JOURNAL OF BACTERIOLOGY, Sept. 1985, p. 973-9820021-9193/85/090973-10$02.0010Copyright © 1985, American Society for Microbiology

Vol. 163, No. 3

Incompatibility Mutants of IncFlI Plasmid NR1 and Their Effect on

Replication ControlRU PING WU,t DAVID D. WOMBLE,* AND ROBERT H. ROWND

Department of Molecular Biology, The Medical and Dental Schools, Northwestern University, Chicago, Illinois 60611

Received 1 April 1985/Accepted 30 May 1985

DNA from the replication control region of plasmid NRl or of the Inc- copy mutant pRR12 was cloned intoa pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine andtransformed into Escherichia col cells that harbored either NR1 or pRR12. After selection for the newlyintroduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmidswere examined further. By this process, 134 plasmids with Inc- mutations in the cloned NRl or pRR12 DNAwere obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertionsin the NRl DNA and were not examined further. Plasmids with apparent point mutations were classified byexamining (i) their ability to reconstitute a functional NRl-derived replicon (Rep' or Rep-), (ii) the copynumbers of the Rep' reconstituted replicons, (iii) the cross-reactivity of incompatability among the variousmutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stableinheritance of a coresident plasmid.

When two closely related plasmids are introduced into thesame bacterial host cell, segregants lacking one plasmid orthe other are produced during subsequent growth and celldivision (9). This inability to maintain both plasmids in thedescendents of the original cell is known as plasmid incom-patibility (9). Plasmids that are not closely related usually aremaintained stably together and therefore are compatible.Traditionally, plasmid incompatibility has been used as ameans of characterizing and classifying various plasmids.Incompatibility is now generally recognized to be a directmanifestation of the mechanisms that regulate plasmid DNAreplication and segregation at cell division.The 90-kilobase-pair (kb) transmissible antibiotic resist-

ance plasmid NR1 belongs to the FII incompatibility group,which also includes plasmids Rl and R6 (9). NR1 has a lowcopy number and is maintained stably in an Escherichia colihost, even in the absence of selection (25, 35, 37, 55). TheNR1 genome (Fig. 1) contains genes for control of autono-mous DNA replication, antibiotic resistance, conjugal trans-fer, and stable inheritance (stb) (24, 25, 44). The genes forNR1 plasmid replication control are contained within twocontiguous PstI restriction fragments 1.1 and 1.6 kb in size(Fig. 1) (25). The structures of the replication regions ofplasmids Rl and R6 are similar to that ofNR1 (16, 27, 39, 42,43, 46). The 33,000-dalton product of the repAl gene isrequired for the initiation ofDNA replication at the plasmidorigin (23, 25, 39, 45). The frequency of initiation of IncFIIplasmid replication is controlled by regulating the synthesisof the repAl initiation protein (11, 12, 18, 19, 21, 38, 52, 53).Transcription of the repAl gene is initiated from two dif-ferent promoters, producing RNA-CX and RNA-A (Fig. 1)(12, 38, 54). The repA2 gene (33), whose 10,000-daltonproduct is a repressor of RNA-A transcription (10, 22, 28,38), also is transcribed from the RNA-CX promoter. There isno known function for the 7,000-dalton product of the repA3gene (5, 33), which is traversed by both RNA-CX and

* Corresponding author.t Present address: Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, Shanghai 20031, China.

RNA-A (Fig. 1). The origin of replication (ori) is in the 1.6-kbPstI fragment (23, 25, 30, 35, 39).At the wild-type copy number of NR1, there is a sufficient

supply of repA2 protein in the cell to repress nearly com-pletely transcription from the RNA-A promoter (10, 38).Transcription of the repAl gene therefore is primarily aresult of the constitutive synthesis of RNA-CX. Because therepA2 repressor is saturating at the wild-type copy number,it plays only a small role in the regulation of wild-typeplasmid replication or incompatibility (10, 20, 22, 32, 38, 52).The product of the inc gene is RNA-E, a 91-base, untrans-

lated transcript (Fig. 1) (8, 12, 34, 41, 53, 54). RNA-E istranscribed from the opposite DNA strand from RNA-CX(12, 34, 53, 54). RNA-E regulates the synthesis of the repAlinitiator by binding to and inhibiting the translation of itsmRNA (21, 53). The inhibition of repAl translation is theprimary mechanism of IncFII plasmid incompatibility andcopy number regulation. Base substitutions in the inc gene,which weaken the complementary binding of RNA-E toRNA-CX, result in altered incompatibility properties andelevated plasmid copy numbers (4, 11, 13, 25, 34, 35, 38, 39,41, 53).

Previously, most mutants of IncFII plasmids with alteredincompatibility properties (Inc-) were obtained by selectionfor copy number (Cop-) mutants (4, 11, 13, 25, 29, 35, 48).These mutants had plasmid copy numbers that were elevatedcompared with the wild-type plasmid and therefore con-ferred resistance to higher levels of antibiotics. This selec-tion demanded that the Inc- mutations be nonlethal to theplasmid replication system and was limited to selection ofplasmids with elevated copy numbers. In this report, wedescribe the isolation and characterization of mutants withapparent point mutations of the NR1 replication regionwhich were selected only for loss of incompatibility withoutregard to any other replication function. This selectionresulted in plasmid mutants with novel incompatibility andreplication phenotypes.

MATERIALS AND METHODS

Bacterial strains and plasmids. E. coli K-12 strains KP245met trp his thy lac gal (24) and KP435 trp ilv thy rpsL recA

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0Uc

ip.,§

Iat10/C)

0

0.

