Vol. 144, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1980, p. 923-9320021-9193/80/12-0923/10$02.00/0

Location of Two Relaxation Nick Sites in R6K and SingleSites in pSC101 and RSF1010 Close to Origins of VegetativeReplication: Implication for Conjugal Transfer of Plasmid

Deoxyribonucleic AcidALFRED NORDHEIM,t TAMOTSU HASHIMOTO-GOTOH, AND KENNETH N. TIMMIS*

Max-Planck-Institut fir Molekulare Genetik, Berlin-Dahlem, West Germany

A nick-labeling method has been used to localize the relaxation complex nicksites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in theirhost range, deoxyribonucleic acid replication, and conjugal transfer properties.Single specific relaxation sites were located in pSC101 and RSF1010, but surpris-ingly two distinct sites could be identified in the bi-origin plasmid R6K. In allcases, relaxation nick sites, which are thought to be origins of plasmid conjugaltransfer, were shown to be located near origins of vegetative replication. Thisresult suggests a functional interaction between these two types of deoxyribonu-cleic acid loci, and we speculate here that replication events initiated at origins ofreplication may constitute an integral part of the process of conjugal transfer ofsmall plasmids among bacteria. Consistent with this proposal is the finding thatinhibition of vegetative replication of the pSC101 and ColEl plasmids results ina severe inhibition of their conjugal transfer ability.

Most bacterial plasmids can be isolated fromhost cells as supercoiled DNA-protein "relaxa-tion complexes" which are converted to opencircular nicked forms by in vitro treatment withprotein-denaturing agents (8). In vitro relaxationresults in the introduction ofa "nick" at a uniquelocation in the plasmid molecule in one specificDNA strand. The relaxation nick sites of ColEland R6K have been located in regions of theplasmid genomes containing an origin of vege-tative replication, oriV (28, 30). These findingsled to the suggestion that relaxation complexesplay a role in plasmid replication (21). Recentexperiments with ColEl (13, 22) and RK2 (17)indicate, however, that relaxation complexes areinvolved in the conjugal transfer of plasmidDNA from donor bacteria to recipients.

Definitive elucidation of the biological func-tion of relaxation complexes requires precise lo-calization of relaxation nick sites in plasmid ge-nomes to correlate such sites with sequences ofknown function, such as origins of replicationand conjugal transfer.We have recently developed methods that

allow the rapid and precise localization of plas-mid relaxation nick sites (A. Nordheim, Ph.D.thesis, Freie Universitiit Berlin, Berlin, WestGermany, 1979) and are using these to investi-gate the relaxation complexes of three plasmids

t Present address: Department of Biology, MassachusettsInstitute of Technology, Cambridge, MA 02139.

(pSC101 [9], RSF1010 [14], and R6K [25]) thatdiffer markedly from one another in their repli-cation and/or conjugal transfer properties. Inthis communication we show that R6K, a plas-mid possessing two distinct functional oriV's (11,25), contains two nick sites, whereas the singleorigin plasmids pSC101 (7) and RSF1010 (14)each contain a single relaxation nick site. In allcases, the relaxation nick sites were found to belocated in the neighborhood of origins of vege-tative replication. The striking proximity of re-laxation nicks and oriV's in these plasmids, andin ColEl (5, 28), has led us to postulate a func-tional relationship between these two types ofDNA sites.

(The work presented here was submitted byA.N. to the Freie Universitat Berlin in partialfulfillment of the requirements for a Doctor ofNatural Sciences degree.)

MATERIALS AND METHODSRestriction endonucleases and proteinase K were

purchased from New England Biolabs and Merck,respectively. DNA polymerase I and DNA polymeraseI-Klenow fragment, dATP, dGTP, dCTP, and dTTPwere obtained from Boehringer-Mannheim, whereas[methyl-3H]thymidine (40 Ci/mmol) and [a-32P]dATP(400 Ci/mmol) were obtained from Amersham-Buch-ler.Preparation of relaxation nick-labeled plas-

mid DNA. Escherichia coli K-12 strains containingplasmids pSC101 (9), RSF1010 (14), and R6K (25)were grown in Tris-buffered glycerol minimal (TBM)

923

924 NORDHEIM, HASHIMOTO-GOTOH, AND TIMMIS

medium (8), and cleared lysates of bacterial cultureswere prepared as reported (26). The isolation of spe-cifically relaxed plasmid DNA molecules and the ra-dioactive labeling of their relaxation nicks will bedescribed in detail elsewhere. Briefly, plasmid mole-cules in cleared lysates were relaxed by treatment withsolutions of ethidium bromide (26) or sodium dodecylsulfate, purified by passage through a Sepharose 2Bcolumn, and treated with proteinase K. After phenol-chloroform extraction and ethanol precipitation, re-laxed plasmid molecules were radioactively labeled ina limited polynucleotide chain extension reaction byusing the free 3'-OH at the nick as a primer for DNApolymerase I (Klenow fragment) and [a-32P]dATP asone of the substrate triphosphates (see Fig. 1). Nick-labeled DNA was separated from unincorporated tri-phosphates by chromatography and cleaved with therestriction endonucleases indicated in the figure leg-ends under conditions that are recommended by theenzyme suppliers. The fragments thereby generatedwere then separated by electrophoresis through aga-rose gels containing Tris-borate buffer and ethidiumbromide as previously described (2).

