Vol. 162, No. 2

Comparison of 10 IncP Plasmids: Homology in the Regions Involvedin Plasmid Replication

GARY K. CHIKAMI, DONALD G. GUINEY,* THOMAS J. SCHMIDHAUSER, AND DONALD R. HELINSKI

Department of Medicine, University of California, San Diego Medical Center, San Diego, California 92103, andDepartment of Biology, University of California, San Diego, La Jolla, California 92093

Received 17 December 1984/Accepted 22 February 1985

We have examined the DNA homology in the replication regions of 10 IncP plasmids independently isolatedfrom several different countries. Two regions of RK2, the best-studied plasmid of this group, are required forvegetative DNA replication: the origin of replication (oniV) and the trfA* region, which codes for a gene productnecessary for replication. Six of nine IncP plasmids studied were identical to RK2 in the oriV and trfA* regionsas shown by Southern hybridization. Three P plasmids, R751, R772, and R906, showed weaker homology withthe RK2 trfA*, region and hybridized to different-sized HaeII fragments than the other six plasmids. R751,R772, and R906 hybridized to the region of the RK2 replication origin which expresses P incompatibility butdiffered markedly from RK2 and the other six plasmids in the GC-rich region of the origin required forreplication. These data indicate that the P-group plasmids can be divided into two subgroups: IncPoa, whichincludes the RK2-like plasmids, and IncPI8 which includes the R751-like plasmids.

Plasmids of the P incompatibility group are able to transferamong and be maintained in most gram-negative bacteria.This broad-host-range property is important in both thespread of antibiotic resistance and the utility of these plas-mids as cloning vehicles in many gram-negative organisms.P-group plasmids have been found in a variety of bacterialhosts from many different geographic locations. Recentstudies have investigated the structural relationship of theplasmids of this incompatibility group. Ward and Grinsted(24) found sequence homology between RP1 and two otherIncP plasmids, R751 and R906. Similarly Azaryan et al. (1)have shown homology between RK2 and the IncP plasmidsR702, R751, and R906. Villarroel et al. (23), in an extensiveheteroduplex analysis, found large regions of sequence ho-mology between RK2 and the P plasmids R702, R839, R938,R995, R1033, and pUZ8. However, three plasmids, R751,R906, and R772, showed only partial or no homology withRK2 by heteroduplex analysis, indicating that they mayhave diverged early in evolution from the RK2-like plas-mids. This finding is supported by the results of homologystudies of the transfer origins (ori7) of the IncP plasmids.Using the cloned RK2 origin of conjugal transfer as a labeledprobe for hybridization studies, Yakobson and Guiney (25)have shown that all the IncP plasmids tested share homologyin the oriT region, but three plasmids (R772, R751, and R906)had a hybridization pattern different from that of RK2 andsix other IncP plasmids. These workers designated theRK2-like plasmids IncPa. and the non-RK2-like plasmidsIncPI3.To extend these observations, we have studied the rela-

tionship between 10 naturally occuring IncP plasmids byexamining homology in specific regions involved in vegeta-tive DNA replication. Two regions of RK2, a 700-base-pair(bp) HaeII fragment containing the origin of replication(oriV) and a 1.5-kilobase-pair region designated trfA* (fortrans-acting replication function), together encode stablereplication and maintenance functions in Escherichia coli (4,14, 17, 19, 20). Minimal RK2 derivatives containing trfA*and oriV also replicate the strains of Pseudomonas, Rh-

* Corresponding author.

izobium, Acinetobacter, Caulobacter, Azotobacter,Agrobacterium, and Rhodopseudomonas (manuscript inpreparation). To study whether these replication regionswere present in other P-group broad-host-range plasmids,the restriction fragments which contain oriV and trfA* fromRK2 were labeled and used as probes in Southern hybridi-zations with nine other P-group plasmids.

