discussed.

The use of Bacillus subtilis as a system forrecombinant DNA technology has been ad-vanced by the observation by Ehrlich (5) thatseveral antibiotic resistance plasmids fromStaphylococcus aureus can be transformed intoB. subtilis and are stably maintained and ex-pressed in this host. Subsequent studies havedemonstrated the use of these and other S.aureus plasmids as cloning vehicles in B. subtilis(6, 9, 11, 12, 15).This laboratory has previously described a

number of useful plasmid cloning vehicles and aseries of multiple antibiotic resistance chimericplasmids constructed by in vitro manipulation(9). Inactivation of plasmid markers by the clon-ing of foreign DNA into a given restriction en-donuclease cleavage site (insertional inactiva-tion) is a useful property since it permits thedetection of recombinant plasmid molecules.This report presents restriction endonucleasecleavage site maps of the B. subtilis chimericplasmids previously described (9) and demon-strates several plasmid antibiotic resistancemarkers which undergo insertional inactivation,making these plasmids useful for molecular clon-ing experiments in B. subtilis.

MATERIALS AND METHODSBacterial strains. For the experiments, we used

the B. subtilis strain BD170 (trpC2 thr-5) and theminicell producer BD474 (thyA thyB metB divIVB1).

Bacterial plasmids. The parental plasmids arelisted in Table 1, and a catalog of chimeric plasmidderivatives is presented in Table 2. The derivations of

the latter plasmids are described in a previous publi-cation (9), except for pBD64 which is described in thisreport. Physical maps of the chimeras are shown inFig. 1. Isolation of plasmid DNA was previously de-scribed (8).

Restriction endonuclease digestions. Restric-tion endonuclease digestions were performed as pre-viously described (8) with the addition of digestionswith MboI (7), PvuII (16) (New England Biolabs), andTacl (13) (Bethesda Research Laboratories) whichwere according to manufacturer instructions. BclI (1)(New England Biolabs) digestions were performed at50°C for 30 min (A. G. Bingham, personal communi-cation). Multiple restriction endonuclease digestionswere as previously described (8).

Restriction fragment mapping. Agarose gelswere run as previously described (8). Polyacrylamidegel electrophoresis was performed by using a modifi-cation of the procedures of Bolivar et al. (2). Acryl-amide gels (7.5%) were made by mixing 2.4 ml of lOxTris-borate buffer, 15.6 ml of water, 6 ml of 30%acrylamide (Sigma Chemical Co.), 0.8% N,N'-meth-ylenebisacrylamide (Eastman Organic Chemicals) and12 pl ofN,N,N',N'-tetramethylethylenediamine (East-man Organic Chemicals). After degassing, 120 td of10% ammonium persulfate was added, and the mixturewas cast into a standard vertical gel apparatus (20).Gels were electrophoresed at 120 V for 2 h, stainedwith ethidium bromide (2 yg/ml) for 15 min, destainedin water for 5 to 10 min, and photographed. Thefragments were sized by comparison with HindIIIdigests of A (14) and PM2 (2) DNA. Multiple digestionsand partial restriction digests were analyzed to deter-mine restriction maps.

Ligation conditions. Conditions for plasmid-plas-mid and plasmid-chromosome ligations were describedpreviously (9).

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VOL. 141, 1980

TABLE 1. Parental plasmidsOther useful prop-

ertiesResist- Single

Plasmid Mol wt ance restric- Segre-con- tion gates Tmsnferred sites into sitivity

cell

pUBllO 3.0 x 106 Kmr EcoRI Yes ResistantBamHIXbalTacIBglIIIPvuII

pE194 2.4 X 106 Emr HpaI Yes SensitiveBcllPstIXbaIHaeIII

pC194 2.0 x 106 Cmr HaeIII Yes ResistantHindlIlHpaIIHhaI

pSA0501 2.7 x 106 Smr EcoRI NT8 SensitiveHindIIIHindIIXbaI

a NT, Not tested.

Plasmid transformation. Transformation ofcom-petent cells and minicells was performed by usingpreviously described procedures (3, 4).

Protein analysis. Procedures for the analysis ofplasmid-encoded proteins in B. subtilis minicells bysodium dodecyl sulfate-polyacrylamide gel electropho-resis were described previously (18).

RESULTSInactivation of kanamycin resistance.

