The repA2 gene of the plasmid R100.1 encodes a represser of plasmid replication

10, 148-155 (1983)

PLASMID

The repA

Gene of the Plasmid R100.1 Encodes a Repressor of Plasmid Replication

CHENG-PINLIU,* GORDON CHURCHWARD,AND LUCIEN CARO lhirersity

qf Geneva. Department oJMolecular

Biology. 30. Quai Ernest .-lnsermet. 121 I Geneva 4, Switzerland

Received March 21, 1983 We have constructed two miniplasmids, derived from the resistance plasmid RIOO.1. In one of these plasmids 400 bp of R 100.I DNA have been replaced by DNA from the transposon Tn 1000 (gamma-delta). This substitution removes the amino-terminal end of the repA coding sequenceof RIOO.I and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of RIOO.I. The repA gene thus encodesa trans-acting repressorfunction involved in the control of plasmid replication.

wild type Rl plasmid. Such a reduction of mutant plasmid copy number to normal levels is not observedin the presenceof R 100(Molin et al., 198I). In view of the similarity between the DNA sequencesof the two plasmids this result indicates that the site of action of the copB gene product probably overlaps the region of sequence divergence, assuming that copB interacts with the DNA sequence. The copB gene product of Rl acts to represstranscription from a promotor that overlaps the carboxyl terminus of the gene. This transcription, which proceeds in the same direction as copB transcription, results in the production of a protein, repA, that is required for replication (Light and Molin, 1982). The region of sequencedivergence between R 100 and R I within the replication genesends between the -35 and -10 regions of promotor-like sequences in both plasmids (Rosen et al., 1980; Stougaard et al., 1981a; Ryder et al.. 1982). We show that the repA coding sequence et al., 1982). It has been observed in Rl that the copB of R 100, like the copB gene of RI, encodes a gene, analogous to rep‘42, encodesa repressor function involved in the regulation of plasmid of plasmid replication (Molin et al., 198 1). A replication. A mutation in the repA gene reshort deletion within the copB gene results in sults in an increased copy number: it can be an increased plasmid copy number which is efficiently complemented by RlOO, but not reduced to normal levels in the presence of a by RI, to restore the normal copy number. Thus, the rep,42 and copB genesof R 100 and * Presentaddress:Institute of Radio-medicine, Chinese RI encode analogous repressors of plasmid replication. Academy of Medical Sciences.Beijing, China. The plasmids R 100 (also known at NR 1) and R 1 are resistance plasmids that belong to the incFI1 incompatibility group. They are closely related and share extensive sequence homology (Sharp et al., 1973). Their replication genes are clustered in a small, 3-kb region of the plasmid (Taylor and Cohen, 1979;Molin et al., 1979).This region has been sequenced in both plasmids (Rosen ef al., 1980, Stougaard et al., 198la; Ryder et al., 1982)and the DNA sequencesare very similar. For each plasmid the replication region contains three coding sequences,denoted repAl. rep‘42, and repA in the caseof RlOO (Rosen et a/., 1980).The synthesisof proteins by these coding sequenceshas been observed in vitro (Brawner and Jaskunas, 1982). The only extensive sequencedivergence between the two plasmids in the replication region begins within the rep‘42 coding sequenceand extends 26 bp beyond the carboxyl terminus (Ryder

0147-619X/83 $3.00 Cop)r,ght Ml n&s

c I983 hy Academtc Press. Inc. of reproducuon m any form wsencd.

