Vol. 176, No. 24JOURNAL OF BACrERIOLOGY, Dec. 1994, p. 7735-77390021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

The IncN Plasmid Replicon: Two Pathways of DNAPolymerase I-Independent Replication

HONG-YEOUL KIM, S. K. BANERJEE, AND V. N. IYER*

Department of Biology and Institute of Biochemistry, Carleton University,Ottawa, Ontario, Canada KJS 5B6

Received 23 June 1994/Accepted 5 October 1994

The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a

region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PoIA- hosts and a

regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region,there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts anda third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. Wenow show that both oriB and oriS can function in a ApoL4 strain but that in such a strain, only oriB has an

absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteronregion is deleted and that in this circumstance, it is the only origin, the usage of which is detected. Therequirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can berecovered as a plasmid only ifRepA is provided in trans. These complex features of this replicon are also shownto be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable originsin a small replicon may be a way of endowing such a replicon with a broad host range.

A group of plasmids that has often been isolated fromseveral gram-negative species of eubacteria and which has arelatively broad host range is plasmids of the incompatibilitygroup N, among which the 39-kb plasmid pCU1 is a charac-terized member (13). The basic 2,053-bp replicon of pCU1 hasnearly the same broad host range as the parental plasmid (19)and has been described previously (20) (GenBank accessionno. M18262). Its relevant features are displayed in Fig. 1. Theyshow some similarity to the broad category of iteron-bearingplasmid replicons, among which are the intensively studiedFIA replicon of F, the P1 plasmid replicon, and the repliconsof pSC101 and R6K. All these replicons have been shown tofunction in vivo either in the absence of Escherichia colipolymerase I (Poll) or in the presence of greatly reducedamounts of this polymerase. Each specifies a protein withreplication initiation (or initiation and inhibition) activity.However, previous studies (3, 18) have indicated that thepCU1 replicon is more complex. Analysis of this complexity isof interest because it may reveal the molecular basis of itsbroad host range.

Previous structural and functional analyses (18, 20) indi-cated that the basic replicon is composed of two regions, aregion of about 1,200 bp that is sufficient for replication ineither PoMA' or PolA- E. coli and an adjacent region, the mostprominent feature of which is a group of iterons called thegroup I iterons (Fig. 1). A regulatory role has been ascribed tothese iterons because they express IncN incompatibility whencloned into a compatible vector and the complete or partialdeletion of the iterons is associated with an increase in theaverage copy number of the resulting replicon (20, 23a). Onthe other hand, the 1,200-bp replication component was foundto contain three origins of replication (3). Two of these, calledoriB and MnS, were found to function in both PolA' and PoIAdeletion mutant strains of E. coli while a third, orV, functionedonly in the PoIA+ host. Also contained in the 1,200-bp regionis an open reading frame (ORF) encoding 239 amino acids,

* Corresponding author. Phone: (613) 788-3864. Fax: (613) 788-4497.

shown as repA in Fig. 1. The product of repA has been detectedpreviously (18), and its N-terminal amino acids have thepredicted sequence (26a). Krishnan et al. (18) used the basicreplicon containing the iteron region and introduced twodifferent mutations near the N- and C-terminal regions ofRepA. The mutations resulted in the loss of detectable RepAprotein and in the detection of a truncated protein, respec-tively. Both plasmid mutants could transform PolAW strainsbut not a ApolA strain. These observations suggested that atleast in the absence of Poll, oriB and oriS were both dependenton the plasmid RepA protein. Unexpectedly in the presentstudy, we observed that when the iteron region is deleted fromeither of the repA mutant plasmids, the resulting plasmids dotransform the APolA strain, indicating that at least one of thetwo Poll-independent origins was not dependent on the avail-ability of the RepA protein. We used electron microscopy ofreplicating DNA molecules to determine unambiguously whichof the three clustered origins is utilized in this circumstance.We find that only onS is used and oriB is not. The requirementof RepA for oriB usage was confirmed by deriving a plasmidthat carries oriB as the only origin and showing that itsmaintenance in the ApolA strain is dependent on providingrepA in trans. Thus, the group I iteron region is shown here tohave an inhibitory effect on MrS usage. The evidence for thisand related conclusions and their implications for furtherstudies are described and discussed. We also present results toindicate that the features that we describe are not a specialattribute of the pCU1 replicon but are likely to be shared by allor most members of the IncN group of replicons.