3_7.

ar 0Zv

H

I-1H

acI

262inc

E

=> cxA

FIG. 1. Maps of the self-transmissible antibiotic resistance plasmid NR1 and its replication control region. NR1 confers resistance totetracycline (tet), chloramphenicol (cat), streptomycin and spectinomycin (strlspc), sulfonamides (sul), and mercuric ions (mer). The stb locusis responsible for stable plasmid inheritance. In the replication region, sites for Pstl and Sau3A (S) restriction cleavages are indicated, as arethe distances, in kb and bp, respectively, between them. Coding regions for proteins repAl, repA2, andrepA3 are indicated by boxes. Thetranscription promoters for rightward transcripts RNA-CX and RNA-A (PCX and PA, respectively) and for the leftward transcript RNA-E (PE)are indicated, as are the locations of the incompatibility gene (inc) and origin of replication (ori).

(24) were used for plasmid construction and incompatibilitytests, respectively. Strains JG112 met thy rpsL polA (26) andDKlOOts214 his arg met leu thy xyl lac rpsL polA(Ts) (15)were used for reconstitution of NR1-derived replicons frompBR322 clones and for testing the polA dependence ofchimeric plasmids composed of both pBR322 and NR1replicons. The plasmids used in this study are listed in Table1. The structure of plasmid NR1 (and pRR12) is shown in Fig.1. Plasmids pRR403 and pRR634 have deleted the tetracyclineresistance determinant, but are otherwise identical to NR1and pRR12, respectively (Fig. 1). Minireplicator plasmidspRR933 and pRR942 (Fig. 2) are composed of the 1.1-kb incPstI fragment and the 1.6-kb ori PstI fragment from thereplication control region (Fig. 1) of NR1 and pRR12,respectively, joined to a 2.2-kb fragment encoding resistanceto chloramphenicol (cat). The minireplicator plasmids retainthe incompatibility and copy number phenotypes of their90-kb parental plasmids (11, 25). Plasmid pFYRR1 (Fig. 2)was obtained by partial digestion of pRR942 DNA with PstI,treatment with excess S1 nuclease and blunt end ligation(Y. L. Fan and R. H. Rownd, unpublished data). Approxi-mately 700 base pairs (bp) of DNA, including the PstI sitebetween the ori and cat fragments, was deleted to produce aplasmid with a new orilcat PstI fusion fragment of 3.7 kb (datanot shown). Each of the individual PstI restriction fragmentsfrom pRR933, pRR942, and pFYRR1 was cloned into the PstIsite of the vector plasmid pBR322 (Fig. 2). Plasmids pRR945and pRR931 have both the 1.1- and 1.6-kb fragments, in theirnative orientation, from NR1 and pRR12, respectively (Fig.2). In these plasmids, both the pBR322 and NR1 or pRR12replicons are functional.

Culture media. L broth (17) was used for culturing cells

after transformation with plasmid DNA and for plasmid copynumber determinations. Penassay broth (Difco Laborato-ries) was used for all other cultures. Thymine was added toall culture media to a final concentration of 20 ,ug/ml.Antibiotics (Sigma Chemical Co.) were included in somemedia to select for cells harboring various plasmids: tetra-cycline hydrochloride (10 pug/ml) or chloramphenicol (25,ug/ml). Agar media contained 15 g of Bacto-agar (Difco) perliter. Chloramphenicol acetyltransferase (CAT) indicatoragar (31) contained the rosanilin dye basic fuchsin (2 ,ug/ml)from Sigma. Cells were cultured at 37°C, except strainDKlO0ts214, which was cultured at 30 or 42°C. Bacterialgrowth was monitored by turbidity at 650 nm with a Gilfordmodel 260 spectrophotometer.DNA isolation and manipulation. Plasmid DNA isolation,

restriction endonuclease digestion, gel electrophoresis, liga-tion of restriction fragments, and transformation of E. colicells with plasmid DNA were performed as previouslydescribed (11, 25). The alkaline minilysate method (1) wasused for quick screening of plasmid sizes and restrictionanalysis.

Mutagenesis of plasmid DNA. Plasmid DNA was mutagen-ized with hydroxylamine in vitro (14) or with 4-nitroquinoline-1-oxide (NQO) in vivo (7). Plasmid DNA (2,ug) was treated with 0.4 M hydroxylamine in a volume of 0.2ml (0.05 M sodium phosphate [pH 6.0], 0.1 M EDTA) at 75°Cfor 30 or 45 min, followed by extensive dialysis against 20mM CaCl2 at 4°C. For NQO mutagenesis, 5 ml of exponen-tially growing cells harboring the plasmid to be mutagenizedwas treated with NQO at 40 or 80 jig/ml at 32°C for 2 h. Afterthe addition of sodium thiosulfate to inactivate the NQO, themutagenized cells were washed twice and used as the

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IncFll INCOMPATIBILITY MUTANTS 975

inoculum for a 200 ml Penassay broth culture from whichplasmid DNA was isolated.

Plasmid incompatibility assay. Incompatibility tests wereperformed as described previously (11, 25). The donorplasmid DNA was introduced by transformation into recAcells (KP435) harboring a resident plasmid. After selectionfor the antibiotic resistance of the donor plasmid only,retention of the resident and donor plasmids was tested byreplica plating for each antibiotic resistance.

Plasmid replicon reconstitution. The ability of the mutant1.1-kb PstI inc fragments to reconstitute functional repliconswas tested by ligating the fragments to 1.6-kb PstI ori plus2.2-kb PstI cat fragments (3-PstI-fragment minireplicatorplasmids) or to the 3.7-kb PstI orilcat fusion fragment(2-PstI-fragment minireplicator plasmids) as illustrated inFig. 3. The pBR322 clones containing the appropriate PstIfragments were digested with PstI, mixed, ligated, andintroduced into JG112 (polA) by transformation, selectingfor chloramphenicol resistance. Because the pBR322replicon is not active in a polA host (15), only reconstitutedNR1 replicons were present in the chloramphenicol-resistant, tetracycline-susceptible transformants. For the3-PstI-fragment minireplicators, the cat fragment was ob-tained in both orientations. Those plasmids with the catfragment in the same orientation as pRR933 and pRR942were retained for further examination. All 2-PstI-fragmentminireplicators had the native orientation of the inc andorilcat fragments, as in plasmid pFYRR1 (Fig. 2).

