Vol. 147, No. 3JOURNAL OF BACTERIOLOGY, Sept. 1981, p. 736-7430021-9193/81/090736-08$02.00/0

Complementation of Replication-Deficient DeletionDerivatives of Plasmid Mini-FJURGEN EBBERS AND RUDOLF EICHENLAUB

Ruhr- Universitat Bochum, Lehrstuhl fur Biologie der Mikroorganismen, 4630 Bochum 1, Federal Republicof Germany

Received 16 December 1980/Accepted 3 June 1981

Deleted mini-F plasmids with defects in replication were constructed and testedto see whether they could be rescued through complementation by a helperplasmid. This allowed us to identify two genetic loci determining trans-actingfunctions required for stable maintenance of plasmid mini-F, one encoded by thePstI fragment from 45.7 to 47.3 F-coordinates (F) and the other most probablylocated in the region from 43.1 to 43.8 F. The smallest mini-F plasmid that couldbe established through complementation consists of the PstI fragment 44.0 to45.7 F, encoding origin II and the incB locus.

The replication of a plasmid element in abacterium is a process involving numerous host-specified /functions. This is demonstrated by thefact that a number of mutations in dna genes ofEscherichia coli also affect plasmid replication(4, 12, 21, 29, 32). Assuming that all enzymaticand regulatory functions necessary for replica-tion are supplied by the host cell, the minimalrequirement that a plasmid has to meet in orderto exist as a replicon is the presence of an originof replication. This seems to be the case forplasmid ColEl in which replication has beenshown to be independent of any plasmid-en-coded protein (6, 18). However, replication ofseveral other plasmids depends on plasmid-spec-ified proteins, since conditional plasmid mutantsaffecting replication have been reported (5, 8,10, 14, 15). Recently such a protein, the r proteinof R6K, has been characterized by in vitro rep-lication and complementation studies (17, 23,24).There is evidence that the replication of plas-

mid mini-F also involves plasmid-encoded pro-teins (8, 10, 20, 26). Experiments to further sub-stantiate these observations are described in thepresent communication. We examined whetherplasmid mini-F, deleted for DNA segments func-tioning in replication, can be maintainedthrough complementation by a helper plasmidwhich supplies the missing functions. Thismethod may allow determination of how manysuch functions exist and location of their positionon the mini-F genome. Provided that all proteinsrequired for replication are available, the behav-ior of defective plasmids may also indicate theregions which are important for maintenance ofplasmid mini-F other than the origin.

MATERIALS AND METHODSBacterial strains and plasmids. E. coli C600 thr-

1 leu-6 thi-1 supE44 lacYl tonA21 was as described(1). E. coli C600 AtrpE5 recA (syn. MV12) was ob-tained from D. R. Helinski (6).

Plasmids used were pML31 (mini-F Km; 25);pDF41, consisting of mini-F joined to a DNA fragmentcarrying the trpED genes (19); and pRE13, consistingof RSF2124 (28) in which the trpED fragment (16)was cloned into the EcoRI site. Plasmids pJE421 (34)and pHW30 (34) are deleted derivatives of mini-Finserted into the EcoRI site of cloning vector pBR322(2). pJE421 lacks the KpnI fragment (43.8 to 46.8 F-coordinates [F]), and pHW30 carries a PstI-generateddeletion from 43.6 to 47.3 F. pJE181 consists ofpBR322 with a 4.6-megadalton fragment carrying thetrpED genes inserted into the PstI site.Media. Bacterial cultures were grown in L-broth or

M9 medium supplemented with 0.4% Casamino Acidsas described previously (8). Selection for mini-F plas-mids determining kanamycin (Km) resistance was onagar plates or liquid medium containing 50 ,ug of kan-amycin per ml.Enzymes. Restriction endonucleases EcoRI and

BamHI were obtained from Boehringer (Mannheim,Germany), KpnI was purchased from New EnglandBiolabs (Beverly, Mass.), and the recBC enzyme (13)was a gift of A. Prell. PstI was prepared according toSmith et al. (27). Phage T4 polynucleotide ligase wasprepared from E. coli 1100(XNM983) kindly providedby K. Murry.Plasmid DNA isolation. Plasmid DNA was iso-

lated by equilibrium centrifugation in dye-cesium chlo-ride density gradients (8). DNA fragments generatedby digestion with restriction endonucleases were pur-ified either by sedimentation in neutral 15 to 20%sucrose gradients or by elution from 1% agarose gelsafter electrophoretical separation (33).