148

PLASMID

MATERIALS

REPLICATION

AND METHODS

Bacterial Strains and Plasmids

The bacterial strains used were C600 thr leu thi lacy supE and CSHSO pro lac rpsL (Miller, 1972).The plasmids RlOO.1 (TcR SpcR SmRCmR HgR) and Rl. 16 (KanR) were from our collection. These are derivatives of RIO0 and Rl that are derepressedfor transfer. Bacteria were grown on LB medium (Lennox, 1955) supplemented with 40 pg/ml streptomycin, 25 pg/ml spectinomycin, 25 pg/ml ampicillin, or 50 pg/ml kanamycin as appropriate. The pLC682 plasmid was constructed as follows. A plasmid, pLC64, carrying the EcoRI B fragment of R 100.1, which contains the origin of replication (Taylor and Cohen, 1979), constructed by D. Lane and M. Chandler (unpublished results), was partially digested with PstI and completely digested with EcoRI. A second plasmid, pBR322 (Bolivar et al., 1977) carrying a large PstI fragment consisting of half the resistancedeterminant of R 100.1,was cleaved with EcoRI and PstI. One of the products of such a digestion should be an EcoRIPstI, RlOO.1 fragment that carries the gene(s) specifying resistance to streptomycin and spectinomycin (Taylor and Cohen, 1979).The two digestswere mixed, ligated, and following transformation, plasmids specifying resistance to streptomycin were isolated. One such plasmid, pLC68, that consisted of the R1OO.l minimal replication region ligated to the streptomycin/spectinomycin-resistance fragment, was purified and an EcoRI fragment specifying resistanceto ampicillin (Timmis et al., 1975) was inserted into its unique EcoRI site to yield pLC68 1. This plasmid contained a unique BamHl site near the streptomycin resistance gene, and a Hind111 site within the EcoRI ampicillin-resistance fragment. A small BamHI-Hind111 fragment cloned from the bacteriophagelambda which contains attX was inserted between these two sites to yield pLC682. A 200-bp Hind111 fragment was deleted from the streptomycin resistancesegment of pLC68 1 during this last step.

REPRESSOR

149

The plasmid pLC600 is pBR322 carrying two PstI fragments that contain all the information required for replication of RlOO. 1 (Taylor and Cohen, 1979). A plasmid carrying an insertion of the transposon TnlOOO (gamma-delta) into the repA coding sequence of pLC600 was isolated following conjugal mobilization of pLC600 by the F plasmid (Guyer, 1978).The site of insertion of Tn 1000 in 50 pLC600 plasmids was determined by restriction analysis. One of these insertion plasmids is pLC62 1 (Fig. 1). DNA of this plasmid was digestedwith BglII and recircularized to delete most the Tn 1000sequencesresulting in pLC633 (Fig. 1). To construct pLC683, DNA of pLC682 was digested with PstI and mixed with a lo-fold excessof P&digested pLC633 DNA. Following ligation and transformation, DNA from SpcRApR transformants was prepared and examined for the presence of the 0.9-kb PstI fragment from pLC633. Estimation

of Plasmid Copy Number

The percentageof covalently closed,circular plasmid DNA was estimated by cesium &IOride-ethidium bromide density gradient centrifugation of sheared lysates as described by Womble et al. (1977). Cultures were grown in M9 glucose medium (Adams, 1954) supplemented with 0.4% casamino acids (Difco) and 25 pgg/mlampicillin at 37°C. Single-cell resistance to ampicillin was determined by plating dilutions of fresh overnight cultures on plates containing different concentrations of ampicillin. Heteroduplex Analysis

The formation of heteroduplexes and their analysis by electron microscopy was performed as described previously (Chandler et al., 1977). Other Techniques

Restriction enzyme digestswere performed in 10 tnM Tris-HCl, pH 7.4, containing 10

150

LIU, CHURCHWARD,

Rsd

a

AND CAR0

PstI

EcoRI t

HindIll

BornHI

EaRI

pLC683

ECORI I

Pstl

PslI

:

Bg/U

repA2: EcoRt

Pstl

--

EcoRI

PstI

ECORI

I

La’

PSlI

---.__ : PstI

-. Bglu

pLC600

‘. -_ “--@gin

repn2::1n1000 : Psll

b

PSlI

ECORI

I pLC621 ( pLC600 : : TnlOOO )

,A _’

ECORI pLC633 I (pLC621

ABgUI )