Plasmid copy number determinations. The relative copynumbers of plasmids that carry the cat gene were estimatedfrom gene dosage effects by measuring the CAT enzymespecific activity in cell extracts prepared from exponential-phase cultures as previously described (11, 40), except thattotal protein was measured by the method of Bradford (3).Continuous selection for the plasmids was maintained duringgrowth of the cultures. Comparative copy numbers also

TABLE 1. Plasmids used in this study

Plasmid Description' Referenceor source

NR1 Natural isolate, Cmr Tcr (36)pRR12 Inc- Cop- mutant of NR1, Cmr Tcr (29)pRR403 Tcs mutant of NR1, Cmr (11)pRR634 Tcs mutant of pRR12, Cmr (11)pRR933 Minireplicator of NR1, Cmr (25)pRR942 Minireplicator of pRR12, Cmr (25)pFYRR1 Deletion mutant of pRR942 (3.7-kb oril This study

cat), CmrpBR322 Cloning vector, Tcr Apr (2)pRR935 pBR322 plus 1.1-kb PstI inc fragment of (11, 25)

NR1, TcrpRR939 pBR322 plus 1.1-kb PstI inc fragment of (11, 25)

pRR12, TcrpRR936 pBR322 plus 1.6-kb PstI ori fragment of (11, 25)

NR1, TcrpRR714 pBR322 plus 2.2-kb PstI cat fragment, (25)

Cmr TcrpWFRR1 pBR322 plus 3.7-kb PstI orilcat This study

fragment, Cmr TcrpRR945 pBR322 plus 1.1- and 1.6-kb PstI (11, 25)

fragments of NR1, TcrpRR931 pBR322 plus 1.1- and 1.6-kb PstI (11, 25)

fragments of pRR12, TcrpACYC177 Cloning vector, Kmr Apr (6)

a Cmr, chloramphenicol resistance; Tcr, tetracycline resistance; Apr, ampi-cillin resistance; Kmr, kanamycin resistance.

pRR933 (NRI)pRR942 (pRR12)

pRR 936 (NRI) pRR935INRI)pRR 939 (pRR12)

pFYRRI (pRR12)

pR322

1.1kb 1.6kb

pRR 945 (NRI)pRR 931 (pRR12)

Inc- Mutants Inc- Mutants

FIG. 2. Structure of minireplicator plasmids and the pBR322clones with the PstI fragments inserted into the amp (ampicillinresistance) gene.

were estimated on CAT indicator medium (31), on which theamount of red coloration of the colonies was proportional tocat gene dosage.

Plasmid stability assay. Cells harboring one or more plas-mids were cultured in Penassay broth containing antibiot-ic(s) to which resistance was conferred by the plasmid(s).These cells were then repeatedly subcultured by 106-folddilution into drug-free medium followed by overnight incu-bation. After each subculture, appropriate dilutions of thecultures were spread on drug-free agar plates, and theantibiotic resistance of each colony was tested by replicaplating (25).

In vitro transcription assay. In vitro transcription experi-ments were carried out essentially ad described by Winkleret al. (51). Reaction mixtures (25 ,ul) contained 36 mM Trisacetate (pH 7.8), 0.1 mM disodium EDTA, 0.1 mM dithio-threitol, 4 mM magnesium acetate, 150 mM KCl, 5%(vol/vol) glycerol, 150 ,uM ATP, 150 ,um CTP, 150 ,uM UTP,approximately 0.2 ,ug of template DNA, and 0.036 R&g (0.07pmol) of RNA polymerase holoenzyme. Transcription wasstarted by adding 20 p.M [c-32P]GTP (15 ,uCi) to eachreaction tube. After 40 min at 37°C, transcription reactionswere stopped by the addition of 25 ,ul of 0.09 M Trisborate-2.5 mM EDTA (pH 8.3) (TBE) containing 50%(wt/vol) urea, 4 mg of bromphenol blue per ml, 4 mg ofxylerne cyanol per ml, and 1 p.g of sodium dodecyl sulfate perml. The reaction mixtures were loaded onto 6% polyacryl-amide-7 M urea gels containing TBE and subjected toelectrophoresis. Transcription products were detected byautoradiography. The amount ofRNA-E synthesized in each

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pRR936

Inc- Mutants

pRR714

3 - PstI - fragment 2 - PstI - fragmentminireplicator plosmids minireplicator plosmids

FIG. 3. Schemes for replicon reconstitution experiments.

reaction was estimated from densitometer tracings of theautoradiographs by comparison with the amount of RNA-Itranscribed from the pBR322 vectors (47). (Sample autora-diographs are shown in Fig. 3 of reference 53.)