Superhelical Relaxed DNARelwation Complex

Klenow

[[,4-32P] dXTP

FIG. 1. Schematic representation of the procedureused to localize plasmid relaxation nick sites. Thebox on the supercoiled plasmid molecule representsrelaxation proteins, one ofwhich becomes covalentlyattached to the 5' end ofthe nicked strand during therelaxation event (29). This and other proteins asso-ciated with theplasmid molecuk after relaxation areremoved by treatment with proteinase K. The free 3'-OH end of the nicked strand is then labeled asdescribed in the text.

RESULTSLocalization of unique relaxation nick

sites in plasmids pSC101 and RSF11O. Itwas recently demonstrated that the two small,nonconjugative but mobilizable plasmidspSC101 and RSF1010 may be isolated from hostbacteria in the form of supercoiled plasmidDNArelaxation complexes which can be specificallyconverted in vitro to relaxed plasmid forms bytreatment with ethidium bromide or sodium do-decyl sulfate (Nordheim, Ph.D. thesis). To lo-calize the relaxation nick sites in the genomes ofthese plasmids, we prepared relaxed moleculesand carried out the nick labeling reaction asoutlined in Fig. 1. Labeled plasmid preparationswere then treated with individual restriction en-donucleases or combinations of restriction en-donucleases, and the fragments thereby gener-ated were separated by electrophoresis throughan agarose gel.As can be seen in Fig. 2, a single relaxation

site is located on the large HpaI/BamHI frag-ment (tracks 1 and 2), the second largest HincIIfragment, H-2 (tracks 3 and 4), a 0.87-kilobase(kb) Hinfl fragment (tracks 5 and 6), and a 0.57-kb HincII/Hinfl fragment (tracks 7 and 8) ofplasmid pSC101. From the restriction endonu-clease cleavage map of pSC101 (36; Nordheim,Ph.D. thesis), we can conclude that its relaxationsite is located within the 0.57-kb Hinfl terminalsubfragment of HincII fragment H-2 that is con-tiguous with H-1 (Fig. 2).

Cabello et al. reported that the pSC101 originof vegetative replication is located 50% of themolecule length from the unique EcoRI cleavagesite (7). Allowing for ±5% in origin mapping byelectron microscopy, we may conclude that thepSC101 replication origin is contained within a0.9-kb DNA segment which includes about 450base pairs of each of the two adjacent HincIIfragments H-1 and H-2. Recently, it was re-ported that the H-1 fragment is able to form anautonomous replicon (31) and hence presumablycontains the pSC101 vegetative origin. Takentogether, these data indicate that the pSC101relaxation nick site and oriV are adjacent butphysically distinct loci, as has been found pre-viously for ColEl (5), separated by a DNA seg-ment that is 0.45 to 1.1 kb long.Using a similar approach to analyze RSF1010,

we could localize the relaxation nick site on thelargest AvaI fragment (not shown) and on a0.85-kb HaeII fragment of this plasmid (Fig. 2,tracks 9 and 10). Endonuclease cleavage sitemapping experiments have allowed us to locatethe nick site-containing HaeII fragment on thegenome of RSF1010 (3) in a region that has alsobeen shown to contain the RSF1010 origin of

J. BAc-rzIoL.

RELAXATION COMPLEX NICK SITES 925

1 2 3 4 5 6 7 8

...~~~~~~~~~....;1. .:.m.W I..}..

::.. ..-~

9 10

...

d"

EcoRI

2 '

IcII (Hpal)

-300 bp 570 bp- NIC---

FIG. 2. Localization of relaxation nick sites in plasmids pSC101 and RSFIO1O. Top: Labeled DNApreparations were mixed with unlabeled preparations, treated with restriction endonucleases, and analyzedby electrophoresis through a 1.2% agarose gel. Tracks 1 (pSC1O1/BamHI + HpaI), 3 (pSC101/HincII), 5(pSC101/HinfI), and 7 (pSC101/HincII + HinfI) contain relaxation nick-labeled pSC1l1 DNA preparations,whereas tracks 2, 4, 6, and 8 contain uniformly labeled DNA preparations that were treated with theendonuclease combinations indicated for 1, 3, 5, and 7, respectively (HincII fragments 2 and 3, and 4 and 5,ran as doubles in this gel). Tracks 9 and 10 show the photograph and autoradiograph of HaeII-digestedrelaxation nick-labeled RSF1010 DNA. Bottom: Physical maps of pSC101 and RSF1010. These maps arebased upon information published in references 7, 9, 31, and 36 and upon our own data reported here and tobe published. Abbreviations: Sm and Su, resistance to streptomycin and sulfonamide; cII, fI and I arecleavage sites for HincII, Hinfl, and AvaI, respectively.