Construction of plasmid derivatives. Restric-tion endonuclease-generated DNA fragments were in-cubated with phage T4 ligase under conditions as

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COMPLEMENTATION OF DEFECTIVE MINI-F's 737

described by Dugaiczyk et al. (7). The ligation buffercontained 66 mM Tris-hydrochloride (pH 7.5), 33 mMNaCl, 10 mM MgCl2, 10 mM dithiothreitol, and 1 mMATP. Incubation was at 15°C overnight and was ter-minated by both heat treatment (10 min, 65°C) andthe addition of EDTA (final concentration, 20 mM).The DNA was then used for transformation of E. coliaccording to the procedure of Cohen et al. (3), exceptthat the CaCl2 concentration was 100 mM and theheat shock was extended to 3 min at 42°C. Aftertransformation cells were plated on supplemented M9agar plates and incubated for 48 h at 37°C. The EcoRItrpED fragment (16), which has a single PstI recogni-tion site, was used as selective marker for the construc-tion of mini-F derivatives. This fragment can be con-

verted into aDNA segment with PstI-specific cohesiveends by circularization using the EcoRI ends, followedby digestion with PstI. This DNA was then insertedinto the PstI site of plasmid vector pBR322. Thehybrid plasmid, designated pJE181, was used for thepreparative isolation of the PstI trpED fragment.

Test for plasmid segregation. E. coli harboringmini-F derivatives defective in replication togetherwith the wild-type "helper" plasmid pML31, pJE421,or pHW30 was grown in supplemented M9 mediumcontaining kanamycin or penicillin, which selects forboth plasmids. At a cell titer of 2 x 108 cells per ml,cells were pelleted by centrifugation, suspended inprewarmed L-broth medium, and grown for 7 h at37°C with repeated dilutions to keep the culture in theexponential growth phase. Samples were taken fromthe cultures at certain time intervals and plated on

supplemented M9, L-broth, L-broth-kanamycin(pML31) and L-broth-penicillin (pJE421, pHW30)agar plates. After incubation of the plates at 37°C forabout 48 h, the frequencies of Trp', KMr, and Penrclones were calculated.

RESULTS

Deletion of PstI fragment B (45.7 to 47.3F). How far can a plasmid be reduced in sizewithout affecting autonomous replication? Ef-forts to identify this minimal region in plasmidmini-F were made by several groups (11, 20, 26,34). Many of the mini-F derivatives describedare not stably maintained. When comparing theproperties and molecular composition of mini-Fplasmids, it appears that stable maintenancerequires (i) the PstI fragment from 44.0 to 45.7F (A fragment), encoding origin II and an incom-patibility locus termed incB (19, 22); (ii) the PstIfragment from 45.7 to 47.3 F (B fragment), withthe second incompatibility locus ineC (19, 22);and (iii) an extension on the left side, possiblyup to the BamHI site at 43.1 F.

In the first experiment we asked whether de-letion of the B fragment would cause a defect inmaintenance. If so, would the defect be sup-pressed through complementation by an intactmini-F plasmid, or not? Complementation wouldthen indicate that the B fragment encodes a

trans-acting function required for mini-F main-tenance. To construct a mini-F plasmid with adeletion of the B fragment, plasmid pDF41(mini-F trp), which has four PstI recognitionsites, was subjected to a limited digestion byPstI. This was followed by incubation with T4polynucleotide ligase to recircularize the linearmolecules. TheDNA was then used to transformE. coli MV12 carrying the mini-F Kmr plasmidpML31. In addition the strain is recA to preventrecombination between plasmids. After transfor-mation the cells were plated on supplementedM9 agar which selects only for the Trp marker.Due to the strong incompatibility between twomini-F plasmids it was expected that Trp+ cellsharboring intact pDF41 would lose pML31 bysegregation, rendering the cells Km8. However,cells containing derivatives ofpDF41, dependingon complementation by plasmid pML31, shouldalways carry the markers of both plasmids (Trp+and Km'), even if selection is primarily for Trp+only.A large number of Trp+ KMr clones were