REStJLTSIsolation and characterization of incompatibility (Inc-) mu-

tants. The pBR322 clones (Fig. 2) that have the 1.1-kb PstIinc fragment from NR1 (pRR935) or its Inc- Cop- mutantpRR12 (pRR939) or that have both the 1.1-kb PstI incfragment and 1.6-kb PstI ori fragment from NR1 (pRR945) or

pRR12 (pRR931) were treated with hydroxylamine in vitroor NQO in vivo as described above. The mutagenizedpRR935 and pRR945 plasmid DNA was used to transformKP435 harboring pRR933 (Fig. 2, from NR1), whereas themutagenized pRR939 and pRR931 plasmid DNA was used totransform KP435 harboring pRR942 (Fig. 2, from pRR12).Transformants were selected on CAT indicator mediumcontaining tetracycline. The tetracycline selected the TcrpBR322 donor plasmids, and the CAT indicator was used toscreen for colonies that retained the Cmr resident plasmids.On CAT indicator medium, Cms colonies are white, whereasCmr colonies are pink to red, with the amount of redcoloration proportional to cat gene dosage (plasmid copynumber). By this method, thousands of colonies can bescreened easily to find compatible donor and resident plas-mids. No pink or red colonies were observed after transfor-mation with the nonmutagenized pBR322 clones, and nodifference in the frequency of white, pink, and red colonieswas observed with treated and nontreated pBR322 DNAalone. Plasmid DNA was isolated from the pink and redtransformants by the alkaline minilysate method, and KP245cells were transformed to separate the Tcr Cms mutant donorplasmids from the Tcs Cmr resident plasmids. The incompat-

ibility phenotypes of these possible Inc- mutants were thencharacterized by transformation of purified plasmid DNAinto KP435 harboring pRR403 (Tcs NR1) or pRR634 (TcspRR12). Fourteen independent Inc- mutants were obtainedfrom pRR935, 43 were obtained from pRR939, 61 wereobtained from pRR945, and 16 were obtained from pRR931.The 134 mutants could be further divided into 11 classes(Table 2), based upon additional characterization as de-scribed below. Class III mutants, although no longer incom-patible with NR1, were now incompatible with pRR12.Mutants of all other classes were compatible with both NR1and pRR12 (Table 2).To identify insertion or deletion mutants, DNA of each

mutant plasmid was digested with PstI, and the fragmentsizes were determined by gel electrophoresis. Seven mutantsfrom pRR935 (class IV) and five mutants from pRR945 (classIX) contained deletions or insertions in the 1.1-kb incfragment; these mutants were not examined further. None ofthe other Inc- mutant plasmids had obvious alterations insize, and they were retained as possible point mutants (datanot shown).To confirm that the Inc- phenotypes resulted from muta-

tions of the cloned 1.1-kb NR1 or pRR12 PstI fragments,these fragments from representatives of each mutant classwere cloned into the vector plasmid pACYC177 and thenback into pBR322. In every case examined, the Inc- pheno-type was associated with the 1.1-kb PstI fragment (data notshown).

Replication properties of Inc- mutants. To determine whateffects, if any, the mutations had on the replication controlproperties of the 1.1-kb PstI fragments, their ability toreconstitute functional NR1-derived replicons was tested.For the mutants from pRR935 (classes I, II, and III) andpRR939 (classes V and VI), the mutant 1.1-kb PstI fragmentswere ligated to wild-type 1.6-kb PstI ori and 2.2-kb PstI catfragments, or to the 3.7-kb PstI orilcat fusion fragment (Fig.3) as described above. This resulted in the reconstruction ofminireplicator plasmids having three PstI fragments, similarto pRR933 and pRR942, or of minireplicator plasmids havingtwo PstI fragments, similar to pFYRR1 (Fig. 2 and 3). The3.7-kb orilcat fragment was used because the frequency offruitful in vitro recombination is 10-fold higher when onlytwo fragments must be joined (1.1 plus 3.7 kb) compared tothe joining of three fragments (1.1 plus 1.6 plus 2.2 kb).

Viable reconstituted minireplicator plasmids were ob-tained from mutant classes II, III, V, and VI. Both 3-PstI-fragment and 2-PstI-fragment plasmids were obtained fromeach class. These mutant plasmids therefore were classifiedInc- Rep' (Table 2). After multiple attempts, side-by-sidewith the other mutant or other wild-type 1.1-kb PstI frag-ments, no reconstituted minireplicator plasmids were ob-tained from any class I mutant. These mutants thereforewere classified Inc- Rep- (Table 2).For the mutants from pRR945 (classes VII, VIII, and IX)

and pRR931 (classes X and XI), the function of the mutantreplicons could be tested directly, because both 1.1- and1.6-kb PstI fragments were present in the parental plasmids(Fig. 2). Plasmids pRR945 and pRR931 are able to transforma polA recipient, JG112, in which the pBR322 repliconcannot replicate, whereas the NR1 and pRR12 replicons can(25). Mutants of classes VII and X also were able totransform a polA recipient and were designated Inc- Rep+(Table 2). These Inc- Rep' mutants also were retained atthe non-permissive temperature in the polA(Ts) host,DKlOOts214, as were their pRR945 and pRR931 parentalplasmids (data not shown). Mutants of classes VIII, IX, and

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TABLE 2. Properties of Inc- mutants

Parent Mutant No. Incompatibility Test Replication Copy No. RNA-Eplasmid class isolated Example (Inc) function control synthesisd

NRl pRR12 (Rep)b (COPY~pRR935 Wild type + - + + +

I 3 pWNRR3 - - - NT -II 2 pWNRR2 - - + - +III 2 pWNRR5 - + + - +IVe 7 pWNRR29 - - NT NT NT

pRR939 pRR12 - + + - +V 41 pWRRR8 - - + - +VI 2 pWRRR3 - - + - NT

pRR945 Wild type + - + + NTVII 45 pWNRR101 - NT + NT NTVIII 11 pWNRR124 - - - NT +IXe 5 pWNRR156 - NT - NT NT

pRR931 pRR12 - + + - NTX 15 pWRRR101 NT - + NT NTXI 1 pWRRR105 - - - NT +

'Tested by transformation into KP435 recipients harboring Tcs derivatives of NR1 or pRR12, selecting only the Tcr donor plasmids: +, plasmids wereincompatible (approximately 0%o retention of the resident plasmid); -, plasmids were not incompatible (approximately 100lo retention of the resident plasmid);NT, not tested.b+, Viable autonomous minireplicator plasmids were reconstructed with the mutant 1.1-kb (inc) and the wild-type 1.6-kb (ort) plus 2.2-kb (cat) PstI fragments

or with 3.7-kb orilcat PstI fragment or with both; -, no minireplicator plasmids were obtained in multiple cloning attempts. For classes VII through XI, the + alsoindicates that the mutant plasmids were able to transform JG112 polA and were retained at 42'C in DK100ts214 polA(Ts); the - indicates that no transformantswere obtained in multiple attempts or the plasmids were lost at 42'C in DK100ts214 or both.