FE. I. Restriction maps of plasmids. Heavy black regions denote the replication region of R 100.I ; white regions denote TnlOOO DNA. (a) The structures of pLC682 and pLC683 are shown demonstrating the DNA substitution that replaces part of the repA coding sequence. The asterisks indicate that only those RsaI sites used in the mapping of the substitution are shown. (b) The structures of the pLC600 plasmid and its derivatives used in the isolation of a PslI fragment containing a mutated repA coding sequence are shown. An insertion of Tn too0 into the repA gene of pLC6OO was isolated resulting in pLC621. Digestion of pLC62 I with BgrIl followed by recircularization resulted in the deletion of most of the Tn 1000 DNA of pLC62 I to give pLC633. Only the extremities of TnlOOOare presented in the structure of pLC62 I.

mM MgC&, 50 mM NaCI, and 6 mM /3-mercaptoethanol. This buffer was supplemented with 0.5 tnM ATP for ligation reactions. Transformations were performed according to the method of Cohen et al. ( 1972). Gel electrophoresis was as described previously (Chandler et al., 1977). RESULTS

Isolation of a repA Deletion Mutant The structures of two miniplasmid derivatives of RlOO. 1 are presented in Fig. 1a. The pLC682 plasmid, of 11.2 kb, consists of a 4.1kb segment containing the replication functions of R 100.1 ligated to DNA fragments encoding resistance to the antibiotics streptomycin, spectinomycin, and ampicillin. It also contains a small DNA segment carrying the prophage attachment site, attX, cloned from the bacteriophage X. The construction of pLC682 is described under Materials and Methods. The replication region consists of two PstI fragments of 1.6 and 1.1 kb that con-

tain all the information necessaryfor regulated replication of R100.1 (Taylor and Cohen, 1979; Miki et al., 1980). The repA coding sequence lies entirely within the 1.I-kb PstI fragment. In order to study the function of this putative replication control gene, we constructed a plasmid, pLC683, in which approximately one-third of the l.l-kb PstI fragment of pLC682 had been replaced by DNA from the transposon Tn 1000(gamma-delta) beginning at a site within the repA coding sequence. An insertion of TnlOOO into the cloned repA genecarried by pLC600 was isolated, resulting in pLC621 (Fig. 1b). Following deletion of most of the Tn 1000 sequencesfrom this plasmid (pLC633; Fig. lb) a 0.9~kilobase PstI fragment carrying part of the repA coding sequencewas used to replace the 1.1-kb PsrI fragment of pLC682. The details of these constructions are given under Materials and Methods. From the published sequence data (Rosen et al., 1980) cleavage of pLC682 with RsaI

PLASMID REPLICATION

and BglII should produce an RsaI-BglII fragment of 5 10 bp which contains the entire repA gene (Figs. la; 2a, top arrow). The second RsaI-BglII fragment of pLC682 is probably the third band in lane 2 (Fig. 2) as this seems slightly smaller than the equivalent band of pLC683. Since TnlOOO contains an RsaI site 30 bp from each end of the transposon (Reed et al., 1979) cleavageof pLC683 with RsaI should result in a new fragment that carries part of the rep42 gene (Figs. la; 2a, bottom arrow). This fragment is 320-bp in length as determined by electrophoresis in polyacrylamide gels (Fig. 2a). No cleavage of pLC683 is observed with BglII (data not shown) consistent with the replacement of part

151

REPRESSOR

of the l.l-kb PstI fragment of pLC682 by TnlOOO DNA in pLC683. The size of this new RsaI fragment allows us to define the extent of the substitution in pLC683. A segment of DNA of some 440 bp in pLC682 has been replaced by approximately 230 bp of TnlOOO DNA (A PstI site is located 230 bp from one end of TnlOOO, G. Churchward, unpublished results). This substitution removes some 27, out of 86, codons from the amino-terminal end of repA and sequencesupstream of the gene. To ensure that no other rearrangementshad occurred during the construction of pLC683, DNA of pLC682 and pLC683 was digested with EcoRl, denatured. and renatured. The

a

3.8OtO.16

622

0.392 0.06 1 n u f 0.29* 0.05

7.00r0.25

527

309 FIG. 2. Structure of pLC682 and pLC683. (a) Restriction enzyme digests of both plasmids. Lane I, pBR322 digested with MspI; Lane 2, pLC682 digested with BgU and R.raI; Lane 3, pLC683 digested with Rsd. The upper arrow indicates the 510-bp RsuI-&/II fragment of pLC682 that contains repA2. The lower arrow indicates the new RsuI fragment present in pLC683. (b) Heteroduplex between EcoRldigested pLC682 and pLC683 DNA. The interpretative drawing below gives the size of the various features in kilobases (kb).