Construction and partial characterization of thechimeric plasmid pBD9 has been previously de-scribed (9). This plasmid, which confers resist-ance to kanamycin (KMrn) and erythromycin(Emr), has single cleavage sites for the restrictionendonucleases EcoRI, BamHI, Bgill (from thepUBilO parent), HpaI, PstI, and BclI (from thepE194 parent). A restriction endonuclease mapof this plasmid is shown in Fig. 1. Previousstudies have shown that the single XbaI site oneither parental plasmid does not inactivate re-sistance to kanamycin and erythromycin, sinceligation at these sites was used to construct thecomposite double resistance plasmid (9). Simi-larly, cloning of foreign DNA into the EcoRIsite of pUB110 does not inactivate kanamycinresistance (9, 11). Cloning and deletion analysis(Gryczan and Dubnau, unpublished data) have

CHIMERIC PLASMIDS IN B. SUBTILIS 247

shown that the BamHI site on this plasmid isnot located in the Km' gene.

Since the BglII site on pBD9 is relativelydistant from the EcoRI and BamHI sites, it wasdesirable to test whether cloning into the BglIIsite will inactivate kanamycin resistance. Theplasmid DNA was cut with BglII and ligated toa fivefold excess of Bacillus lichenifornis chro-mosomal DNA. The ligation mixture was usedto transform B. subtilis competent cells withselection for erythromycin resistance. Ten Emrtransformants were screened for kanamycin re-sistance, and two transformants with the phe-notype Emr Km' were isolated. Analysis of theplasmid carried by one of these transformantsindicated the presence of a new plasmid, largerthan the original pBD9 plasmid (Fig. 2). Diges-tion of this new plasmid with BglII yielded tworestriction fragments on agarose gels, one cor-responding to linear pBD9 and a second frag-ment with a mass of -0.62 megadaltons (Mdal).The inactivation of Kmr by insertion into thisBglII site has been confirmed by the use ofpBD9, pBD12, and pBD64 for several cloningexperiments in our laboratory (Gryczan andDubnau, unpublished data; T. Gryczan et al.,Mol. Gen. Genet., in press).Inactivation of erythromycin resistance.

The effect of interference with the three singlerestriction enzyme sites on the pE194 moiety ofpBD9 was tested for the effect on erythromycinresistance. Plasmids pBD10 and pBD11 (9) lackthe PstI site of pE194 but express erythromycinresistance, indicating that loss of sequences in-cluding the PstI site has no effect on erythro-mycin resistance. Cloning of foreign B. licheni-formnis chromosomal DNA into the Bcll site onpBD9 results in the formation ofKmr Em8 trans-formants, one of which was tested and found tocontain a new larger plasmid. Bcll digestion ofthis plasmid revealed two BclI fragments, onethe size of linear pBD9 and a second fragmentof -0.3 Mdal (Fig. 3A).

Since the HpaI site on pBD9 is located onlyabout 0.25 Mdal from the BclI site, it was rea-sonable to test whether the HpaI site is situatedin an essential erythromycin resistance gene.pBD9 was digested with HpaI, and the linear-ized plasmid was rejoined with a T4 DNA ligasepreparation which we knew contained contami-nating exonuclease. Kmr Em' transformantswere isolated, and nine of these were screenedfor their plasmid DNA content. Four of theseplasmids were indistinguishable in size frompBD9, whereas the other five plasmids weremissing from 0.9 to 2.3 Mdal of DNA. All of thefour plasmids which appeared similar in size topBD9 were found to have lost their HpaI sites.

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248 GRYCZAN, SHIVAKUMAR, AND DUBNAU

TABLE 2. Properties of chimericplasmidsOther useful properties

Inacti-

Plasmid Sou (ref oi wt Resistance con- Single vated re- Segre-erence) ferred sitee sistance Amplifi- gates Temp sensi-marker ableb into tivity