The copy numbers of the Rep+ recombinant minireplicator plasmids were compared with the low wild-type copy number of pRR933: +, wild-type copynumber; -, elevated copy number.

d +, RNA-E was synthesized in vitro; -, 50-fold or greater reduction in RNA-E synthesis in vitro.These mutants contained insertions or deletions in the 1.1-kb PstI inc fragment.

XI were not able to transform JG112 polA and were lost atthe nonpermissive temperature in DKlO0ts214. In addition,no viable 2-PstI-fragment minireplicator plasmids could bereconstructed with the 1.1-kb PstI fragments of mutantclasses VIII and XI. These mutants therefore were classifiedInc- Rep- (Table 2).Copy numbers of Inc- Rep' minireplicator plasmids. The

reconstituted minireplicator plasmids of mutant classes IU,III, V, and VI were transferred to strain KP245, and theircopy numbers were measured relative to the equivalentplasmids with wild-type or pRR12 1.1-kb PstI fragments(Table 3). The 3-PstI-fragment minireplicator plasmid de-rived from NR1 has the sam,e low copy number as NR1 (11,25), whereas the copy number of the 3-PstI-fragmentminireplicator plasmid derived from Inc- Cop- pRR12 wasabout six- to sevenfold higher than the wild type (Table 3).The 3-PstI-fragment minireplicators from all four mutantclasses were elevated compared with the wild type (Table 3).These mutants therefore can be classified as Inc- Rep'Cop- (Table 2). Although the copy numbers of both class Vand VI mutants were higher than the wild type, those of classV mutants were about twofold higher even than that of theirparental-type pRR12 minireplicator plasmid, whereas thoseof class VI mutants were a little lower (Table 3).The 2-PstI-fragment minireplicator plasmids (Fig. 3) gen-

erally had lower copy numbers than the equivalent 3-PstI-fragment plasmids (Table 3). However, the Cop phenotypeof the mutant plasmids was still evident. The nature of thedefect that causes a decrease in plasmid copy number afterdeletion of DNA downstream from ori (Fig. 1 and 2) is notunderstood.RNA-E synthesis by Inc- mutants. We have previously

reported in vitro and in vivo transcription experiments that

showed that RNA-E was sypthesized in a normal amount byInc- Cop- pRR12 DNA template (53, 54). In vitro transcrip-tion of RNA-E was examined by using representatives of thevarious classes of Inc- mutants. The amount of RNA-Esynthesis was estimated from densitometer tracings of theautoradiographs. In vitro RNA-E synthesis from all classesexamined, except class I, was indistinguishable from thewild-type case of pRR935. Both of the class I mutants tested(pWNRR3 and pWNRR21) had a 50- to 100-fold reduction inthe amount of RNA-E synthesized (Table 2).

Incompatibility properties of Inc- mutant plasmids. Recip-ient strains that harbored the reconstituted minireplicatorplasmids of classes II, III, V, and VI were transformed withthe pBR322 clones that contained the various 1.1-kb PstI incfragments. Plasmid pBR322 itself was compatible with all of

TABLE 3. Copy numbers of mutant minireplicator plasmidsa

Source of Relative copy no.1.1-kb PstI inc Mutant1.1-kbmPstI inC class 3 PstI 2 PstIfragment fragments fragments

NR1 Wild type 1.0 0.3 ± 0.2pWNRR2 II 8.8 ±2.7 NTpWNRR5 III 8.1 ± 1.0 4.3 ± 0.3pRR939 pRR12 6.4 ± 1.5 2.5 ± 0.3pWRRR8 V 14 ± 5 16 ± 10pWRRR3 VI 4.1 ± 0.8 2.2 t 0.3

a The construction of minireplicator plasmids composed of three PstIfragments (1.1-kb inc plus 1.6-kb ori plus 2.2-kb cat) or two PstI fragments(1.1-kb inc plus 3.7-kb orilcat) is shown in Fig. 3.

b Copy numbers were measured by cat gene dosage relative to the low wild-type copy number of the 3-PstI-fragment minireplicator plasmid from NR1,pRR933. NT, Not tested.

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TABLE 4. Incompatibility properties of Inc- mutant plasmids% of transformants retaining both donor and indicated resident plasmidsa

Donor plasmid 3-PstI-fragment minireplicators 2-PstI-fragment minireplicatorsb(class) pR3 FRpRR933 pWNRR2-1 pWNRR5-1 pRR942 pWRRR8-1 pWRRR3-1 pWFRR2 pWNRR5- pFYRR1 pWRRR8-OC pWRRR3-OC

(wild (11) (1II) (pRR12) (V) (VI) (wild OC (III) (pRR12) (V) (VI)type) type)None 47 100 100 100 100 100 4 100 97 84 87pBR322 66 100 100 100 99 100 10 100 98 85 90pRR935 (wild 0 100 100 100 100 100 0 74 69 0 85

type)pWNRR2 48 34 NT 100 NT NT NT 98 96 NT NT

(II)pWNRR5 48 100 0 0 NT NT 0 0 0 NT 0

(III)pRR939 65 100 0 0 0 98 0 0 0 0 0(pRR12)

pWRRR8 (V) 57 100 NT 100 100 100 NT 90 79 0 78pWRRR3 69 NT NT 100 73 34 NT 6 4 0 0

(VI)a Transformants (100) were picked and replica plated to test for resistance to tetracycline (donor) and chloramphenicol (resident); 100% retained the donor. In

the line marked none, the percentage of competent cells that retained the resident plasmid is indicated. NT, not tested.b A designation such as pWNRR5-OC indicates that the orilcat fusion PstI fragment was used for the construction of the minireplicator plasmnid.