VOL. 144, 1980

926 NORDHEIM, HASHIMOTO-GOTOH, AND TIMMIS

vegetative replication (14; Fig.that the relaxation nick siiRSF1010 are within 850 baseother.Plasmid R6K contains t

sites. Plasmid R6K originallyutilize a single origin of vegetwhose location was indistinguiofthe plasmid relaxation site (3(R6K was shown to contain anbut two origins of replicationa-ori and fl-ori, which are locfragments H-4 and H-2, respecmine with which vegetative orijnick is associated, we preparecules of plasmid R6K, performing reaction, digested labeledHindIII endonuclease, and selments on an agarose gel. As ca3 (track 2), most of the radioaiporated into HindIII fragmersome was also introduced intand H-2.R6K HindIII fragment H-1

35% of the total plasmid gen(

1 2 3 4 5 6 f

**43...... a...- -

0

4-0

FIG. 3. Identification oftwo disR6Kplasmid that contain relaxaidioactively labeled R6K DNA wostriction endonucleases and analyresis through a 1% agarose gel.uniformiv labeled R6K DNAfA- Iano

2). This indicates pected to contain a high proportion of the totalte and oriV of random nicks present in the molecules of thepairs of one an- plasmid preparation and, consequently, a high

proportion of the unspecifically incorporated ra-two relaxation dioactivity. To determine whether the radioac-was reported to tivity present in fragments H-1 and H-2 was duetative replication to labeling of specific or random nicks, nick-ishable from that labeled R6K DNA was subjected to double0). Subsequently, digestion with HindIII and HpaI endonucleasesd utilize not one, (Fig. 3, track 5). This generated three HindIII/(11), designated HpaI subfragments from H-4, one of which con-

-ated on HindIII tained all of the label originally present in theAively. To deter- latter, and four subfragments of H-1, all ofwhichgin the relaxation contained only background levels of label. Al-ed relaxed mole- though the H-2 fragment, which contains noed the nick label- HpaI sites, is smaller than the two largest[ molecules with HindIII/HpaI subfragments of H-1, it neverthe-parated the frag- less contains much more radioactivity. Thus,in be seen in Fig. radioactive label is incorporated specifically intoctivity was incor- two HindIII fragments of R6K, H-2 and H-4.at H-4, although Fragments H-2 and H-4 are not contiguous in;o fragments H-1 the R6K genome and cannot be labeled specifi-

cally in a single DNA synthesis reaction fromcomprises about one nick site; both of them, therefore, containDme and was ex- relaxation sites. Because these sites are located

on HindIII fragments that contain the a and ,B8 9 10 origins of vegetative replication, we have desig-

nated the nick sites a-nic (on H-4) and /-nic (onH-2), respectively.Localization of the relaxation nick sites

of plasmid R6K. According to Kolter and Hel-* 13-4! inski (25), R6K fragment H-4 is cleaved twice by

HpaI endonuclease, generating three subfrag-W* & - ments having approximate sizes of 1.85,1.25, and

41 f.p1*4g 0.68 kb, and twice by HaeII, generating threesubfragments whose sizes we determined byHaeII digestion of isolated H-4 fragment DNAto be 0.51, 0.605 (terminal subfragments), and

.1a .* * gf?2 >2.5 kb. The radioactivity introduced intoHindIII fragment H-4 by nick labeling of R6K

,larelaxed DNA was found to be located on the1.85-kb HindIII/HpaI subfragment (Fig. 3, track5), a 0.51-kb HindIII/HaeII subfragment (Fig. 3,tinct regions in the track 7), a 0.60-kb HinfI fragment, and a 0.55-kb

sintrenck tes.tRae HaeII/HinfI subfragment (Fig. 3, track 10). Thisszed by electropho- localizes a-nic to the 0.51-kb right-hand terminal' Lanes 1 and 4: segment of H-4 on the R6K map shown in Fig..s 2- fi 7 Q .9 and 4.&t&UC,,U&, A&VAX&.ss u^sca X, Up ,,,O., ESIM

10: nick labeled relaxation complex preparations ofR6K; lanes 3 and 6: supercoiled, noncomplexed R6KDNA treated with the relaxation nick-labeling pro-cedure. Preparations were cleaved with HindIII(tracks 1 to 3), HindIII/HpaI (tracks 4 to 6), HindIIIIHaeII (track 7), HaeII/HincII (track 8), HincII/HinfI(track 9), HaeII/HinfI (track 10). a and ,8 indicate therespective positions of labeled DNA fragments con-taining a-nic and ,B-nic relaxation sites (sizes givenin kilobases). From densitometer tracings of tracks 4and 5 (not shown), we estimate that a-nic was labeled10 to 15 times more efficiently than fl-nic.