minicells

pBD6 9 5.8 x 106 Kmr Smr BamHI None NT NT Sensitivem" IF vlrTacIBglIIHindIH

pBD8 9 6.0 x 10' Km' Sm' Cm' EcoRIHindIIIBglIIBamHIXbaI

5.4 x 106 Kmr Emr EcoRIBamHITaclBglIIBcllHpaIPstI

4.4 x 106 Km Cmr Emr BglIIBamHIXbaIHpaIBcll

4.4 x 106 Kmr Em1

4.5 x 10i Km' Cmr

XbaIBamHIBglIIHpaIBclI

EcoRIXbaIBamHITacIBglIIHindu

Nonie

Smr

SmrSm1KmrNoneNone

NoneNoneNoneKmrEm1

K,r

Em1None

Km'NoneNoneEmrEmr

NoneNoneKm1Em'Emr

NoneNoneNoneNoneKnmrNone

NT NT Sensitive

Yes Yes Resistant

NT Yes Resistant

NT Yes Resistant

Yes Yes Resistant

pBD64 Thisreport

3.2 x 10i Kmr Cmr EcoRIXbaIBamHITacIBglII

NoneNoneNoneNoneKmr

NT NT Resistant

a These unique restriction sites are available for cloning since their removal by insertion of foreign DNA or

by deletion does not interfere with replication of the chimeric plasmid (Gryczan and Dubnau, unpublisheddata).

b This refers to amplification of plasmid copy number in a Ts dnaC30 host strain during incubation at thenonpermissive temperature (Shivakumar and Dubnau, unpublished data). pBD6 and pBD8 were not tested(NT) because they are cured at elevated temperature. Although pBD10, pBD11, and pBD64 were also nottested, they are most likely amplifiable since their parental plasmids are.

The results with one ofthem (pBD40) are shownin Fig. 3B. pBD40 may have been generated bya small deletion or even by a point mutation,altering the HpaI site with simultaneous inacti-vation of erythromycin resistance. Acrylamide

gel analysis indicates that if the alteration inpBD40 causing erythromycin sensitivity is a

deletion, it must be composed of less than 20base pairs (Gryczan and Dubnau, unpublisheddata). These results strongly suggest that the

pBD9 9

pBD10 9

pBDll 9

pBD12 9

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VOL. 141, 1980

Km'BMHI I I-

Xbe Tee I Sol 11I . -

CHIMERIC PLASMIDS IN B. SUBTILIS 249

PetI

EcoRI XboI I

Em'

Hpa Bcl XboI I

I IpBD9

Xbo I Hpo 11

EcoR I BomHII I __

Xbo I Hpo II_

EcoRi IBomHI HI III,t --I I

Cm'

Hind llI HpoI I

,_-I-mmm_

1mI

EcoRI Xba

-41-1

1pa I I

Sm'

Hind IlIl EcoRI Xbo I

- - - - --H- -

Cm' Sm'

Hind III EcoRI Xbo

BoimHIIHo I

Xbe I Toc I

K m' Cmf Em'I _ _ _ _ _

1II1 Hpo I Bcl Xbo I

-- I I I1C~~m' E m'rIMHI Hpo Il-

Xbo I l 4c B II Hpo Bc I Xbo I

I-- I ----- II I

0 2 3 4 5 6I I I I I I . I I

pBDIO

pBD 11

MDoI

FIG. 1. Restriction endonuclease cleavage maps. Maps of the chimeric plasmids used in this study areshown in a linear form cleaved at a specific restriction enzyme site. Derivation of chimeric plasmid DNA isas follows: -, pUB110; -, PE194; -----, pC194; ------ pSA0501. In pBD8, pBD10, andpBDI1, boundariesbetween parental plasmid DNAs are uncertain and are given as approximate values. Locations of specificantibiotic resistance markers are listed above the linear restriction enzyne cleavage maps.

HpaI site is located in an erythromycin-resistantgene of pBD9 and that insertional inactivationof erythromycin resistance at this site could beemployed for molecular cloning.Inactivation of streptomydin resistance.

It has been previously shown that cloning ofDNA into the single EcoRI site of pSA2100results in the inactivation of streptomycin re-sistance (Smr) (12). Restriction mapping dataindicate that there is a HindIII site about 0.1Mdal away from the EcoRI site on all of the Smrplasmids derived from pSA0501 includingpSA2100 (Fig. 1) (8). To test the effect of cloninginto this HindIII site, pBD6, a chimeric plasmidwhich confers resistance to kanamycin andstreptomycin, was used. A 1:1 mixture ofHindIII-cleaved pBD6 and HindIIl-cleavedpHVll (6) was ligated and used for transforna-