the minireplicators, but the wild-type 3-PstI.fragmentminireplicator displayed- an inherent instability (Table 4),owing to the lack of the stb region (Fig. 1) required for stableinheritance of low-copy-number IncFII plasmids. The mu-tant minireplicator plasmids were more stable than thewild-type minireplicator (Table 4), probably because of theirelevated copy numbers (Table 3). The 2-PstI-fragmentminireplicators were generally less stable than the equivalent3-PstI-fragment plasmids (Table 4), probably because of thelower copy numbers of the 2-PstI-fragment plasmids (Table3).When the 3-PstI-fragment minireplicators were the resi-

dent plasmids, wild-type donor pRR935 was incompatibleonly with the wild-type resident plasmid (Table 4), and thisincompatibility reaction was very strong: 0% retention of theresident plasmid. In contrast, class II mutant donor plasmidssuch as pWNRR2 did not exclude wild-type or pRR12resident plasmids, but they did exhibit a weak incompatibil-ity reaction against their own 3-PstI-fragment minireplicatorplasmids (Table 4). The behaviors of the class III mutantswere indistinguishable from those of the pRR12 derivatives,both as donors and as residents (Table 4). A class V donorplasmid such as pWRRR8 was apparently compatible withall of the 3-PstI-fragment minireplicators, including its ownreconstituted replicon, whereas a class VI mutant donorplasmid was weakly incompatible with its own reconstitutedreplicon (Table 4). Additional cross-reactivity among theincompatibility groups was detectable when the 2-PstI-fragment minireplicators were used as residents (Table 4),probably because of their lower copy numbers, which shouldresult in more rapid segregation. For example, the class Vdonor plasmid was able to exclude its own 2-PstI-fragmentminireplicator, and class V and VI donors could be distin-guished by the latter's cross-reactivity against the pRR12(and class III) 2-PstI-fragment minireplicators (Table 4).

Trans copy number effects of Inc- mutants. The relativecopy numbers (cat gene dosage) of resident NR1-derivedplasmids were estimated both by CAT specific activity andby color on CAT indicator plates. There was no significantdifference in copy number between stb+ (pRR403) and stb(pRR933) NR1 plasmids in the presence or absence ofpBR322 (Table 5). Plasmid pRR935 completely excluded

both stb+ and stb plasmids, and a slight difference in color onCAT indicator plates between CAT' and CAT- colonieswas evident. Class I mutants not only failed to exclude theresident plasmids, but actually caused an elevation of theircopy numbers, as seen both by CAT specific activity andcolor on CAT indicator plates (Table 5). Mutants of class IIand class III had little effect, but may have slightly reducedthe copy numbers of the resident plasmids (Table 5). Theclass V and class VI mutants also were tested againstresident plasmids derived from pRR12. None caused asignificant increase in the pRR12 copy number, although theclass VI mutants may have caused a slight (30%) decrease incopy number (data not shown). Finally, although the class Vmnutant pWRRR8 was compatible with its own 3-PstI-fragment minireplicator plasmid (Table 4), the presence ofpWRRR8 in the same host cell lowered the copy numnber ofpWRRR8-1 to 6.8 + 0.3, about one-half its normal value(Table 3).

Effects of Inc- mutants on stable plasmid inheritance.Plasmid pRR403 (stb+) was inherited stably in the absence ofselection, whereas the 3-PstI-fragment minireplicator plas-mid pRR933 (stb) was not (Fig. 4), even though both had thesame copy number (Table 5). The 2-PstI-fragmentminireplicator plasmid from NR1, pWFRR2, was even lessstable than pRR933 (Fig. 4), presumably because of its lowercopy number (Table 3). Introduction of pBR322 into thesestrains had no effect on the stability of these plasmids (data

TABLE 5. Trans copy number effects of Inc- mutants

Coresident Plasmid Relative copy no. (color)aName Class pRR403 (stb+) pRR933

None 1.0 (pink) 1.2 ± 0.4 (pink)pBR322 1.1 ± 0.2 (pink) 1.0 ± 0.4 (pink)pRR935 Wild type 0.0 (white) 0.0 (white)pWNRR3 I 2.8 ± 0.6 (red) 3.3 ± 0.7 (red)pWNRR2 II 0.6 ± 0.2 (pink) 1.1 (pink)pRR939 pRR12 (III) 0.7 (pink) 0.7 + 0.3 (pink)

a Copy numbers were measured by cat gene dosage relative to pRR403 (TcsNR1), determined by enzyme specific activity. The color refers to the amountof coloration of colonies on basic fuschsin CAT indicator plates.

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not shown). The introduction of pRR939 (class III) reducedthe stability of pRR933 (Fig. 4), possibly because of theslight reduction in pRR933 copy number (Table 5). Incontrast, introduction of the class I mutant pWNRR3 causedan increase in pRR933 stability (Fig. 4), presumably becauseof the increase in pRR933 copy number (Table 5). PlasmidspRR939 and pWNRR3 had no effect on the stability of stb+pRR403 (data not shown). In each case the pBR322 deriva-tives were inherited stably. Essentially identical results wereobtained with a KP245 recA+ host (data not shown).The 3-PstI-fragment minireplicator plasmid derived from

pRR12 (pRR942) was inherited stably even without the stb+locus, whereas the equivalent 2-PstI-fragment plasmid(pFYRR1) was slightly unstable (Fig. 5). The presence ofpBR322, pRR935, or pWNRR3 had no effect on the stabilityof pRR942 (data not shown). The 3-PstI-fragmentminireplicator plasmid from a class V mutant, pWRRR8-1,was somewhat unstable (Fig. 5), despite its very high copynumber (Table 3). In addition, coresidence of pWRRR8caused pWRRR8-1 to be even more unstable (Fig. 5),presumably because of the reduction of pWRRR8-1 copynumber.