HaeII endonuclease was found to cleave iso-lated R6K fragment H-2 twice, thereby gener-ating subfragments of 0.68, approximately 1.2and >2.0 kb in length (data not shown). The0.68-kb HaeII/HindIII subfragment containsthe right-hand terminal segment of H-2 and ispart of the HaeII fragment that contains H-15and part of H-9 (Fig. 4). The radioactivity incor-porated at the /-nic was found to be located inthe 0.68-kb HaeII/HindIII subfragment (Fig. 3,track 7), a 1.04-kb HaeII/HincII subfragment

J. BACTERIOL.

RELAXATION COMPLEX NICK SITES 927

a -nic

FIG. 4. Physical map ofplasmid R6K. The locations of most endonuclease cleavage sites, the origins ofvegetative replication, a-ori and /3-ori, and the terminus of replication, ter, are taken from reference 30.According to this report, imprecision in origin localization is ±i to 2 kb. ft and cII designate, respectively, theHinfI and HincII endonuclease cleavage sites in the neighborhood of the relaxation nicks.

(Fig. 3, track 8), a 0.445-kb Hinfl fragment (seeHincII/HinfI digest, Fig. 3, track 9; HincII doesnot cleave within this HinfI fragment), and a0.125-kb HaeII/HinfI subfragment (Fig. 3, track10). These data position /3-nic to a 125-base pairHaeII/Hinfl subfragment of H-2, whose terminiare located 0.55 and 0.68 kb from the H-2/H-15junction (Fig. 4).From the published R6K replication origin

mapping data (11, 12, 25), which place a-ori nearthe center of HindIII fragment H-4, and /B-oriclose to or at the H-2/H-15 HindIII fragmentjunction, we conclude that the a-ori and a-nicsites, and the /3-ori and /3-nic sites, are probably

separated by 0.7- to 1.8-kb and 0.5-kb segmentsof the R6K genome, respectively. Thus, al-though there is some margin of error in theorigin mapping data, it seems likely that therelaxation nick sites and origins of replication ofplasmid R6K are physically distinct but closelyassociated loci.Mobilization frequency of pSC101 is re-

duced by inhibiting plasmid vegetative rep-lication. The physical proximity of vegetativeorigins and relaxation nick sites may reflect afunctional interaction between these two typesof loci and a role for vegetative origins in plasmidmobilization. We have therefore investigated the

VOL. 144, 1980

928 NORDHEIM, HASHIMOTO-GOTOH, AND TIMMIS

influence of genetic blocks that inhibit the rep-

lication of pSC101 on the frequency of plasmidmobilization. pSC101 has recently been shownto have an absolute requirement for the dnaAchromosome initiation function (20), which pre-

sumably acts at the level of initiation also inpSC101 plasmid replication. Moreover, pSC101mutants which are temperature-sensitive forreplication have been isolated (19), and theseare probably conditionally defective in initiationof plasmid replication. We have compared theR64drd-11-promoted mobilization of pSC101-derivative plasmids from dnaA+ and integra-tively suppressed dnaA(Ts) donor bacteriagrown under conditions that are either permis-sive or nonpermissive for plasmid vegetative rep-

lication and have found that inhibition ofpSC101 replication results in a two- to three-order-of-magnitude reduction in the mobiliza-tion proficiency of this plasmid (Table 1).

DISCUSSIONPlasmids are extrachromosomal elements that

have been found in most bacteria and some

higher organisms and that confer importantproperties upon their host cells. Their mainte-nance in bacterial populations is achievedthrough stable vertical transmission within a

clone (replication and partitioning), and occa-

sional horizontal transmission to new clones, byinfectious transfer or conjugation. Conjugationis the transfer of a single linear plasmid DNAstrand from a donor bacterium to a recipient,where it is subsequently converted to the normaldouble-stranded form (32, 33). The plasmid lociat which these two basic maintenance mecha-nisms are initiated are termed the origin of veg-etative replication, oriV, and the origin of con-

jugal transfer, oriT, respectively.Plasmid relaxation complexes are ubiquitous

forms of plasmid DNA consisting of supercoiledDNA-protein complexes which can be convertedin vitro to a relaxed form containing a strand-and site-specific nick. Because the relaxationnick site of ColEl is located near both oriV andits presumptive oriT, it is generally believed thatrelaxation complexes are involved in one of thetwo basic plasmid activities of replication andconjugal transfer. These conclusions assumethat (1) the in vitro single-strand cleavage reac-

tion mimics a physiological process, (2) covalentattachment of a relaxation protein to the 5' endof the nicked DNA strand facilitates recircular-ization of this strand and conserves the energyof phosphodiester bond cleavage for the recir-cularization event (16), and (3) relaxation nicksmust be located close to, or be identical with,vegetative or transfer origins.