tion. pHV11 confers resistance to chloramphen-icol (Cmr) and tetracycline (Tcr) and is cleavedby HindIII into two fragments (6). Transforn-ants with the phenotype Kmr Sm5 were isolated,and when plasmidDNA was analyzed by agarosegel electrophoresis, new plasmids larger thanpBD6 were obtained. HindIII digestion of oneof these plasmids produced two HindIII frag-ments, one corresponding to linear pBD6 (5.8Mdal) and a second containing 1.5 Mdal, corre-

sponding to the smaller HindIII fragment ofpHV11 (Fig. 4). This new plasmid (pBD63) alsoexpresses the tetracycline resistance conferredby the smaller HindIII fragment of pHV11 (6).Derivation of plasmid pBD64. A physical

map of pBD12 (9) is presented in Fig. 1. Al-though the BglII site on this plasmid has beenutilized to clone a number of plasmid and chro-

"o Sol 11I I

K.'

"Poll Sal 11I I

DHI 11_____Km

Xbl I Tic I ei il

I. II

mI

pBD12

pBD64

pBD6

pBD8

a I 1__j - -

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250 GRYCZAN, SHIVAKUMAR, AND DUBNAU

a bcd e quite stable. Growth on media containing eitherchloramphenicol or kanamycin and replicationonto media with both drugs indicated no loss ofeither resistance character. Repeated isolationof pBD64 DNA has not revealed the presence ofsmaller derivatives. Restriction enzyme analysisindicates that the deletion event which gener-ated pBD64 occurred entirely within the pC194portion of pBD12. A physical map of pBD64 isshown in Fig. 1. This plasmid exhibits inactiva-tion of kanamycin resistance at the BglII site asexpected, and has been employed as a cloningvehicle for a variety of plasmid and chromo-somal fragments (Gryczan et al., in press). Nocleavage site which is located within the Cmrdeterminant has been identified.Temperature sensitivity ofchimeric plas-

mids. Since the pE194 (21) and pSA0501 (10)plasmids have been shown to be temperaturesensitive (Ts) for replication, it was desirable toknow whether any pE194 or pSA0501-derivedplasmid chixneras would also be temperaturesensitive for plasmid replication. Strain BD170derivatives containing these chimeric plasmidswere grown on tryptose blood agar base plates

a b c a b c d

FIG. 2. Insertional inactivation ofKm' inpBD9 atthe BglII site. Plasmids and restriction digests wereelectrophoresed on an 0.8% agarose gel at 50 V for 3h. (a) A + HindIII; (b) pBD9; (c) pBD9 + BglII; (d)Km8 Emr insertplasmid; (e) Km8 Emr insertplasmid+ BglII.

mosome-derived restriction fragments withscreening for loms of kanamycin resistance (Gry-czan et al., in press), its usefulness as a cloningvehicle was limited by the frequent spontaneousappearance of smaller deletion plasmids. Trans-formation of a plasmid-negative recipient witha pBD12 DNA preparation which containedsuch a smaller derivative resulted in the isolationof Kmr Cmr transformants that contained onlythe smaller pBD12 derivative plasmid. This newplasmid, pBD64, has a mass of 3.1 to 3.2 Mdal,indicating that a deletion of 1.2 to 1.3 Mdal ofpBD12 DNA occurred. pBD64 appeared to be

Ii

AFIG. 3. Insertional inactivation ofEmr inpBD9 at

the BclI site. Plasmids and restriction digests wereelectrophoresed on an 0.8% agarose gel at 50 V for 3h. A: (a) pBD9; (b) pBD9 + BclI; (c) Km' Em8 insertplasmid + BclI. B: (a) pBD9; (b) pBD9 + HpaI; (c)pBD40; (d) pBD40 + HpaI.

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CHIMERIC PLASMIDS IN B. SUBTILIS 251

a b c of the proteins coded by the parental plasmids(pUB110 and pE194), with the-exception of two(Kl and El) (18). A pBD9 derivative whichcontains a B. licheniformis fragment inserted inthe BglII site was transformed into the B. sub-tilis minicell strain, and the plasmid-specific pro-teins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results (Fig.5) indicate that the plasmid showing kanamycinresistance inactivation produces an altered formof a protein which normally has a molecularweight of 38,000. Y. Sadaie (personal comnmuni-cation) has identified this 38K protein as a kan-

a

38K-

FIG. 4. Insertional inactivation ofSmr inpBD6 atthe HindIII site. Plasmids and restriction digestswere electrophoresed on an 0.8% agarose gel at 50 Vfor 3 h. (a) pBD6 + HindIII; (b) pHVII + HindIII;(c) pBD63 + HindIII.