DISCUSSIONThe primary control of expression of the repAl initiator

protein of plasmid NR1 is mediated by the product of the incgene, RNA-E (Fig. 1) (19, 21, 38, 53). RNA-E inhibits thetranslation of repAl mRNA (21, 38) by binding directly tothe target sequence in the upstream portion of RNA-CX(Fig. 1) (21, 53). There is a 20-fold excess of RNA-Etranscription compared with RNA-CX (38, 54). Possiblesecondary structures of these transcripts have been pre-dicted by computer analysis (4, 34, 38), and these structuresfeature complementary single-stranded 6-base loops in bothtranscripts (Fig. 6), which may be the site of the initial

pRR403100 _ 0 ___

90 pRR933 -

806 0 2M3

z

o 50-

Er_ 40rILpR93

0 1 2 3 4 5 6 7 8NUMBER OF SUBCULTURES

FIG. 4. Stability of wild-type NR1 derivatives.

100

90pFYRRI

80 \

z4 70-

60 \ pW\PWRRR8-1

zo 50

40II- pWRRR8-1

30 - pWRRR8

20

I0

0-0 1 2 3 4 5 6 7 8

NUMBER OF SUBCULTURESFIG. 5. Stability of Inc- Cop- mutant plasmids.

contact between them (4, 38). The nucleotide sequences ofseveral Inc- Cop- mutants, such as pRR12, have basesubstitutions that would make complementary changes inthese 6-base loops (Fig. 6) (4, 13, 38, 39, 42). If the 6-baseloops formed a base-paired RNA-RNA duplex (Fig. 6), theG-C pair of NR1 at position 3 would be replaced by a U-Apair for pRR12. Similar changes in sequence have beenobserved for Cop- mutations of plasmid Ri (13, 39, 42) andR6 (4). These mutants all were selected for their increase inplasmid copy number.The wild-type incompatibility expressed by NR1 is strong,

since two differentially marked low-copy-number NR1 plas-mids cannot coexist without continuous dual selection (25,35, 45), and such conditions often lead to the selection ofInc- mutants (45). In contrast, the incompatibility betweenpRR12 plasmids is weak and is not discernible by the testused for Table 4, unless the pRR12 inc gene is cloned onto ahigh-copy-number vector plasmid, such as pBR322 (11, 12,25). This difference must result from the weaker base-stacking free energy when the G-C pair of NR1 at position 3is replaced by the U-A pair of pRR12 (Fig. 6). The higher

RNA- CX UU U t

5' t

G C5' - U U G

1 2 33' - A A C

RNA-EAt AA

C t ;5'G C

G C G - 3'4 5 6C G C - 5'

FIG. 6. Secondary structures of the target region of RNA-CXand the inhibitor region of RNA-E from NR1. The single basesubstitution in pRR12 results in complementary changes at position3 of the single-stranded loops. The possible base pairing between the6-base loops is illustrated.

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copy number of the mutant results from a need for higherconcentrations of the constitutively expressed target andinhibitor transcripts before they reach levels that are regu-lating, whereas regulation is achieved at a low copy numberfor the wild-type plasmid (4, 13, 38). NR1 and pRR12 arecompatible, even when their inc genes are cloned intohigh-copy vector plasmids (Table 4) (11, 25). This is consis-tent with the even lower base-stacking energy that wouldresult from the U-C or G-A base pairing at position 3 (Fig. 6)if the pRR12 target and NR1 inhibitor transcript, or viceversa, were paired.We have examined the properties of 134 independent Inc-

mutants (Table 2), which were selected for the loss ofexpression of NR1 or pRR12 incompatibility without con-comitant selection or demand for any other replicationfunction. The mutants obtained after hydroxylamine treat-ment appeared to result from point mutation, whereas amajority of the mutants obtained after NQO treatment haddeletions or insertions in the 1.1-kb PstI fragment.Mutants of class I, isolated from wild-type pRR935 (Fig.

2), were compatible with both NR1 and pRR12. Thesemutants were incapable of reconstituting a functionalreplicon (were Rep-), and they failed to synthesize theRNA-E inhibitor in vitro. The Rep- phenotype, associatedwith an Inc- point mutation, has not been reported previ-ously. From this class, mutant pWNRR3 has been examinedin greater detail. There is a single base substitution in thenucleotide sequence of the RNA-E transcription promoter ofpWNRR3 (38, 54), resulting in a 50- to 100-fold reduction inRNA-E synthesis. The nucleotide sequence of the incompat-ibility target of RNA-CX from pWNRR3 is unaltered (38,54), which explains the ability of this class of mutant toincrease the copy number of NR1 in trans (Table 5). TheRNA-CX transcribed from pWNRR3 titrates the RNA-Einhibitor transcribed from a coresident NR1 plasmid, and thetitration is Inc group specific (52, 53). The elevated copynumber resulted in increased stability of the 3-PstI-fragmentminireplicator from NR1 (Fig. 4).There would be insufficient RNA-E to regulate the repli-

cation of a reconstituted replicon from a class I mutant. Ifthe resulting uncontrolled plasmid replication were lethal tothe host cell, this could explain the lack of transformants (orapparent Rep- phenotype) from reconstitution experimentswith class I mutant DNA. Conditionally lethal "runaway"replication mutants of IncFII plasmids have been reported(49, 50), but the nature of the defect(s) in these mutants hasnot been determined. We have preliminary evidence that theclass I mutation of pWNRR3 can be complemented in trans.In a replicon reconstitution experiment with pWNRR3DNA, transformants are obtained only when a "helper"plasmid that can synthesize the RNA-E inhibitor is presentin the recipient cells (data not shown).Mutants of classes II and III also were isolated from