152

LIU. CHURCHWARD,

resulting heteroduplexes were examined by electron microscopy. The only region of nonhomology between the two plasmids corresponds to that predicted from restriction enzyme analysis (Fig. 2b). Copy Number of the Wild-Type and Mutant Plasmids

To demonstrate the effect of the substitution, cells carrying either pLC682 or pLC683 were labeled with [3H]thymidine and sheared lysates (Womble et al., 1977), prepared from the cultures, were centrifuged in cesium chloride-ethidium bromide density gradients. The amount of covalently closed, circular (CCC) DNA in each lysate was determined. Representative gradient profiles are presentedin Fig. 3. It is evident that there is more CCC DNA in the lysate prepared from cells containing pLC683 (Fig. 3b). The quantity of CCC DNA in each gradient, normalized to the amount of linear (chromosomal) DNA, indicates an increase in plasmid DNA content of IO-fold in the culture containing pLC683. It seems, therefore, that the deletion of part of the repA gene results in an increased plasmid copy number. This is confirmed by measurements of single-cell resistance to ampicillin conferred by each of the two plasmids. It has been shown, at least for Rl, that the level of resistance to ampicillin reflects the plasmid copy number (Uhlin and Nordstram, 1977). Cells containing pLC682 are resistant to a maximum of

AND CAR0 TABLE

I

SINGLE CELL RESISTANCETO AMPICILLIN Strain CSHSO/pLC68? CSH50/pLC683 CSH50/pLC682 + CSHSO/pLC683+ CSH50/pLC682 + CSHSO/pLC683+

R 100.I RlOO.1 RI. 16 RI.16

AP

Ap + Tc

200 800 200 150 200 600

200 150

ApfKan

200 400

Now The resistancelevel is expressedin microgramsper milliliter (&ml) ampicillin and is that concentration of ampicillin which tint produces a reduced efficiency of plating. Afier growth in selective media (for both plasmids.where appropriate) bacteria were plated for singlecolonies on either ampicillin alone, or on ampicillin + tetracycline. or ampicillin + kanamycin.

200 pg/ml of ampicillin, whereas cells containing pLC683 are resistant to 800 &ml of ampicillin, a fourfold increase (Table 1). If RIOO.l is introduced into the cells, the resistance level of cells carrying pLC683 is reduced to 150 &ml, with or without selection for the RlOO.l plasmid. No effect of RlOO.1 is observed on pLC682. Thus the defect in pLC683 resulting in increased copy number is complemented by RlOO. 1. The lower resistance level of pLC683, compared to that of pLC682, in the presence of RlOO. 1 (Table l), is presumably due to the removal of the repA promotor in pLC683, resulting in a lower level of transcription of the replication genes of the plasmid. A similar effect has been observed in copB deletion mutants of RI (Riise et al., 1982).

FIG. 3. Dye-c&urn chloride density gradient centrifiugation of bacterial lysates. The arrows indicate the position of CCC DNA in the gradients. In both panels the scale of the right-half of the gradient containing the chromosomal DNA peak has been reduced by a factor of 5. (a) pLC682, (b) pLC683.