obtained. Plasmid DNA was prepared fromthese clones and analyzed by agarose gel electro-phoresis. Deletion of the 1.6-kb B fragment inpDF41 should result in a plasmid of 11.2 kb witha single KpnI site at 43.8 F. Cleavage of bulkplasmid DNA from these clones by KpnI re-sulted in a band pattern as shown in Fig. 1, track3. The uppermost band of 11.2 kb represents thenew plasmid, designated pJE1001, and the bandsof 5.7, 4.35, 3, and 2.7 kb correspond to the fourKpnI fragments of pML31 seen in Fig. 1, track2. The identity of pJE1001 was further examinedby KpnI-EcoRI double digest of the bulk plas-mid DNA from one of the clones. pJE1001 wascleaved into two fragments; these were readilyobserved beside the pML31-specific bands (com-pare Fig. 1, tracks 4 and 5). The upper band(track 5) of 7.3 kb represents a composite frag-ment comprising the trpED genes and 800 basepairs of mini-F (40.3 to 40.4 F and 43.1 to 43.8F). The 3.9-kb fragment (third band from thetop) is composed of the KpnI-EcoRI fragment43.8 to 49.3 F, in which the B fragment is deleted.To demonstrate that maintenance of plasmid

pJE1001 requires the presence of a helper plas-mid, the following experiments were performed.E. coli MV12 and E. coli MV12(pML31) weretransformed with a plasmid DNA preparationfrom a Trp+ KMr clone (containing pJE1001 andpML31). In the first transformation with exclu-sive selection for Trp+ (which is the marker ofpJE1001), we obtained only 1 Trp+ clone (per,ug of plasmid DNA and 2 x 108 cells). This clonewas also Kmr, and analysis of plasmid DNAproved that the cells contained both plasmids

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FIG. 1. Agarose gel electrophoresis ofmini-Fplas-mids andpJE1001. Afterpreparation, plasmidDNAswere digested with restriction endonuclease KpnI.After heat inactivation of KpnI (5 min, 650C) therestriction assays were divided, and one half wasfurther digested with restriction endonuclease EcoRI.Reactions were stopped with EDTA (final concentra-tion 20 mM) and incubation for 5 min at 65°C. Sam-ples were loaded onto a vertical agarose slab gel (1%agarose in TBE buffer, consisting of 90 mM Tris, 90mM boric acid, and 2.5mM EDTA, pH 8.3). Electro-phoresis was performed at 120 V, 60 mA for 2 h.Track 1: Phage A DNA-EcoRI (molecular weightmarker); track 2: pML31-KpnI; track 3: pML31,pJE1001-KpnI; track 4: pML31-KpnI and EcoRI;track 5: pML31, pJE1IO1-KpnI and EcoRI; track 6:pDF41-KpnI and EcoRI.

pJE1001 and pML31. The low frequency atwhich transformants containing pJE1001 wereobtained is perhaps best explained by the factthat both plasmids need to be transformed si-multaneously. In contrast, when E. coli MV12harboring pML31 was transformed with thesame plasmid DNA preparation as above, 119Trp+ clones (per jig of plasmid DNA and 2 x 10icells) were obtained which were also Kmr. Thisshows that pJE1001 can only be established ina cell in the presence of the helper plasmid.The generally low transfornation frequencies

in these experiments are not due to low compe-tence of the recipient cells. This was confirmedby control transformations with plasmidpBR322 DNA. Rather, we assume that lowtransformation frequencies result from incom-patibility between the mini-F plasmids, whichmay be especially effective during transforma-tion.