(This research forms part of the graduate dissertation ofHong-Yeoul Kim [14a].)

Bacterial strains and plasmids. The E. coli strains and theplasmids used are listed with their properties and sources inTables 1 and 2. A number of deletion derivatives or clones ofdifferent regions of the basic replicon of plasmid pCU1 wereconstructed as part of this study. The procedures used for theseconstructions are indicated in the legends to the appropriatefigures or in the tables. All E. coli strains were grown inLuria-Bertani broth or Luria-Bertani agar (23), with the

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FIG. 1. Features of the basic replicon and relevant derivatives. The top half shows features deduced from the nucleotide sequence. The arrowsindicate all ORFs present in the sequence. Two of these are shown as thicker arrows because they represent the only ones whose polypeptides havebeen detected. The bent arrows are internal initiation codons within the larger ORFs. The asterisk and dark square over repA indicate the positionsof polypeptide chain-terminating mutations in two mutants (18). The boxed arrowheads indicate the nonessential iteron region, and the shadedbox indicates the essential region. The hexagons below the line indicating the replicon show the positions of bidirectionally replicating origins, twoof which (shown shaded) function in apolA deletion mutant while the third does not. The limits of each origin region are indicated on the verticalaxis of Fig. 2. Potential DnaA protein-binding (dA) and IHF protein-binding (IHF) sites are indicated vertically above the line. The boundedhorizontal lines in the lower part of the figure are sequences present in relevant deletion derivatives (pCU derivatives) or derivatives cloned in pUCvectors (pKI derivatives) (see Table 2).

exception of the ApoU strain SR1672, which was grown intryptone yeast extract broth or agar without added NaCl. Thegrowth temperature was 37TC. Antibiotics were added asrequired to presterilized solid media at the following concen-trations (per ml): ampicillin, 100 pug; kanamycin, 50 jxg;spectinomycin or streptomycin, 50 jig; and tetracycline, 20 Rg.Plasmid DNA was isolated by the procedure of Birnboim andDoly (4) or by using Qiagen affinity columns (Qiagen Inc.,Chatsworth, Calif.) as recommended by the manufacturer.Restriction endonucleases were purchased from New England

TABLE 1. E. coli strains used

Strain Genotype or phenotype reference

HB101 F- hsdS20 recA13 ara-14 proA2 lacYl 7galK2 rpsL20 xyl-5 mitl-i -

C600 F- thi-1 thr-1 leuB6 lacYl tonA21 2supE44 X-

DH5a F- endAl hsdR7 supE44 thi-I- recAI 10gyrA96 reLAl deoR A(lacZYA-argF)U169 (4+80 dlacZ AM15)

C1200 F- rha met his 17C2110 F- pol4l rha met his M. FilutowiczW3110 X- rinD-n-nE 12SF800 poL4l Nalr thy 11WA802 F- A(lac)6 supE44 galK2 galT22 mcrA 28

rfbDl metBI mcrBl hsdR2WA802P,,L41 Similar to WA802 but poL4i 28SR1758 A(gal-bio) thi-1 reLAl spoTi F+ Kanr F+ 25SR1672 Similar to SR1758 but also ApoU 25

BioLabs (Beverly, Mass.) or Promega Canada Ltd. and usedwith buffers as recommended. Electrophoresis was done inhorizontal agarose gels ranging in concentration from 0.8 to1.0% (normal agarose) to 2.0 to 3.0% (low-melting-pointagarose). DNA fragments were recovered from gels as de-scribed by McDonell et al. (22) with Gene clean or Mermaidkits purchased from Bio 101 Inc., La Jolla, Calif., by proce-dures recommended by the manufacturer. Plasmid DNA trans-formation of bacteria and the procedures used for the isolationand analysis of replicating plasmid DNA molecules by electronmicroscopy were as described previously except for the choiceof the restriction enzymes and their sites used to linearize themolecule (3, 20).