b

at 48°C, and the colonies were either replicatedor patched onto tryptose blood agar base platescontaining the appropriate antibiotics and incu-bated at 320C. Results indicate that pBD6 andpBD8 are both temperature sensitive for plas-mid replication since all colonies grown at 480Cwere found to be antibiotic sensitive and to havebeen cured of their plasmids. These two plas-mids thus appear to replicate under the controlof the Ts Sm' parental plasmid. pBD9, whichcontains all ofthe plasmid pE194, is temperatureresistant, indicating that the replication systemof the pUBllO parent is still functional. pBD10and 11, both of which have deletions whicheliminate about 50% of the pE194 derived se-quences, are also both temperature resistant.Chimeric plasmid protein alterations.

pBD9 has been previously shown to specify all

FIG. 5. Protein patterns of B. subtilis minicellstrain carrying pBD9 and Km' Emr insert plasmid.Minicells were labeled, and theproteinproducts wereanalyzed on sodium dodecyl sulfate-polyacrylamidegels, as previously described (18). (a) pBD9; (b) Km'Emr insert plasmid.

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252 GRYCZAN, SHIVAKUMAR, AND DUBNAU

amycin nucleotidyltransferase. We have shownthat a 29K protein specified by pE194 is respon-sible for erythromycin resistance (18). The de-terminant for this protein contains the HpaI siteof pE194 and of its derivatives, since mutationsaffecting the HpaI site of pBD9, including theone in pBD40 described above (Fig. 3), result inthe production of an altered 29K protein (Shi-vakumar, Gryczan, and Dubnau, unpublisheddata). These findings thus identify proteins in-volved in resistance to kanamycin and erythro-mycin, demonstrating that insertional inactiva-tion of these two drug resistance markers altersproteins coded by the resistance components ofthe plasmid chimera.

DISCUSSIONThis report presents restriction endonuclease

cleavage site maps of seven chimeric plasmidsthat are useful as vectors for molecular cloningexperiments in B. subtilis. These plasmids, inaddition to conferring resistance to two or moreantibiotics, exhibit insertional inactivation (19)of several of these drug resistance markers. Thisphenomenon facilitates the construction of plas-mid-foreign DNA recombinant molecules byscreening for the loss of one of the plasmidantibiotic resistance markers. Thus, inactivationofkanamycin resistance by insertion in the BglIIsite of pUBllO-derived vectors, inactivation oferythromycin by cloning in the HpaI and BclIsites of pE194 derivatives, and inactivation ofstreptomycin resistance by cloning into theHindIII and EcoRI (12) sites of pSA0501 deriv-atives make these vectors useful for molecularcloning in B. subtilis. The BglII sites of pBD12and pBD64 have been used for cloning B. Ii-cheniformis chromosomal DNA and for cloningall five MboI fragments ofpE194 (Gryczan et al.,in press).

Cloning into the BglII site of pUB110 and itsderivatives alters a 38K protein. Thus, a frag-ment inserted in this site might be transcribedfrom the Km' promoter which is required for theexpression of kanamycin resistance. Since elim-ination of the HpaI site which is in the Emr geneof pE194 alters a 29K polypeptide which is in-volved with erythromycin resistance, it is likelythat cloning in the HpaI site may fuse the insertwith the Emr promoter of this plasmid. Sincethe 29K protein is inducible by erythromycin(18), it is possible that a gene carried on such afragment will also be controlled (inducible) bythe level of erythromycin. This might be usefulfor a variety of studies, including investigationof the mechanism of induction of erythromycinresistance (21). Mutants have been isolatedwhich produce the 29K protein constitutively