In cells carrying pJE1001 and pML31, segre-gation should be expected due to the incompat-ibility between the two plasmids, since pJE1001is mncB and pML31 is incB incC. To obtainsegregation kinetics for the two plasmids, E. coliMV12(pML31, pJE1001) was grown in the ab-sence of selective pressure in L-broth medium at370C for 14 generations. At time intervals sam-ples were plated (i) on L-broth agar plates toobtain the total cell titer, (ii) on L-broth-kana-mycin plates to determine the titer of cells carry-ing pML31, and (iii) on supplemented M9 agarplates to establish the number of cells harboringpJE1001. Cell titers were identical when deter-mined on L-broth and on L-broth-kanamycinplates, showing that plasmid pML31 did notsegregate. However, titers on the supplementedM9 plates were lower, indicating a loss ofpJE1001 in the population. Plotting of the frac-tion of Trp+ cells (log N/No) versus time ofincubation shows that upon release of selectivepressure about 15% of the cells containedpJE1001. This value dropped to 0.1% after 5.5 h(11 generations) (Fig. 2A). As expected from thedependence of pJE1001 on a helper plasmid, allTrp+ clones were also Kmr.Construction of a plasmid reduced to the

A fragment. Provided that all necessary plas-mid-specified proteins are supplied by a helperplasmid, it might be possible to construct a mini-F plasmid consisting only of the A fragmentcarrying the origin II and the incB locus (11, 20,22). Such a plasmid was constructed in vitro byjoining purified PstI fragment A to the PstI-generated cohesive ends of another purified PstIfragment carrying a selective marker, the trpEDgenes obtained from plasmid pJE181. E. coliMV12(pML31) was transformed with this DNA;cells were plated on supplemented M9 agar toselect for the presence of the new plasmid con-sisting of the 1.7-kb A fragment and the 6.9-kbtip fragment. After incubation of the plates for2 days at 370C, five Trp+ clones were obtained.These clones were also Kmr. To demonstratethe presence of pML31 plus the new mini-Fderivative, plasmid DNA was prepared fromTrp+ Kmr clones, digested by PstI, and analyzedby agarose gel electrophoresis. Four bands wereobtained (Fig. 3, track 3). The band of 6.9 kb,which corresponds in size to the tip fragmentand which is not present in PstI digests ofpML31 (compare Fig. 3, tracks 2 and 3), indi-cates that the cells contain another plasmid be-sides pML31. The A fragment of the tentativeplasmid cannot be identified as an individualband since it comigrates with the A fragmentgenerated from pML31.However, the presence of the tentative plas-

mid can be demonstrated unambiguously by the

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INCUBATION TIME ( hours )

0zz

co

0.

I-

1-

2 6 10 14 18

INCUBATION TIME ( hours)FIG. 2. Segregation kinetics of mini-F derivatives

pJE1(X)1, pJE2001, and pJE3001. (A) Cultures of E.coli MV12(pML31, pJE1001) and MV12(pML31,pJE2001) were grown at 37°C in supplemented M9medium containing kanamycin (50 pg/ml) to a densityof2 x 108 cellsper ml. Bacteria were transferred intofresh, prewarmed L-broth medium and further incu-bated at 37°C with aeration. At the indicated timessamples were removed, diluted, and plated on sup-plemented M9, L-broth, and L-broth-kanamycinagar plates. After 2 days, plates were scored, and thesurvival (log N/No) of Trp+ cells was calculated.Titers on L-broth and L-broth-kanamycin plateswere identical, indicating that plasmid pML31 was

maintained in all the cells. Symbols: (0) pJE1001;(0) pJE2001. (B) A culture ofE. coli MV12(pJE3001)was grown in supplemented M9 medium and testedfor plasmid segregation as described in (A), exceptthat dilutions were plated on supplemented M9 andL-broth media only.

FIG. 3. Agarose ge electrophoresis ofmmii-Fpls-mids and pJE2(X)1 digested with restriction endonu-clease PstI. Ekectrophoresis was as described in Fig.2. Track 1: Phage A DNA-EcoRI; track 2: pML31-PstI; track 3: pML31, pJE2001-PstI; track 4:pML31,pJE2001-BamHI, recBC enzyme, then PstI (see Re-sults); track 5: pRE13-EcoRI.