MATERIALS AND METHODS

Bacterial strains and plasmids. E. coli MV10 is a derivativeof strain C600 that is also AtrpE5 (9). E. coli DF4063 is MV10naiR. E. coli MC1029 is a recA strain (14). The P-groupplasmids used were obtained from D. Figurski and are de-scribed in Table 1. pTJS26 is an RK2 replicon which containsthe RK2 HaeII oriV fragment and the trfA* region (14).pGC3 was derived from pTJS26 by deletion of the tetracy-cline resistance fragment and insertion of the HaeII ampicil-lin resistance fragment from pUC9 (22). pRK233, obtainedfrom R. Meyer, is a derivative ofRK2 with a PstI deletion inthe ampicillin resistance gene.Media and reagents. All bacteria were grown in 1% tryptone

(Difco Laboratories, Detroit, Mich.)-0.5% yeast extract(Difco)-0.5% NaCl. To select for trimethoprim resistance,M9-Casamino Acids-glucose was used with 200 ,ug of tri-methoprim per ml. Other antibiotics were used at the follow-ing concentrations: ampicillin, 200 ,ug/ml; kanamycin sul-fate, 50 ,ug/ml; and tetracycline hydrochloride, 20 ,ug/ml.

Plasmid DNA and enzymes. Restriction endonucleases andT4 ligase were obtained from Bethesda Research Laborato-ries, Gaithersburg, Md. Reaction conditions were as speci-fied by the supplier. Plasmid DNA was purified by themethod of Currier and Nester (3).

Labeling of DNA fragments. Restriction maps of the oriVand trfA* regions of RK2 are shown in Fig. 1. From the oriVregion, three fragments were used as hybridization probes:the 700-bp HaeII fragment (purified from pCT17 [20]), a

393-bp HpaII fragment (purified from pTJS65 [T. Schmid-hauser, unpublished data]), and a 150-bp HaeIII fragment(purified from pDS120 [21]). From the trfA* region, theHaeII A* and D fragments (from pTJS27 [14]) were used asprobes. The restriction fragments were purified by poly-

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HOMOLOGY IN REPLICATION REGIONS OF IncP PLASMIDS

TABLE 1. Properties of the IncP plasmids used in this study

Plasmida (m e ResistancepatternS Original host Geographical Reference(megadaltons) Reitneptengenus originRK2 37 Am Km Tc Klebsiella England 13R751 30 Tp Klebsiella England 11R772 33 Km Proteus United States 3R906 35 Am Sm Su H Bordetella Japan 19pUZ8 33 Km Tc Pseudomonas Spain 8R702 46 Km Sm Su Tc Proteus United States 7R839 46 Am Km Sm Su Tc Serratia England 9R938 53 Am Cm Km Sm Su Tc Sp Serratia England 9R995 31 Km Sm Tc Proteus Hong Kong 6R1033 45 Am Cm Gm Km Sm Su Tc H Pseudomonas Spain 12a Plasmids, except for RK2, were obtained from David Figurski (5).b Abbreviations: Am, ampicillin; Km, kanamycin; Tc, tetracycline; Tp, trimethoprim; Sm, streptomycin; Su, sulfonamide; H, mercury; Cm, chloramphenicol;

Sp, spectinomycin, Gm, gentamicin.

acrylamide gel electrophoresis and labeled with [32P]dCTPby nick translation with DNA polymerase I according to themethod of Rigby et al. (13).

Southern hybridization. Purified DNA samples (1 to 2 ,ug)from each of the 10 IncP plasmids were digested to comple-tion with HaeII and run on a 1.4% agarose slab gel. Theseparated fragments were then transferred to nitrocellulose

ori V REGION

Haell FRAGMENTI

HincId

HpalI FRAGMENT

Hael FRAGMENT IMboI I L I

A/T G/C

Had

INCOMPATIBILITY

trf A* REGION

paper (BA85; Schleicher & Schuell, Inc., Keene, N.H.)according to the method of Southern (16). The labeled DNAfragments were heat denatured and hybridized to the filtersby 4x SSC (lx SSC is 0.15 M sodium chloride plus 0.015 Msodium citrate)-0.06% Ficoll 400-0.06% polyvinyl-pyrolidone-0.06% bovine serum albumin-0.2% sodium do-decyl sulfate-100 ,ug of calf thymus DNA per ml, with either40% (low-stringency) or 55% (high-stringency) formamide.Hybridization was done at 37°C for 14 to 16 h. Afterhybridization the filters were washed five times in thehybridization buffer at 37°C and twice in 2 x SSC at 22°C andautoradiographed.