PLASMID

REPLICATION

If R 1.16 is introduced into cells carrying pLC683 a reduction in resistance level is observed, but this effect is less marked in the absenceof continuous selection for both plasmids. The plasmid function responsible for this is most likely the product of the copA gene that determines plasmid incompatibility (Stougaardet al., 198lb; Danbara et al., 1981), although it is possible that there is some crossreactivity between the copB gene product of Rl and pLC683. In the absenceof selection, incompatibility between the two plasmids probably results in the segregation of Rl and the growth of mixed colonies. These would contain cells with pLC683 alone which would confer an increased level of resistance to ampicillin. Such an effect is not observed with R 100.1, presumably because of a weaker incompatibility between pLC683 and RlOO. 1 due to the lower copy number of pLC683 in cells harboring R 100.1. We conclude from thesedata that the repA gene of Rl00.1 encodes a plasmid function involved in the negative regulation of plasmid replication. DISCUSSION

The results presented here show, by two different criteria, that the copy number of an Rl00.1 plasmid lacking a functional repA gene is increased. The fraction of CCC DNA in lysatesof bacteria carrying pLC682 is 0.8% in the experiment shown in Fig. 3a. In lysates of bacteria carrying pLC683 this fraction is increased to 8% (Fig. 3b). We have previously measured by DNA-DNA hybridization the copy number of R 100.1 at I.5 copies per chromosomal origin of replication, under identical conditions (T. Goebel, G. Churchward, and L. Caro, unpublished data). Assuming a doubling time of 30 min and a time of 40 min for a replication fork to move from the origin to the terminus of replication (Chandler et al., 1975) one can calculate that there are 1.5 origin sequences per genome equivalent of DNA (see, for example, Bremer and Churchward (1977)). Since Rl00.1 has a length of 94 kb and the Escherichia co/i chromosome a length of ap-

REPRESSOR

153

proximately 3200 kb, RlOO. 1 DNA should represent6.6% of the total cellular DNA under these conditions. Since pLC682 is only 11.2 kb rather than 94 kb the expected fraction of CCC DNA in lysates prepared from strains carrying this plasmid would be 0.8%, in good agreement with what is observed. Assuming that most of the plasmid DNA present in the lysates is present in the CCC DNA band in the gradients, pLC682 has the same copy number as the parental RlOO. 1 plasmid. Measurements of single-cell resistance to ampicillin show a fourfold increase in resistance level, specified by pLC683 compared to pLC682. For the P-lactamaseencoded by Tn3, which is present on R 1, Uhlin and Nordstrom ( 1977) have demonstrated a good correlation between resistance level and plasmid copy number. This correlation has not been demonstrated explicitly for the p-lactamase encoded by the Staphylococcus aureus plasmid ~I258 (Timmis et al., 1975), used in the construction of pLC682 and pLC683 but the data of Ogura et al. ( 1980) show that a correlation exists between pI258-derived /3-lactamaseactivity in cell extracts and plasmid copy number in E. coli. In view of the smaller difference in copy number indicated by the single cell resistance level compared to that detected by measurement of CCC DNA it seemspossible that single cell resistanceusing this /3-lactamase doesnot accurately reflect plasmid copy number. However, the single-cell resistance can certainly be usedto detect qualitative changes in copy number. The increased resistance level normally conferred by pLC683 is not observed in cells harboring both pLC683 and RIOO.1 (Table 1). This shows that the defect in replication control of pLC683 can be complemented by R 100.1 since we have not detected significant levels of recombination between the two plasmids during such experiments. The single-cell resistance of cells carrying both pLC683 and R 1.16 is partly reduced in comparison to that of cells carrying pLC683 alone. This might suggestthat the copB gene product of Rl retains some activity on R 100.1 DNA although since the target sites of the two plasmids prob-