following method. Plasmid DNA isolated froma Trp KM clone was digested with BamHIcleaving only pML31 (two sites). The digest wasthen incubated with an exonuclease selectivelydegrading linear duplex DNA. We used therecEC enzyme of E. coli. After extensive hy-drolysis the recEC enzyme was inactivated byheat shock, and the remaining DNA was cleavedby PstI and analyzed on an agarose gel. Therestriction pattern (Fig. 3, track 4) shows the1.7-kb A fragment and the 6.9-kb trp fragment.This was taken as evidence for the presence ofthe desired mini-F derivative, designatedpJE2ool.To confirm whether plasmid pJE2001 de-

pends on complementation by a wild-type helperplasmid, E. coli MV12 and MV12(pML31) weretransformned with purified pJE2001 DNA. NoTrrp+ clones were obtained by transformation ofthe plasmid-free E. coli strain, but transforma-tion of the strain carrying pML31 yielded 306Trp' clones (per t.&g of DNA and 2 x i05 cells).All Trp' clones were also KMr. This demon-strates that pJE2001 can only be established ina bacterium in the presence of another wild-typemini-F plasmid. In the absence of selective pres-sure, pJE2001 segregates with kinetics similar topJE1001 (Fig. 2A).To exclude the possibility that the A fragment

carried a mutation which causes defective main-

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tenance, plasmids identical to pJE2001 havebeen constructed. Three different plasmidDNAs were used as sources for the A fragment:pJE201 (34), pJE401 (34), and pSC138 (30). Inall pJE2001 plasmids obtained, maintenance wasdependent on functions supplied by a helperplasmid. This result is in agreement with pub-lished data which show that the A fragment isdefective in replication when cloned intopBR322; the hybrid plasmid cannot be estab-lished in a polA host (20, 34).

In a series of experiments, which will be de-scribed in detail elsewhere, we tried to reducepJE2001 even further in size. Deletion of theincB locus by removal of the BglII-PstI regionfrom 45.0 to 45.7 F or the XhoI-PstI region from44.8 to 45.7 F always caused defects in mainte-nance which could not be suppressed by com-plementation. Alternatively, integration ofDNAfragments into the XhoI and BglII sites at 44.8and 45.0 F, respectively, had the same effect.This is similar to an observation of Kahn et al.(20), who showed that integration of pBR322into the BglII site at 45.0 F leads to a defect inmini-F replication. Maintenance ofmini-F seemsto require an unimpaired region of 1.7 kb be-tween coordinates 44.0 and 45.7 F. Defects re-sulting from deletion of the incB locus or inser-tions at 45.0 or 44.8 F cannot be complementedby a helper plasmid.Joining of the A and B fragments. The

properties of plasmid pJE2001 document thatthe A fragment encoding origin II and the incBlocus can replicate in the presence of a helperplasmid and that one of the functions suppliedby the helper plasmid maps within the B frag-ment. Therefore, the question arises of whetherthe joining of fragments A and B might result inthe formation of a stable replicon without de-pendence on complementation. For the con-struction of such a plasmid, purified PstI frag-ments A and B were added to PstI trp fragmentand joined in the presence of T4 ligase. Theligated DNA was used to transform E. coliMV12, and cells were plated on supplementedM9 medium to select for Trp+ clones. A singleTrp+ clone was obtained, and analysis of theplasmid DNA confirmed that this clone in factcarried a plasmid which consisted of mini-F frag-ments A and B and the trp fragment (Fig. 4,track 3). The new mini-F plasmid was designatedpJE3001. Restriction analysis with BglII showedthat fragments A and B were joined in the sameorder as in the wild-type plasmid (data notshown).The segregation of pJE3001 in E. coli MV12

in the absence of selective pressure was tested(Fig. 2B). The plasmid segregated with about

FIG. 4. Agarose gel electrophoresis ofmini-Fplas-mids andpJE3001. Plasmid DNAs were cleaved withrestriction endonuclease PstI or EcoRI and furthertreated as described in Fig. 2. Track 1: Phage ADNA-EcoRI; track 2: pML13-PstI; track 3:pJE3001-PstI; track 4: pRE52-EcoRL