Presumably, for relaxation complexes to be

TABLE 1. Inhibition of vegetative replication ofplasmidpSC101 greatly reduces its mobilization by

R64drd-11

Donorcefl Temp (.QMobfiiztion fre-Donor cells Temp (00) quency'

dnaA+pHSG101 30 1.4 x 105dnaA+pHSG101 43 2.5 x 104dnaA+pHSG501 30 3.3 x 105dnaA+pHSG501 43 1.3 x 102dnaA(Ts)pHSG101 30 5.0 x 103dnaA(Ts)pHSG101 43 1.2 x 10'aPlasmids pHSG101 (18) and pHSG501 (T. Hashi-

moto-Gotoh, unpublished data) are kanamycin-resist-ant derivatives of plasmids pSC101 and its tempera-ture-sensitive replication mutant pHSG1 (19), respec-tively. The E. coli K-12 dnaA+ and dnaA(Ts) donorstrains are Oml24(R64drd-11) (Hashimoto-Gotoh, un-published data) and EC8 (4), which is integrativelysuppressed with R64drd-11, whereas the recipientstrain is Om124 Nalr. The conjugative plasmid used tomobilize the pSC101 derivatives was R64drd-11. Over-night cultures of donor and recipient strains in anti-biotic medium no. 3 (Difco) grown at 290C were di-luted in fresh medium and incubated at 290C withgentle shaking for 3.5 h. Log-phase recipient and donorbacterial cultures were then mixed in the ratio 1:5, and50-pl portions of the mixtures were applied to Milliporefilters (HAWP 01300; diameter, 13 mm; pore size, 0.45,um) on antibiotic medium no. 3 agar plates prewarmedto 30 or 43°C. The plates were incubated further at 30or 43°C for 3.5 h, after which the filters were removedand agitated vigorously in 1 ml of cold antibioticmedium no. 3. Cell suspensions were immediately di-luted and plated on antibiotic medium no. 3 platescontaining nalidixic acid (20 ,ug/ml) and nalidixic acidplus kanamycin (50 ,ug/ml) to count total recipientsand transconjugants, respectively. The mobilizationfrequency is the number of transconjugants per 108recipient bacteria.

involved in vegetative replication, they mustintroduce nicks precisely at oriV, if involved ininitiation, or some nucleotides upstream of thissite, if involved in unwinding of the moleculeduring unidirectional plasmid replication. InColEl, the relaxation site is some 300 nucleo-tides downstream of oriV (5). For this reason,and because a functional relaxation complex isnot obligatory for ColEl replication (22, 39), itseems unlikely that the complex plays a role inthe normal vegetative replication of this plas-mid. Recently, Warren et al. showed that theColEl relaxation nick site is located on a 300-bpDNA fragment, which also carries a DNA se-quence (bom), that is essential for conjugaltransfer of this plasmid (39). Although neithersite was accurately localized in this study, War-ren et al. proposed that the ColEl nick site isoriT and that the relaxation complex plays anessential role in plasmid conjugal transfer. It isevident that precise mapping of the loci of relax-ation nicks, vegetative origins, and transfer

J. BACTERIOL.

VOL. 144, 1980

origins of several different plasmids exhibitingdistinct replication and transfer characteristicswill be important for the elucidation of the bio-logical role of relaxation complexes.

In this communication we report the locali-zation of the relaxation sites of plasmids pSC101,RSF1010, and R6K. pSC101 (9) is a small (9-kb), nonconjugative plasmid that can be mobi-lized by a coexisting conjugative plasmid. It hasa low copy number (six copies per chromosome),replicates unidirectionally (7), and has a veryrestricted host range. RSF1010 (14) is also asmall (8.2-kb), nonconjugative, mobilizable plas-mid but has a high copy number (13 to 25 copiesper chromosome), replicates both uni- and bidi-rectionally (14), and can be transmitted to, andmaintained in, a wide range of bacterial hosts(3). R6K (25) is an unusual, large (38-kb), con-jugative plasmid that has a high copy number(20 copies per chromosome) and that can initiatevegetative replication at either of two origins ofreplication, a-ori and 3-ori (11). Gene cloningexperiments have recently identified a third or-igin of replication in R6K (R. Kolter and D. R.Helinski, personal communication).We have located a single relaxation nick site

on the pSC101 genome, within HincII fragment2, close to the H-1/H-2 fragment junction (Fig.2). Recent cloning experiments suggest that thepSC101 origin of vegetative replication is alsolocated very close to this junction but within theH-1 fragment (31). Thus, the pSC101 relaxationnick site and replication origin are physicallydistinct but adjacent loci separated by a 0.45- to1.1-kb segment of the plasmid genome. The sin-gle nick site of RSF1010 was found to be locatedwithin a 850-bp HaeII fragment on the plasmidgenome (Fig. 2). This site is also very close tothe previously mapped origin of vegetative rep-lication (14). Surprisingly, when we examinedplasmid R6K we were able to identify two dis-tinct relaxation nick sites, a-nic and fl-nic, ontwo noncontiguous HindIII fragments, H-4 andH-2, which also carry the two R6K origins ofvegetative replication, a-ori and /3-ori, respec-tively. These results and origin mapping exper-iments of others (12, 25) indicate that a-nic and/3-nic are located close to, but probably distinctfrom, a-ori and /3-ori, respectively. Thus, al-though we have shown that four relaxation nicksites, two in mobilizable plasmids and two in aconjugative plasmid, are located close to originsof vegetative replication, one is certainly andtwo are probably physically separable from theorigins. These results provide strong support forthe hypothesis that relaxation complexes do notplay an essential role in the initiation of vege-tative replication but that they function in con-jugal DNA transfer (17, 22, 39). Recently, het-