Deletion derivatives and clones of regions from the basicreplicon. Previous observations from this laboratory (20) haveshown that all 2,053 bp of the basic replicon are not essentialfor plasmid replication and that deletion derivatives in whichportions of the basic replicon are linked to antibiotic resistancemarker fragments can be obtained. Eleven derivatives withdeletions from either end of the 2,053-bp pCU1 replicon wereisolated independently in the present study by linking eachfragment with a nonreplicating antibiotic resistance fragment(usually the Spr Smr fi fragment of Prentki and Kirsch [24] butin a few cases the Kmr fragment from pKlink [26] or the Cmror Tcr fragment from pACYC184 [6]). The results obtainedwith these new deletion derivatives were consistent with theresults obtained previously (20). Therefore, only the deletionderivatives that are directly relevant to the conclusions hereare shown in Fig. 1. Plasmid DNA of each of the two deletionderivatives obtained from E. coli HB101 was used to transformtwo or more of the isogenic pairs of E. coli strains: SR1758 and

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TABLE 2. Bacterial plasmids used

Plasmid Antibiotic resistance Plasmid replicon(s), nucleotide sequence(s) from the replicon Referencemarker(s) of pCU1 (if any), and other relevant information

pBR329 Apr Cmr Tcr pMB9 7pACYC184 Cmr Tcr plSa 6pUC18 Apr pMB9pUC1318 Apr pMB9 14pUC1813 Apr pMB9 14pKlink Apr Kmr pIC19R 26pHP45fl Spr Smr pBR322 24pCU1 Apr Spr Smr Parental plasmid 20N3 Smr Tcr Sur Parental plasmid 1R15 Smr Sur Hgr Parental plasmid 8pHYK2 Spr Smr Minireplicon of N3 21pHYK6 spr Smr Minireplicon of R15 14apCU999fl Spr Smr Kmr pCU1 replicon (nt 1-2053) This studypCU999 Linked to Spr Smr fragment from pHP45Ql (in pCU999fl) or Kmr fragment

from pKlink (in pCU999)pCU718 Kmr pCU1 replicon (nt 1-2053) as above but with a chain-terminating mutation 18

in codon 3 of repApCU717 Kmr As above but with the mutation in codon 174 18pCU996 Kmr Partial pCU1 replicon; HaeII fragment from pCU999 (nt 198-1394 of the This study

pCU1 replicon) linked to the Kmr fragment (pKlink)pCU918 Cmr Same HaeII fragment as above but from pCU718 carrying the mutation in This study

repA linked to the 1.4-kb Cmr HaeII fragment of pACYC184pCU917 Cmr As above but from pCU717 This studypCU949 Tcr Partial pCU1 replicon; nt 382-1315 of the replicon linked to the Tcr frag- This study

ment (DraI-HindIII of pACYC184)pKI336 Apr pUC18 with the oriB region of pCU1 (nt 263-598) cloned into it This studypKI92, pKI93 Tcr pBR329 with nt 382-2053 of pCU1 cloned into it in the two orientations This study

SR1672, W3110 and SF800, WA802 and WA802po1, andC1200 and C2100. Different isogenic pairs were used toconfirm our conclusions and also because some pairs had thesame antibiotic resistance markers as the plasmid to be tested.In each of these pairs, the latter strain carries a polA mutation(Table 1). The first two groups of deletion derivatives shown inFig. 1 (pCU996 and pCU949) transformed both members ofthe pair and were maintained in a structurally and segregation-ally stable manner on the appropriate selection media. Se-quences from the basic replicon were also cloned into suitablevectors of the pUC and pBR families. These cloning vectors bythemselves do not transform the PolA- mutants, and theability of sequences linked to them to confer transformationability provided a second means of identifying replicons thatcan function in the absence of Poll. The results were consistentwith those obtained with the above-described deletion deriva-tives; that is, those sequences that were shown to be present inthe deletion derivatives that transformed the PolA- strainswere the ones that when cloned, enabled pCU or pBRderivatives to transform PolA- strains; when more extensivelydeleted fragments from either end were cloned into thesevectors, they failed to transform the PoIA- strains.The ability of pKI949 and pCU949 (nucleotides [nt] 382 to

1315) to transform the PolA- strains and the inability of pKI92and pKI93 (nt 382 to 2053) to do so were curious. These resultssuggested that the group I iteron region interferes with aPoll-independent pathway of replication.