2% loss per generation. This indicates that theplasmid may still miss some function for stablemaintenance. Therefore, we examined whetherstability of pJE3001 could be improved throughcomplementation with helper plasmids. Plas-mids suitable for this purpose should carry dele-tions corresponding to the region of mini-F pres-ent in pJE3001. Plasmids pJE421 and pHW30,which are pBR322:mini-F hybrids deleted forthe KpnI fragment 43.8 to 46.8 F or the PstIfragment 43.6 to 47.3 F, respectively, meet thiscriterion (34). Both plasmids pJE421 andpHW30 are totally compatible with anothermini-F plasmid (34), and only pJE421 shares a500-base pair homology with pJE3001, whereaspHW30 in addition to PstI fragments A and Blacks the small PstI fragment from 43.6 to 44.0F (Fig. 5). When E. coli MV12(pJE421) andMV12(pHW30) were transformed with pJE3001,numerous Trp+ clones were obtained which phe-notypically expressed both plasmid markers.Presence of both plasmids was demonstrated inthe plasmid preparations. Without any selectivepressure a segregation of plasmid was not de-tected in E. coli MV12(pJE421, pJE3001). How-ever, in the case of MV12(pHW30, pJE3001),segregation of pJE3001 was observed at thesame rate as in MV12(pJE3001). This indicatesthat pJE3001 benefits from complementation bya function supplied through plasmid pJE421

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Pst-A

mini-F

pJE 1001

pJE 2001

pJE 3001

pJE 421

Pst-B

pHW 3 0 t -FIG. 5. Restriction map ofplasmid mini-F and its derivatives pJE1001, pJE2001, pJE3001, pJE421, and

pHW30. Recognition sites for relevant restriction endonucleases are indicated with their corresponding Fcoordinates. "RI" refers to EcoRI; "ori" refers to the origins of replication (9, 11); "inc" defines theincompatibility regions (22). The dotted lines represent the deleted parts ofmini-F derivatives.

which is not expressed by pHW30. It furthersuggests that besides the polypeptide encodedby the B fragment, a second protein is requiredfor stable maintenance of mini-F. Since it isknown which mini-F polypeptides are expressedin pJE421 and pHW30, the second polypeptideinvolved in mini-F maintenance can be identified(see Discussion).

DISCUSSIONThe complementation of a defective plasmid

by a second plasmid requires that the particularprotein is provided in amounts high enough tosatisfy the demands of the defective and thehelper plasmid and that the plasmid to be com-plemented has the structural composition nec-essary for interaction with this particular pro-tein. Plasmid-specified replication proteins havebeen correlated to negative or positive controlfunctions, and experimental evidence for bothfunctions has been provided. The 1r protein ofplasmid R6K seems to be a positive controlelement involved in the initiation of R6K repli-cation (17), whereas the properties ofconditionalreplication mutants of pNK102 suggest that inthis plasmid a negative control element is effec-tive (31). In the case of plasmid mini-F there isevidence that more than one protein may beinvolved in the replication control (10, 34). In

this communication we provide further data forthis notion.The deletion of the B fragment of mini-F

results in a defective plasmid which can be res-cued by complementation. This indicates that atrans-acting function maps within the B frag-ment. The B fragment carries a gene locus whichexpresses incompatibility; it is termed incC (19,22) and encodes a 44K protein (34). It hasbeen postulated that the 44K protein may act asa negative control element in replication (34).Absence of the repressor might result in anuncontrolled replication ("runaway replica-tion"), eventually killing the host cell. This kill-ing phenomenon may be the reason whypJE1001 cannot be established in a bacteriumwithout the presence of a wild-type plasmidproviding the negative control element. Evenunder complementation pJE1001 is still some-what unstable. Possible explanations are: (i) theparticular protein acting in complementation ispresent only in low amounts; (ii) deletion of theB fragment results in the loss of a second func-tion which cannot be complemented; or (iii) thewild-type plasmid dominates in the competitionfor functions required for replication and parti-tioning.The smallest mini-F derivative obtained in

our laboratory is plasmid pJE2001, consisting of

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the A fragment. It lacks essential functions andcan only be maintained in a cell in the presenceof a helper plasmid. Defectiveness of replicationof the A fragment is also observed when it iscloned into pBR322; the hybrid cannot be estab-lished in a poU mutant host (20, 34). Experi-ments designed to delete the incB locus inpJE2001 or to insert DNA sequences at 45.0 and44.8 F have shown that defects resulted consis-tently and that they could not be complemented(manuscript in preparation). This indicates thatthe A fragment contains sequences of structuralimportance such as recognition sites for protein-DNA interactions. Apparently such sites are notrestricted to the origin of replication, but extendinto the incB locus.