wild-type pRR935, but were Rep', and their reconstitutedreplicons had elevated copy numbers compared with thewild-type case (Table 3). Mutants of both classes still syn-thesized RNA-E in vitro, and both were compatible withNR1. However, classes III mutants were incompatible withpRR12 (Table 2), and the Inc properties of the class IIIpBR322 clones and the reconstituted minireplicator plasmidswere indistinguishable from the equivalent pRR12-derivedplasmids (Table 4). Therefore, class III mutants appear to bereisolations of the pRR12 mutation, whereas class II mutantsrepresent a new incompatibility group. Mutants of bothclasses may have caused slight reductions of the copynumber of NR1 in trans (Table 5), resulting in decreased

stability of the 3-PstI-fragment NR1 minireplicator plasmid(Fig. 4). Because class II mutant replicons had elevated copynumbers, one can speculate that a substitution ofU or A forG or C in the 6-base target loop (Fig. 6), other than thepRR12 (class III) substitution, might be responsible for theclass II mutations. Both class II and class III mutants stillregulated the replication of their respective reconstitutedreplicons, although the regulation was weaker than that ofthe wild type, and each class was self-incompatible (Table4).

Mutants of classes V and VI were isolated from pRR939(Fig. 2), which already has the pRR12 base substitution inthe 6-base target loop (Fig. 6). Both classes were compatiblewith both NR1 and pRR12 (Table 2) and were Rep+. Theirreconstituted replicons had elevated copy numbers com-pared with the wild type (Table 3), although they could behigher (class V mutants) or lower (class VI mutants) than thepRR12 minireplicators. Mutants having phenotypes of thesetwo classes have not been reported previously. The originalmutation of pRR12 resulted in the formation of a newincompatibility group, and the additional changes present inthe class V and VI mutants also have resulted in theformation of additional new incompatibility groups. Class Vmutants were still able to synthesize RNA-E in vitro. Per-haps class V mutants have a second base substitution in theregion of the 6-base loop (Fig. 6). Even class V mutants wereable to control their replication, although very weakly.However, the reconstituted minireplicator plasmids wereunusually unstable (Fig. 5). This could have resulted fromdetrimental effects to the host cells from the unusually highplasmid copy number or from some other defects not yetunderstood.Because the class VI mutant minireplicator plasmids had

copy numbers lower than those of their pRR12 parent, yetwere higher than wild-type NR1, one can speculate that oneof the U's at position 1 or 2 of the 6-base target loop wasreplaced by G or C (Fig. 6), which would produce anintermediate level of base-stacking free energy. Mutations atpositions 1 or 2 have not been reported, probably becauseonly "copy-up" mutation's were selected previously.

Mutants of classes VII through XI were isolated frompRR945 and pRR931 (Fig. 2), which contain intact NR1 andpRR12 replicons, respectively. Mutants of classes VII and Xwere Rep' and possibly were similar to the mutants inclasses II and III or V and VI, respectively.Mutants of class VIII were Rep-. However, unlike the

Rep- class I mutants, class VIII mutants were able tosynthesize RNA-E in vitro. Whereas the Rep- phenotype ofclass I mutants is likely to be the result of uncontrolledplasmid replication, which is lethal, this is not likely to be theexplanation for class VIII mutants, which contain an intactNR1 replicon. Instead, these plasmids may have "copy-down" mutations; considering that NR1 is already a lowcopy number plasmid, such mutations could be phenotypi-cally lethal to plasmid replication. Such mutants have notbeen reported previously. Recently, we have selected aseries of Rep' revertants from several of the class VIIImutants. Most of these revertants remain compatible withboth NR1 and pRR12 (data not shown), and therefore theyare likely to be second-site revertants. Further investigationof all of the mutants and revertants should help to revealmore of the details of the molecular switch that regulates theinitiation of plasmid DNA replication.

stb- plasmids such as pRR933 exhibit characteristic pat-terns of instability. When the copy number of pRR933 isdecreased or increased, either by mutation or by the influ-

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ence of a second plasmid present in the same cell, pRR933becomes more or less unstable, as expected (Tables 4 and 5,Fig. 4 and 5).Plasmid pFYRR1 was obtained by deleting approximately

700 bp of pRR942 DNA at the PstI site between ori and cat(Fig. 2), including about 300 bp from the right end of the1.6-kb PstI ori fragment (Fig. 1). The resulting 3.7-kb PstIorilcat fusion fragment was used in the replicon reconstitu-tion experiments (Fig. 3). An interesting result of this studyis that the deletion, which occurred about 500 bp down-stream from the ori and about 1.6 kb away from inc (Fig. 1),caused a reduction in plasmid copy number (Table 3). Thiscaused the plasmids to be less stably inherited (Figs. 4 and 5)and more sensitive to cross-reaction between incompatibilitygroups (Table 4), probably because of the higher frequencyof segregation of plasmids at the reduced copy number. Thenature of the defect which reduced the copy number of thesedeletion mutants is not understood, but it may be that theyhave a reduced efficiency of fruitful initiation at ori.

ACKNOWLEDGMENTSThis work was supported in part by Public Health Service research

grants GM14398 and GM30731 from the National Institutes ofHealth,and by generous grants from the Otho S. A. Sprague MemorialInstitute and the Arlo M. Bane Fund.We thank Malcolm E. Winkler and Verne A. Luckow for assist-

ance with the in vitro transcription experiments and Alan M.Easton, Gary A. Huffman, Jerrold Greenberg, Verne A. Luckow,and Yun-Liu Fan for gifts of various plasmids.

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