RELAXATION COMPLEX NICK SITES 929

eroduplex and complementation analysis ofR6Kplasmid deletion mutants identified two series oftransfer functions located near the two EcoRIsites on the R6K genome (see Fig, 4), and atransfer origin at the position of a-nic (A. I.Stepanov, personal communication). These ex-periments, although preliminary, are consistentwith ours and suggest that R6K contains twotransfer origins. The reason for this is still ob-scure but may well reflect the evolution ofbackup systems for gene transfer among bacte-ria, or indicate that R6K is a double replicon, orboth.The question that now must be addressed is:

is there a functional basis for the striking phys-ical proximity of plasmid relaxation nick sitesand origins of replication? Warren et al. (39)have proposed a model for ColEl mobilizationthat is analogous to one proposed for the syn-thesis of viral DNA strands of bacteriophage4X174 (15). They suggested that DNA transferis initiated by specific relaxation of the ColElmolecule at its nick site, followed by orientedtransfer of the nicked strand to a recipient bac-terium and its concomitant replacement by roll-ing-circle-type replication. A second nickingevent at the ColEl nick site releases the trans-ferred strand from the rolling circle as a unitlength molecule, which recircularizes in the re-cipient. This single-stranded circular DNA mol-ecule is converted to the double-stranded fornby the synthesis of a complementary strand(CS). The synthesis of CS DNA in the recipientrequires priming by a short segment of RNA orDNA. Experiments with the conjugative Ia plas-mid R144 have shown that this primihg is notdependent upon the activity of either of theusual priming enzymes, RNA polymerase andthe dnaG primase. R144 appears to solve thepriming problem by means of a new plasmid-encoded primase that is synthesized in donorbacteria (6, 27), but a similar product encodedby other types of conjugative or mobilizableplasmids has not thus far been detected.

In Fig. 5 we suggest a modification of themodel of Warren et al. in which we speculatethat CS synthesis in the conjugative plasmidR6K and nonconjugative, mobilizable plasmidsis primed by DNA synthesis events initiated attheir vegetative origins. In this scheme, a donor-specific DNA synthesis reaction, initiated atoriVand terminated near the oriT/nic site, pro-vides a primer that is base paired to the 5' endof the DNA strand which is transferred to recip-ient bacteria. After recircularization of the trans-ferred strand, complementary strand synthesisproceeds by extension of the 3' end of the co-transferred CS primer.This proposal for CS primer synthesis, which

UE

Tin

FIG. 5. Suggested roles ofrelaxation nick sites andorigins ofvegetative replication in the conjugal trans-fer of plasmid DNA. In some or all of the cellularplasmid relaxation complexes, initiation events atthe origin of vegetative replication ORI V(V), resultin the synthesis of a short DNA sequence, comple-mentary strand (CS) primer, on the DNA strand thatis nicked during relaxation. Synthesis of CS primerproceeds in the direction of, and up to, the location ofthe relaxation proteins (RP), i.e., the transfer origin,ORI T(T). When donor and recipient bacteria forma mating couple (B), a mating signal in the donor cellresults in activation of the relaxation proteins, nick-ing of the template strand, and covalent linkage ofarelaxation protein to the 5' terminus of the nickedstrand (C). The 5' terminus ofthe nicked strand, plusassociated relaxation protein(s) and CS primer, arethen transported through the two cell envelopes (M)into the recipient cell (D). After the oriented transferof the remainder of the strand (E), its 3' and 5' endsare rejoined by the relaxationprotein(s) (F). Synthesisof a DNA strand complementary to the transferredstrand then occurs by extension of the CSprimer (G).In this scheme we have represented DNA transfer asa simple peeling off of the nicked strand from itscomplement, rather than involving a rolling-circle-type replicating intermediate which requires a secondnicking event to generate unit length transferredstrands. Recent findings with the F sex plasmid sug-gest that DNA transfer and resynthesis of the trans-ferred strand are not obligatorily coupled reactions(24).