Incompatibility testing. E. coli MC1029 containing a resi-dent plasmid was transformed with the test plasmid DNAand plated on medium selective for the incoming plasmid.One hundred of the transformants were screened for reten-tion of the resident plasmid. Transformants which containedboth plasmids were grown without antibiotic selection for 50generations. One hundred colonies were then screened forthe presence of the original resident plasmid or the trans-forming plasmid.

RESULTS

Homology in the trfA* region. The trfA* region of RK2consists of two HaeII fragments designated A* and D (Fig.1). The A* and D fragments were labeled and used as probes

Hael Hinfl Hind HadH

0 FRAGMENT_ _

TRANSCRIPTIONFIG. 1. (Top) Map of the HaeII fragment of RK2, which con-

tains the origin of replication (taken from reference 17). The threefragments used as probes for hybridization are indicated by thebrackets. The other HpaII and HaeIII sites are not shown. Thesmall arrows show the positions of the eight direct repeats which areinvolved with the expression of incompatibility. The large arrowindicates the direction of replication, but the exact location of theinitiation of DNA synthesis is not known. AJT and G/C refer to therelatively AT- and GC-rich regions identified by Stalker et al. (17).(Bottom) Map of the trfA* region of RK2 (taken from reference 15and 19 and T. J. Schmidhauser, unpublished data). A small fragmentof the end of TnS remains at the site of integration into the trfAregion in pCT87. The proposed direction of transcription of the trfAgene products is indicated (15) and spans the junction between theHaeII A* and D fragments used as probes in this study.

Low _ __

- -

HIGH

FIG. 2. Homology in the trfA* region of selected IncP plasmids.HaeII digests of the indicated plasmids were electrophoresed,transferred to nitrocellulose, and hybridized to the 32P-labeled A*fragment (A) or D fragment (B) from RK2. Each probe washybridized under low and high stringency, as defined in Materialsand Methods.

Hae[

Hael

Tn5DNA

Hinfl HinflI II

A* FRAGMENT

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658 CHIKAMI ET AL.

as outlined in Materials and Methods. Figure 2A shows thehybridization of the labeled A* fragment with representativeIncP plasmids under low and high stringency. The A* probehybridized with two separate HaeII fragments of RK2. Themore intense upper band is the native HaeII fragment fromthe A* region; the lower band represents another region inthe plasmid with weak homology that disappeared underhigh stringency. The pattern of hybridization for pUZ8,R702, R839, R938, R995, and R1033 was identical to thepattern seen for RK2, and the strong bands persisted at highstringency (data not shown for pUZ8, R702, and R938). Forthree plasmids, R751, R772, and R906, the probe hybridizedweakly to smaller HaeII fragments, and there was no

hybridization at high stringency.The remainder of the trfA* region is contained in the

HaeII D fragment shown in Fig. 1. Hybridizations using thelabeled D fragment as a probe are shown in Fig. 2B. As withthe A* fragmeht, pUZ8, R702, R839, R938, R995, and R1033have the same homologous band a RK2, while R751, R772,and R906 show a different pattern of hybridization. For R772and R906, one of the homologous bands disappeared at highstringency. These results indicate that the trfA* region hasbeen highly conserved in six of these IncP plasmids and thatfor the other three naturally occuring plasmids (R751, R772,and R906) there has been divergence in the sequence of thatregion, particularly in the region of the HaeII A* fragment(Fig. 2A, high-stringency conditions).Homology in the oriV region. Three RK2 oriV fragments