Construction and properties of pCU918 and pCU917. Theprevious designation of ORF239 (Fig. 1) as Rep (now calledRepA) as a plasmid-specified replication protein essential forPolI-independent replication was based on the following ob-servations (18). In an in vitro coupled transcription-translationsystem of E. coli, only the RepA and ORF46 polypeptides weredetected. When polypeptide chain-terminating codons wereintroduced by in vitro mutagenesis, into either the third codon

of repA or codon 174 (shown as an asterisk and black square inFig. 1), the resulting derivatives were found to produce,respectively, no detectable RepA polypeptide or a truncatedpolypeptide. The mutations were designed not to affect theoverlapping ORF46 (Fig. 1). Neither derivative pCU717 norpCU718 transformed thepolA deletion strain SR1672, and thishas been confirmed in the present study (Table 3). The HaeIIfragment (nt 198 to 1394) from the derivatives was linked tothe fl fragment carrying SmrSpr and transformed into HB101.The resulting derivatives were called pCU917 and pCU918,respectively. The sequences present in these plasmids areidentical to those present in pCU996fl (Fig. 1) except for therespective base substitutions in the repA gene. Both pCU917DNA and pCU918 DNA transformed the PolA- strains(Table 3), and the plasmids were maintained stably in thetransformants. Thus and unexpectedly, the deletion of the first195 bp and the terminal nt 1395 to 2053 region led to thesuppression of two independent mutations that had initiallymade the plasmid replicon Poll dependent.

Electron microscopic study of origin usage in pCU918replicating in a PoIA- strain. Plasmid pCU996 has sequencessimilar to those of pCU918 except that it has no mutation inrepA. Previously (3), we have shown that when this plasmidreplicates in a PolA- strain, either oriB or oriS but not oriV isused. By using pCU918 and the same PolA- strain, originusage was examined in the absence of RepA. The results areshown in Fig. 2 and indicate that oriS is used exclusively and bya theta mode in which the rates of movement of the tworeplication forks are similar. This result indicated that at leastin the absence of Poll, oriB is dependent on functional repAwhile MrS is not.

Construction of an oriB plasmid and confirmation that itrequires RepA. Previous electron microscopic analysis (3) hadplaced upper limits to the oriB region. By using appropriaterestriction sites, 336 bp containing oriB were cloned into

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TABLE 3. Ability or inability of derivatives of the basic replicon to transform isogenic pairs of strains carrying mutant polA alleles

Ability to transform polAPlasmid(s) Relevant feature(s) deletion or amber

mutantsa

pCU999 Basic 2,053-bp replicon of pCU1 +pUC717 Basic 2,053-bp replicon with a chain termination mutation in codon 174 of repApCU718 As above but with mutation in the third codon of repApCU917 nt 198-1395 of pCU717 (contains the same mutation as in pCU717) +pCU918 nt 198-1395 of pCU718 (contains the same mutation as in pCU718) +pCU949 nt 382-1315 of the basic replicon +pKI949 As above but cloned into the Poll-dependent pACYC184 +pKI92, pKI93 nt 382-2053 of the basic replicon cloned into the Poll-dependent pBR329 in either orientation

a +, ability to transform mutant strains; -, inability to transform mutant strains. All derivatives could transform strains carrying the wild type.

pUC18 and pUC1813, which are both Poll-dependent vectors.The constructed clones were then used to transform theplasmid-free ApolA strain and the ApolA strain carrying eitherpCU918 or pCU996fk. Only the pCU996fl-carrying strainyielded transformants. To confirm that these transformantscarried pKI336, their total plasmid DNA was analyzed. Theresult indicated that they carried a small plasmid in addition topCU996fl, called pKI336. pKI336 DNA was next digested withHaeII, and a 370-bp fragment containing the origin of pUC18was removed. The remaining DNA fragment recovered fromthe gel was religated under conditions favoring intramolecularligation and transformed into SR1672(pCU996fQ), selectingfor the resistance markers of both plasmids. Plasmid DNA wasextracted from 1 of 35 transformant colonies that were ob-tained and digested with EcoRI to yield three fragments, oneof which was linear poriB and the other two of which were frompCU996fl. The oriB fragment was extracted from the gel and

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ligated. Upon digestion of this preparation with EcoRI andHind1II and analysis in a 3% agarose gel, an expected fragmentof 336 bp was released and could be detected.