930

ARECIPIENT

B

C

V..,

E

F

G

,M

DONOR

VOL. 144, 1980

assumes a fimctional relationship betweenorigins of transfer and replication, makes spe-cific, testable predictions. First, it should bepossible to isolate a short, double-stranded (Si-resistant) DNA segment representing the plas-mid oriV-oriT region from recipient cells thathave been mated with donors under conditionswhich prohibit DNA and RNA synthesis in therecipient. Secondly, because initiation events atthe vegetative origin are postulated to be essen-tial for conjugal transfer, the specific inhibitionof the initiation of plasmid replication shouldreduce or abolish DNA transfer, especially sec-ond-round transfer. This was found to be thecase for the mobilization of pSC101 by the con-jugative plasmid R64drd-11 (Table 1). This re-sult, and the finding that conjugal transfer of theColE1 plasmid is greatly reduced if donor cellsare deficient in DNA polymerase I (23), an es-sential enzyme in the initial stage of ColEl veg-etative replication (34), is consistent with thesecond prediction of the priming mechanism wehave proposed. Experiments are currently inprogress to test the first prediction.

Finally it should be emphasized that not allplasmids need necessarily utilize the CS primingmechanism outlined above. In particular, largeconjugative plasmids, such as F, R6-5 and RK2,whose transfer and replication origins are widelyseparated on the plasmid genomes (1, 10, 17, 35,37) may utilize an entirely different mechanism,perhaps involving an R144-type primase (6, 27).Altematively, vegetative origins which havethus far not been identified may be located closeto the transfer origins (38) and provide CSprimers.

ACKNOWLEDGMENTSWe thank W. L. Staudenbauer and P. M. Slocombe for

advice and stimulating discussions and W. L. Staudenbauerand C. Franklin for critically reading the manuscript.

LITERATURE CITED1. Achtman, M., B. Kusecek, and K. N. Timmis. 1978.

Tra cistrons and proteins encoded by the Escherichiacoli antibiotic resistance plasmid R6-5. Mol. Gen. Genet.163:169-179.

2. Andr6s, I., P. M. Slocombe, F. Cabello, J. K. Timmis,R. Lurz, H. J. Burkardt, and K. N. Timmis. 1979.Plasmid replication functions. II. Cloning analysis ofthe RepA replication region of antibiotic resistanceplasmid R6-5. Mol. Gen. Genet. 168:1-25.

3. Bagdasarian, M., M. M. Bagdasarian, S. Coleman,and K. N. Timmis. 1979. New vector plasmids for genecloning in Pseudomonas, p. 411-422. In K. N. Tinmisand A. Piihler (ed.), Plasmids of medical, environmentaland commercial importance. Elsevier/North Holland,Amsterdam.

4. Bagdasarian, M. M., M. Izakowska, and M. Bagda-sarian. 1977. Suppression of the DnaA phenotype bymutations in the rpoB cistron of ribonucleic acid polym-erase in Sabnonella typhinurium and Escherichia coli.J. Bacteriol. 130:577-582.

RELAXATION COMPLEX NICK SITES 931

5. Bastia, D. 1978. Determination of restriction sites andthe nucleotide sequence surrounding the relaxation siteof ColEl. J. Mol. Biol. 124:601-639.

6. Boulnois, G. J., and B. M. Wilkins. 1979. A novelpriming system for conjugal synthesis of an incIa plas-mid in recipients. Mol. Gen. Genet. 175:275-279.

7. Cabello, F., K. N. Timmis, and S. N. Cohen. 1976.Replication control in a composite plasmid constructedby in vitro linkage of two distinct replicons. Nature(London) 259:285-290.

8. Clewell, D. B., and D. R. Helinski. 1969. Supercoiledcircular DNA-protein complex in E. coli: purificationand induced conversion to an open circular DNA form.Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166.

9. Cohen, S. N., and A. C. Y. Chang. 1977. Revised inter-pretation of the origin of the pSC101 plasmid. J. Bac-teriol. 132:734-737.

10. Crisona, N. J., and A. J. Clark. 1977. Increase in con-jugational transmission frequency of nonconjugativeplasmids. Science 196:186-187.

11. Crosa, J. H., L. K. Luttrop, and S. Falkow. 1976. Modeof replication of the conjugative R-plasmid RSF1040 inEscherichia coli. J. Bacteriol. 126:454-466.

12. Crosa, J. H., L. K. Luttrop, and S. Falkow. 1978.Molecular cloning of replication and incompatibilityregions from the R-plasmid R6K. J. Mol. Biol. 124:443-468.

13. Dougan, G., and D. J. Sherratt. 1977. The transposonTnl as a probe for studying ColEl structure and func-tion. Mol. Gen. Genet. 151:151-160.

14. de Graaff, J., J. H. Crosa, F. Heffron, and S. Falkow.1978. Replication of the nonconjugative plasmidRSF1010 in Escherichia coli K-12. J. Bacteriol. 134:1117-1122.

15. Eisenberg, S., J. Griffith, and A. Kornberg. 1977.0X174 cistron A protein is a multifunctional enzyme inDNA replication. Proc. Natl. Acad. Sci. U.S.A. 74:3198-3202.

16. Guiney, D. G., and D. R. Helinski. 1975. Relaxationcomplexes of plasmid DNA and protein. J. Biol. Chem.250:8796-8803.