were used as probes: the 700-bp HaeII oriV fragment, a

393-bp HpaII oriV fragment, and a 150-bp HaeII fragment(Fig. 1). Both the HaeII and HpaII fragments are functionalorigins of replication and are incompatible with other RK2replicons. However, only the HaeII fragment, when clonedon a high-copy-number vector, is able to displace a residentRK2 plasmid (21). The HaeIII fragment lies outside theincompatibility region but is required for oriV function. Thehybridization of the 700-bp HaeII oriV fragment to the IncPplasmids is shown in Fig. 3A. Eight of the nine P plasmidsshowed hybridization to the same HaeII fragment as RK2(pUZ8, R702, and R839 [not shown] were identical to RK2),but the homologous R751 fragment was considerably largerin size (Fig. 3A). A very faint band appears in the R751 laneat the same position as the RK2 HaeII fragment, but thesignificance of this is not clear. The homology to the HaeIIoriV probe persisted at high stringency for all the plasmids.Similar results were obtained when the 393-bp HpaII oriV

A ,

B0

;.~~~~..I_ _

FIG. 3. Homology in the oriV region of selected IncP plasmids.HaeII digests of the indicated plasmids were electrophoresed,transferred to nitrocellulose, and hybridized to the 32P-labeledHaeII oriV fragment (A) or HaeIII oriV subfragment (B) of RK2.Each probe was hybridized under low and high stringency, as

defined in Materials and Methods.

TABLE 2. Incompatibility of selected P plasmids with pGC3

Incoming Resident Loss of

pncomin Reasid Selection residentplasmid plasmid plasmid"

R751 pGC3 Tp 75/100R772 pGC3 Km 31/100pUZ6 pGC3 Tc 3/100R995 pGC3 Tc 6/100pTJS26 pGC3 Tc 3/100pRK233 pGC3 Tc 3/100pRK35 pGC3 Km 0/100" See text for details of incompatibility testing. Transformants were

screened with penicillin for loss of the resident plasmid after transformation.See Table 1, footnote b, for abbreviations.

fragment was used as a probe, except that the minor faintband in the R751 lane was not present (data not shown).The incompatibility function of the RK2 oriV fragment is

contained in the region of the eight 19-bp direct repeats (Fig.1). These direct repeats might be expected to occur in allmembers of the P incompatibility group. To examine homol-ogy in the region of oriV which does not express incompat-ibility, a 150-bp GC-rich HaeIII fragment (Fig. 1) was usedas the labeled probe for the hybridization shown in Fig. 3B.Again, pUZ8, R702, R839, R938, R995, and R1033 showedstrong hybridization to the same HaeII fragment as RK2,even at high stringency. In contrast, there was relativelylittle hybridization, even under low-stringency conditions, toplasmids R751, R772, and R906. In these latter three plas-mids, the GC-rich sequence in oriV showed significantdivergence from the RK2 group of plasmids.

Incompatibility testing. Since the hybridization data indi-cated substantial differences between RK2 and certain otherP plasmids, four of the nine plasmids (R751, R772, pUZ8,and R995) were tested for the strength of their incompatibil-ity with a derivative of RK2 (Tables 2 and 3). This RK2derivative, pGC3, is composed of the HaeII oriV region, thetrfA* fragment, and an ampicillin resistance marker. Thepositive controls were pTJS26 (an RK2 derivative which hasthe HaeII oriV region, trfA*, and a tetracycline resistancemarker) and pRK233 (a nearly fulled-sized RK2 plasmidwith a PstI deletion in the ampicillin resistance gene). Thenegative control was pRK35, a derivative of R6K (IncX)which contains the EcoRI B fragment (the replication region)and a kanamycin resistance marker. Tables 2 and 3 showthat all of the P-group plasmids were incompatible withpGC3, although there was considerable variation in thestrength of incompatibility. Surprisingly, the IncPp plasmidsR751 and R772 displaced pGC3 at a higher frequency thanthe RK2 replicons pTJS26 and pRK233 or the other IncPaplasmids, pUZ8 and R995 (Table 2). When plasmid combi-nations were tested (Table 3), R772, pUZ8, R995, and

TABLE 3. Determination of the plasmid lost by incompatibility

combination Loss of pGC3a Loss of test plasmid

R772-pGC3 99/100 1/100pUZ8-pGC3 99/100 0/100R995-pGC3 99/100 0/100

pTJS26-pGC3 54/100 12/100pRK233-pGC3 80/100 0/100pRK35-pGC3 8/100 1/100

" See text for details of incompatibility testing. Cells were screened for theloss of plasmid after 50 generations of growth without antibiotic selection.