If the only requirements for replication andmaintenance ofplasmid mini-F were a functionalorigin II, the incB and incC locus, and the 44Kprotein, then joining of the A and B fragmentshould result in a stable replicon. We have con-structed such a plasmid (pJE3001) and haveshown that it can establish itself in a cell withouta helper plasmid. However, its segregation ki-netics showed that it is still slightly unstable.This behavior of pJE3001 can be attributed toa dependency on a second mini-F-encoded pro-tein. Indeed, totally stable maintenance was ob-tained through complementation by plasmidpJE421; this was not achieved by pHW30. Thequestion of which of the proteins expressed bypJE421 might be involved in the complementa-tion of pJE3001 can be answered by a compari-son of the polypeptides expressed by pJE421and pHW30. It was shown that pJE421 expressesthree mini-F-specific proteins of 36K, 34K, and25.3K, whereas pHW30 does not express the25.3K protein (34) (the promoter for this proteinis located within the PstI-KpnI fragment 43.6 to43.8 F, which is missing in pHW30). Therefore,we believe that the 25.3K protein mapping at43.1 to 43.8 F (31) accomplishes the complemen-tation of pJE3001 by pJE421. This assumptionagrees well with the recent suggestion that thisprotein acts as a positive control element forreplication starting at origin II (34).

In conclusion, our experiments suggest thatfunctions required for stable maintenance ofplasmid mini-F are confined to a 4.2-kb segmentbordered by 43.1 and 47.3 F. This region coversorigin II, the incB and incC locus, and genesencoding two polypeptides of 25.3K and 44K.

ACKNOWLEDGMENTSThis work was supported by a grant from the Deutsche

Forschungsgemeinschaft.We thank W. Wackernagel and R. Foelix for critical reading

of the manuscript, and Rita Worttmann for her expert tech-nical assistance.

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Betlach, H. L. Heyneker, and H. W. Boyer. 1977.Construction and characterization of new cloning vehi-cles. II. A multipurpose cloning system. Gene 2:95-113.

3. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972.Nonchromosomal antibiotic resistance in bacteria: ge-netic transformation of Escherichia coli by R-factorDNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114.

4. Collins, J., P. Williams, and D. R. Helinski. 1975.Plasmid DNA replication in Escherichia coli strainstemperature-sensitive for DNA replication. Mol. Gen.Genet. 136:273-289.

5. Cuzin, F., and F. Jacob. 1967. Mutations de l'episome Fd'Escherichia coli K12. II. Mutants a replication ther-mosensible. Ann. Inst. Pasteur Paris 112:398-418.

6. Donaghue, D. J., and P. A. Sharp. 1978. Replication ofcolicin El plasmid DNA in vivo requires no plasmid-encoded proteins. J. Bacteriol. 133:1287-1294.

7. Dugaiczyk, A., H. W. Boyer, and H. M. Goodman.1975. Ligation of EcoRI endonuclease-generated DNAfragments into linear and circular structures. J. Mol.Biol. 96:171-184.

8. Eichenlaub, R. 1979. Mutants of the mini-F plasmidpML31 thermosensitive in replication. J. Bacteriol. 138:559-566.

9. Eichenlaub, R., D. Figurski, and D. R. Helinski. 1977.Bidirectional replication from a unique origin in a mini-F plasmid. Proc. Natl. Acad. Sci. U.S.A. 74:1138-1141.

10. Eichenlaub, R., and H. Wehlmann. 1980. Amber mu-tants of plasmid mini-F defective in replication. Mol.Gen. Genet. 180:201-204.

11. Figurski, D., R. Kolter, R. Meyer, M. Kahn, R. Ei-chenlaub, and D. R. Helinski. 1978. Replication re-gions of plasmids ColEl, F, R6K, and RK2, p. 105-109.In D. Schlessinger (ed.), Microbiology-1978. AmericanSociety for Microbiology, Washington, D.C.

12. Goebel, W., and H. Schrempf. 1972. Replication ofplasmid DNA in temperature sensitive DNA replicationmutants of Escherichia coli. Biochim. Biophys. Acta262:3241.

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