17. Guiney, D. G., and D. R. Helinski. 1979. The DNA-protein relaxation complex of the plasmid RK2: locationof the site-specific nick in the region of the proposedorigin of transfer. Mol. Gen. Genet. 176:183-189.

18. Hashimoto-Gotoh, T., and J. Inselburg. 1979. ColElplasmid incompatibility: localization and analysis ofmutations affecting incompatibility. J. Bacteriol. 139:608-619.

19. Hashimoto-Gotoh, T., and M. Sekiguchi. 1977. Muta-tions of temperature sensitivity in R plasmid pSC101.J. Bacteriol. 131:405-412.

20. Hasunuma, K., and M. Sekiguchi. 1977. Replication ofplasmid pSC101 in Escherichia coli K-12: requirementof dnaA function. Mol. Gen. Genet. 154:225-230.

21. Helinski, D. R. 1976. Plasmid DNA replication. Fed.Proc. 35:2026-2030.

22. Inselburg, J. 1977. Studies of colicin El plasmid func-tions by analysis of deletions and TnA insertions of theplasmid. J. Bacteriol. 132:332-340.

23. Kingsbury, D. T., and D. R. Helinski. 1973. Tempera-ture-sensitive mutants for the replication of plasmids inEscherichia coli: requirement for deoxyribonucleic acidpolymerase I in the replication of plasmid ColEl. J.Bacteriol. 114:1116-1124.

24. Kingsman, A., and N. Willetts. 1978. The requirementsfor conjugal DNA synthesis in donor strain during Flactransfer. J. Mol. Biol. 122:287-300.

25. Kolter, R., and D. R. Helinski. 1978. Construction ofplasmid R6K derivatives in vitro: characterization ofthe R6K replication region. Plasmid 1:571-580.

26. Kupersztoch-Portnoy, Y. M., M. A. Lovett, and D. R.Helinski. 1974. Strand and site specificity of the relax-

932 NORDHEIM, HASHIMOTO-GOTOH, AND TIMMIS

ation event for the relaxation complex of the antibioticresistance plasmid R6K. Biochemistry 13:5484-5490.

27. Lanka, E., E. Scherzinger, E. Gunther, and H. Schus-ter. 1979. A DNA primase specified by I-like plasmids.Proc. Natl. Acad. Sci. U.S.A. 76:3632-3636.

28. Lovett, M. A., D. G. Guiney, and D. R. Helinski. 1974.Relaxation complexes of plasmids ColEl and ColE2:unique site of the nick in the open circular DNA of therelaxed complexes. Proc. Natl. Acad. Sci. U.S.A. 71:3854-3857.

29. Lovett, M. A., and D. R. Helinski. 1975. Relaxationcomplexes of plasmid DNA and protein. II. Character-ization of the protein assOciated with the unrelaxed andrelaxed complexes of plasmid ColEl. J. Biol. Chem.250:8790-8795.

30. Lovett, M. A., R. B. Sparks, and D. R. Helinski. 1975.Bidirectional replication of plasmid R6K DNA in Esch-erichia coli; correspondence between origin of replica-tion and position of single-strand break in relaxed com-

plex. Proc. Natl. Acad. Sci. U.S.A. 72:2905-2909.31. Meacock, P. A., and S. N. Cohen. 1979. Genetic analysis

of the interrelationship between plasmid replication andincompatibility. Mol. Gen. Genet. 174:135-147.

32. Ohki, M., and J. L. Tomizawa. 1968. Asymmetric trans-fer ofDNA strands in bacterial conjugation. Cold SpringHarbor Symp. Quant. Biol. 33:651-657.

33. Rupp, W. D., and G. Ihler. 1968. Strand selection duringbacterial mating. Cold Spring Harbor Symp. Quant.Biol. 33:647-650.

34. Staudenbauer, W. L 1976. Replication of small plasmidsin extracts of Escherichia coli: Requirement for bothDNA polymerases I and III. Mol. Gen. Genet. 149:151-158.

35. Synenki, R. M., A. Nordheim, and K. N. Timmis. 1979.Plasmid replication functions. III. Origin and directionof replication of a "mini" plasmid derived from Rb-5.Mol. Gen. Genet. 168:27-36.

36. Tait, R. C., and HI W. Boyer. 1978. On the nature oftetracycline resistance controlled by the plasmidpSC101. Cell 13:73-81.

37. Timmis, K. N., F. Cabello, and S. N. Cohen. 1975.Cloning, isolation, and characterization of replicationregions of complex plasmid genomes. Proc. Natl. Acad.Sci. U.S.A. 72:2242-2246.

38. Timmis, K. N., F. Cabello, and S. N. Cohen. 1978.Cloning and characterization of EcoRI and HindIIIrestriction endonuclease-generated fragments of anti-biotic resistance plasmids R6-5 and R6. Mol. Gen. Ge-net. 162:121-137.

39. Warren, G. J., A. J. Twigg, and D. J. Sherratt. 1978.ColEl plasmid mobility and relaxation complex. Nature(London) 274:259-261.

J. BACTERIOL.