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HOMOLOGY IN REPLICATION REGIONS OF IncP PLASMIDS

FIG. 4. Agarose gel of pGC13 containing the oriV region ofR906. Lane A, HaeII digest of pGC13 isolated from E. coli CT3;

lane B, HaeII digest of pBR322 as molecular weight markers.

pRK233 all expressed similar incompatibility toward pGC3,which was preferentially lost in each case. These resultsconfirm that the incompatibility functions have been con-

served in the P plasmids and that the IncP,B plasmids R751and R772 express stronger incompatibility toward the mini-mal RK2 replicon, pGC3, than does RK2 itself.

Interaction between the R906 oriV and trf regions of RK2.To see if the oriV region from an IncPp plasmid wouldreplicate with the support of the trfA gene product fromRK2, the HaeII oriV fragment from R906 was cloned linkedto a nonreplicating resistance gene. A complete HaeII digestof R906 was ligated to the 1.9-kilobase-pair HaeII fragmentcontaining the ampicillin resistance gene from pUC9 (22).The ligated mixture was transformed into E. coli CT3, a

derivative of strain C2110 polA carrying the RK2 trfA andtrfB regions in the chromosome (21). All of the plasmidsextracted from the ampicillin-resistant transformants con-

tained a HaeII fragment which comigrated with the RK2HaeIl oriV fragment, as well as other HaeII fragments fromR906. The R906 HaeII oriV fragment was purified on a

polyacrylamide gel and then religated to the 1.9-kilobase-pair ampicillin resistance fragment. The resulting plasmid,pGC13, contained only the two HaeII fragments and repli-cated in CT3 but not in the parent C2110 strain (Fig. 4).These results show that although there has been sequence

divergence in the oriV and trfA regions of the P plasmids, theRK2 trfA gene product can drive replication of the R906 oriYregion.

DISCUSSION

The results of the hybridizations with the trfA* fragmentsand the GC-rich 150-bp HaeIII fragment of oriV divide the10 IncP plasmid studied here into two groups. One group,

consisted of pUZ8, R702, R839, R938, R995, and R1033,shows patterns of hybridization which are identical to thepattern shown by RK2, indicating that these replicationregions have been highly conserved. The other group, con-

sisting of plasmids R751, R772, and R906, gives a different

pattern of hybridization with these probes, and the hybridi-zation was much weaker, indicating that these P plasmidshave diverged from the group of P plasmids typified by RK2.

In the trfA region, the greatest sequence homology wasfound by using the HaeII D fragment as a probe (Fig. 2B),since hybridization bands persisted for all the plasmids evenat high stringency. Shingler and'Thomas (15) have proposedthat two overlapping proteins, with molecular weights of43,000 and 32,000, are made from a transcript reading fromleft to right as shown in Fig. 1, but only the 32-kilodaltonprotein is required for RK2 replication in E. coli. Theseproteins have different N-terminal ends in the HaeII A*fragment and overlap primarily in the D fragment. Thehybridization results indicate that the C-terminal portions ofthese coding sequences, located in the D fragment, arehighly homologous among the P plasmids and suggest thatthis conserved region may represent the functional domainof the proteins.