(21). These might be advantageous in some clon-ing experiments. This BclI site, on the otherhand, appears to be within the Emr promoter,and interference with this site eliminates syn-thesis ofthe 29K protein (Y. Kozlov, T. Gryczan,A. G. Shivakumar, and D. Dubnau, unpublisheddata). Thus, cloning into the Bcll site may be auseful way to insure that a foreign gene is underthe control of its own promoter. The expressionof genes cloned into the HpaI and BclI sites isunder investigation.The temperature sensitive property of plas-

mids pBD6 and pBD8 may give these vectorsadded utility in molecular cloning experiments.For example, if a putative clone carrying a givenDNA fragment has been isolated and it is desir-able to know if the fragment is plasmid bome orhas integrated into the host chromosome, thetemperature-sensitive property of these vectorscould be a decided advantage. If the fragmentwere plasmid borne, growth at the nonpermis-sive temperature would result in the loss of thefragment and its associated phenotype, whereasa fragment which has integrated into the chro-mosome and is under its replication controlwould not be lost at the high temperature. Thus,any plasmid vector which is temperature sensi-tive may have additional usefulness in the anal-ysis of the replication and expression of clonedDNA fragments.

In addition to analysis of cloned fragmentexpression, the temperature-sensitive propertyof plasmid vectors may give added insight intothe replication control of these plasmids. Plas-mids pBD9, pBD10, and pBD1l are derivedfrom a temperature-sensitive parental plasmid(pE194) and are temperature resistant, whereaspBD6 and pBD8 retain the temperature-sensi-tive property of one of their parental plasmids(pSA0501). Since some of our chimeric plasmidsare additive combinations of two parental plas-mids, they may be dual replicons, potentiallycontaining intact replication genes of both plas-mids. As a result, replication may be controlledby either or both replicons, depending on thegrowth temperature and the restriction sitesused to form the chimera.We have previously shown that pUB110 con-

tinues to replicate in several dna(Ts) hosts whenchromosomal replication is restricted by incu-bation at a nonpermissive temperature. In thisway copy numbers of about 1,000 were reached(17). This observation has been extended topC194 and to several chimeric derivatives ofpUB110 and pC194 (Table 2). In addition toproviding a useful aid for the isolation ofplasmidDNA, this amplification system permits contin-ued protein synthesis under conditions of ele-

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vated plasmid-gene dosage. In this way we havebeen able to detect and study plasmid-specificproteins without recourse to minicells (Shivak-umar and Dubnau, unpublished data). We antic-ipate that this method will be useful for thestudy of both plasmid-specific proteins and theexpression of cloned DNA fragments.

ACKNOWLEDGMENTSWe acknowledge with gratitude continual discussions with

I. Smith, E. Dubnau, and S. Contente, as well as the expertsecretarial assistance of A. Howard.

This work was supported by National Institutes of Healthgrant AI-10311 and American Cancer Society grant VC300,both awarded to D.D.

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CHIMERIC PLASMIDS IN B. SUBTILIS 253

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9. Gryczan, T. J., and D. Dubnau. 1978. Construction andproperties of chimeric plasmids in Bacillus subtilis.Proc. Natl. Acad. Sci. U.S.A. 75:1428-1432.

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

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12. Lofdahl, S., J. E. Sjostrom, and L. Philipson. 1978. Avector for recombinant DNA in Staphylococcus aureus.Gene 3:161-172.

13. McConnell, D. J., D. C. Searcy, and J. G. Sutcliffe.1978. A restriction enzyme ThaI from the thermophilicmycoplasma Thermopolasma acidophilum. NucleicAcids Res. 5:1729-1739.

14. Murray, K., and N. E. Murray. 1975. Phage lambdareceptor chromosomes for DNA fragments made withrestriction endonuclease HI of Haemophilus influenzaeand restriction endonuclease I of Escherichia coli. J.Mol. Biol. 98:551-564.

15. Niaudet, B., and S. D. Ehrlich. 1979. In vitro geneticlabeling of Bacillus subtilis cryptic plasmid pHV400.Plasmid 2:48-58.

16. Roberts, R. J. 1978. Restriction and modification en-zymes and their recognition sequences. Gene 4:183-193.

17. Shivakumar, A. G., and D. Dubnau. 1978. Plasmidreplication in dna TS mutants of Bacillus subtilis.Plasmid 1:405-416.

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20. Studier, F. W. 1973. Analysis of bacteriophage T7 earlyRNA's and proteins on slab gels. J. Mol. Biol. 79:237-248.

21. Weisblum, B., M. Y. Graham, T. Gryczan, and D.Dubnau. 1979. Plasmid copy number control: isolationand characterization of high-copy-number mutants ofplasmid pE194. J. Bacteriol. 137:635-643.

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