Other IncN group plasmid replicons. Plasmid pCU1 is oneof a large group of naturally occurring plasmids of the Nincompatibility group (5). A minimal replicon of one otherplasmid of this group, called pR (a derivative of R46), hasrecently been isolated and sequenced (9). It contains nt 1 to1315 of the pCU1 replicon. This complete sequence conserva-tion implies functional conservation of both the origins andtrans-acting functions. The restriction maps of two otherplasmids of this group, N3 and R15, have been reported (1, 8).The replicons of both these plasmids have been isolatedpreviously (pHYK2 and pHYK6) and shown to be also polAindependent (2). Both pHYK2 and pHYK6 were able torescue the oriB plasmid pKI336, indicating that they have repAor a repA analog.

0 10 20 30 40 50 60

% ReplicationFIG. 2. Analysis of the branch points of partially replicated plasmid molecules of pCU918 (the derivative not producing RepA), replicating in

thepolA deletion strain SR1672. The procedures used were similar to those described previously (3). Measurements were from the Ball site to thebranch points (replication forks) and are shown relative to the length of the molecule linearized with Ball and normalized to 100%. For eachmolecule, the relative distances to each branch point of a linearized theta structure are shown by different symbols and the lines drawn by linearregression analysis of the points (Sigma Plot, version 5). The point at which the two lines converge is the origin (0% replication). The relevantportion of the linearized molecule is shown alongside the ordinate with the origin regions (thick bars) as determined previously (3). The bracketsenclose the chloramphenicol resistance gene (cat).

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An attractive feature of the N group replicon is that itcombines a broad host range with an economical use ofsequence information. Relating this information to originusage is useful in determining whether and how origin usage isrelated to broad host range. oriB and oriS were of particularinterest; the fact that both can function in the absence of DNAPoll of E. coli raised the possibility that their requirementswere identical or at least indistinguishable in E. coli. Theresults of this study indicate this is not the case; oiB requiresthe replicon-specified RepA protein, while onS does not.Recent in vitro studies on the binding of the RepA protein toreplicon sequences are supportive of this conclusion (23b).The oriB region has not only RepA binding sites but also the

9-bp sequences known to be DnaA protein-binding sites, anAT-rich sequence and a sequence similar to the bacteriophagelambda integration host factor protein-binding site. These arefeatures of other Poll-independent but DnaA- and Rep-dependent replicons such as those of the relatively narrow-host-range plasmid replicons of P1 and F (15, 27). Such originsmay be activated by a mechanism essentially similar to themechanism of initiation proposed for the E. coli chromosome(16) with the proviso that they require in addition a plasmidreplicon-specific Rep protein. It is likely that the MnS originalso requires the DnaA protein because the entire repliconcontaining all three origins has been shown to require afunctional dnaA gene (20) and there are four dnaA boxeswithin MnS (Fig. 1). MnS may belong to a class of origins amongwhich the DnaA protein instead of being abetted by a proteinlike the RepA protein is abetted by some other factor orprocess such as the small ORF46 protein and/or transcriptionalactivation. This would endow versatility to the IncN repliconwhich in turn may be related to its broad host range. Furtherstudies on oriS, the requirements for its usage, and themechanism by which its usage is inhibited by the group I iteronregion will be necessary to test such speculations. As can beobserved from Fig. 1, the group I iteron region contains notonly the iterons themselves but also ORF245 with an internalinitiation site (Fig. 1). Although we have not detected theproducts of these two ORFs and the survival of pCU949(which is unlikely to have oiB; Fig. 1) in the APolA mutantsuggests that ORF68 and ORF245 are not essential for MnSusage, the role of the N-terminal regions of these putativepolypeptides in inhibiting MnS usage is not ruled out. Neitherdo we know at the present whether the observed inhibition ofMnS usage by the iteron region occurs only in the absence ofDNA Poll and RepA.

This study was supported by the Medical Research Council ofCanada.We thank the several persons who provided some of the bacterial

strains used in this study and Peter Papp for critical comments on themanuscript.

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