Similarly, the hybridization results obtained by using theoriV probes show that the region of the direct repeats isconserved in all the P plasmids, while the GC-rich region,downstream from the direct repeats in RK2, has divergedconsiderably in certain plasmids, most notably R772 andR906 (Fig. 3B). Since the RK2 trfA gene product can stilldrive replication of the R906 oriV region, these resultssuggest that the specific interaction between trfA and oriVmay involve the region of the direct repeats and that majorsequence changes in the GC-rich region still allow oriVfunction.These homology results for the replication regions corre-

late very well with the hybridization data obtained byYakobson and Guiney (25) for the region of the origin ofconjugal DNA transfer (oriT). Using an oriT DNA probefrom RK2, six of the nine plasmids showed strong hybridi-zation in the same HaeII fragment as RK2. These are thesame six which are identical in the replication regions usedin this study (Fig. 2 and 3). However, the three'plasmids,R751, R772, and R906, which differ from RK2 in the repli-cation regions showed only weak homology to oriT in asmaller HaelI fragment. In addition, Yakobson and Guiney(25) found that the RK2-like plasmids were able to mobilizea plasmid carrying the RK2 oriT region at high frequency butthat R751 and R772 were not, indicating that there has beenfunctional divergence which correlates with the sequencedivergence shown by hybridization. From the studies of thekey replication and transfer regions, the IncP plasmids canbe divided into two subgroups: IncPa, consisting of theRK2-like plasmids, and IncPp, comprising the non-RK2-likeplasmids.The homology in both the replication and transfer regions

suggests that all the IncP plasmids evolved from the sameprimordial conjugative P plasmid. The IncP evolutionaryline diverged some time ago into the a and 1 subgroups. Thesequences that have been most highly conserved are in theregion of oriV that contains the direct repeats and specifiesincompatibility. In addition, the ability of the RK2 trfAregion to complement the R906 oriV region indicates consid-erable conservation of function in the replicati'on regions ofthe P plasmids. Similar conservation is also seen in the korAand korB regions (5) but not in oriT, since the IncP,plasmids are not able to mobilize the RK2 oriT sequence(25). The heteroduplex analysis of the P plasmids by Vil-laroel et al. (23) shows that the IncPa plasmids are all closelyrelated to each other and differ mainly by simple insertionsand deletions. The relationship of the 1 plasmids is lessclear, since R906 and R751 did not form heteroduplexes with

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660 CHIKAMI ET AL.

each other. However, the Southern hybridization data indi-cate that R751, R772, and R906 have HaeII fragments ofidentical size determined by using the trfA* and oriT frag-ments as probes (Fig. 2 and reference 25). R751 differs fromR772 and R906 in the size of HaeII fragments defined by theD and oriV fragment probes (Fig. 2 and 3). Further clarifi-cation of the relationships among the IncP,B plasmids willrequire direct comparison between members of the group.

ACKNOWLEDGMENTSThis work was supported by grants to D.G.G. and D.R.H. from

the National Institutes of Health and the National Science Founda-tion. G.K.C. was supported by training grant AI-07036 from theNational Institute for Allergy and Infectious Diseases. D.G.G. wasa Teaching and Research Scholar of the American College of Phy-sicians.

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3. Currier, T. C., and E. W. Nester. 1976. Isolation of covalentlyclosed circular DNA of high molecular weight from bacteria.Anal. Biochem. 76:431-441.

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5. Figurski, D. H., R. F. Pohlman, D. H. Bechhofer, A. S. Prince,and C. A. Kelton. 1982. Broad host range plasmid RK2 encodesmultiple kil genes potentially lethal to Escherichia coli hostcells. Proc. Natl. Acad. Sci. U.S.A. 79:1935-1939.

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15. Shingler, V., and C. M. Thomas. 1984. Analysis of the trfAregion of broad host-range plasmid RK2 by transposon muta-genesis and identification of polypeptide products. J. Mol. Biol.175:229-249.

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17. Stalker, D. M., C. M. Thomas, and D. R. Helinski. 1981.Nucleotide sequence of the region of the origin of replication ofthe broad host range plasmid RK2. Mol. Gen. Genet. 181:8-12.

18. Terakado, N., and S. Mitsuhashi. 1974. Properties of R factorsfrom Bordetella bronchiseptica. Antimicrob. Agents Chemo-ther. 6:836-840.

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22. Vieira, J., and J. Messing. 1982. The pUC plamids: an M13mp7-derived system for insertion mutagenesis and sequencing withsynthetic universal primers. Gene 19:159-168.

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