154

LIU, CHURCHWARD,

ably have extensive differences in nucleotide sequence (Light and Molin, 1982; Ryder et al., 1982) this seemsunlikely. Both R 100 and R 1 encode a separatefunction, copA, involved in plasmid incompatibility and copy number control (Miki et al., 1980; Danbara et al., 1981; Stougaard et al., 1981b). As has been observed for the copB gene product of R 1 (Riise et a/., I982), the repA gene is not involved in the expression of incompatibility. The pLC62 1 plasmid carrying an insertion of TnlOOO into the repA gene still expressesthe same incompatibility toward R 100.1 as pLC600 as measured by segregation, and by the inhibition of replication from an integrated R 100 plasmid in a dnuA’” strain (Molin and Nordstram, 1980: data not shown). The pLC600 and pLC62 1 plasmids are both high copy number pBR322 plasmids and thus expressstrong, copA-mediated incompatibility toward RlOO and Rl. The copy number of these plasmids would not be expected to be affected by the presenceof R 100 or R 1 in the same cell. In contrast, the presence of RlOO or Rl would be expected to affect the copy number of pLC682 or pLC683 due to the action of copA. This presumably explains the effect of Rl on the copy number of pLC683. We cannot explain why the presence of Rl or RIO0 does not seem to affect the copy number of pLC682. The repA2/copB system seemsto act as a switch between low and high copy number modes of replication, whereas the copA product acts to maintain a constant copy number in either mode (Riise et al., 1982). Because these plasmids are transmissible, one would expect a zygotic induction effect on plasmid replication upon the introduction of the plasmid into a new host cell. Plasmid replication would commence at a high rate until sufficient repA2/copB product had accumulated to repress the rate of replication. The two modes could be viewed as establishment and maintenance replication. The fact that the repA copB functions seem to possessplasmid specificity means that, for example, an RlOO plasmid might establish itself at the expense of a

AND CAR0

resident Rl plasmid, which could be preferentially lost due to a lower replication rate and cop.4-mediated incompatibility between the two plasmids. In this regard it is interesting to note that a similar dual control of plasmid replication exists in the caseof the mobilizable plasmid colEI (Cesareni et al., 1982). ACKNOWLEDGMENTS We are grateful to Edith Gallay for performing the electron microscopy. We also thank Otto Jenni for preparing the figures and Monique Visini for typing the manuscript. This work was supported by Grant 3.169.08 I from the SwissNational Science Foundation. Chengpin Liu was the beneficiary ofa fellowship from the World Health Organization. REFERENCES ADAMS, M. H. (1959). “Bacteriophages.” p. 446. Interscience, New York. BREMER,H., .&ND CHURCHWARD,G. (1977). An examination of the Cooper-Helmstetter theory of DNA replication in bacteria and its underlying assumptions. J. Theor. Biol. 69, 645-654.

BOLIVAR,F.. RODRIGUEZ,R. L.. GREENE,P. J., BETLACH, M. C., HEYNEE;ER,H. L.. BOYER, H. W., CROSA. J. H., AND FALKOW,J. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-I 13. B~WNER, M. E., AND JASKUNAS,S. R. (1982). Identification of polypeptides encoded by the replication region of resistance factor RIOO. J. Ifool. Biol. 159, 3555.

CESARENI,G., MUESING,M. A., AND POLISKY.B. (1982). Control of colE I DNA replication: the rop gene product negatively affects transcription from the replication primer promoter. Proc. Nat/. .dcad. SC?.L!S.4 79,63 I36317. CHANDLER,M., BIRD, R. E., AND CARO, L. (1975). The replication time of the Escherichia coli K-12 chromosome as a function of the cell doubling time. J. Mol. Biol. 94, 127-132.

CHAPIDLER.M., ALLET, B., GALLAY, E., BOYDELATOUR, E., AND CARO, L. (1977). Involvement of ISI in the dissociation of the r-determinant and RTF components of the plasmid RIOO.I. Mol. Cm. Genef. 153, 289295.

COHEN,S. N., CHANG, A. C. Y., AND Hsu, L. (1972). Non-chromosomal antibiotic resistancein bacteria: genetic transformation of E. coli by R factor DNA. Proc. Natl. Acad. Sci. USA 69, 21 IO-21 14.

DANBARA,H., BRADY, G.. TIMMIS, J. K., AND TIMMIS, K. N. ( I98 I). Regulation of DNA replication: “target” determinant of the replication control elements of plas-

PLASMID REPLICATION mid R6-5 lies within a control element gene.Proc. Nut/. Acad. Sci. USA 78,4699-4103.

GIJYER,M. S. (I 978). The yb sequenceof F is an insertion sequence.J. Mol. Biol. 126, 341-365. LENNOX, E. S. (1955). Transduction of linked genetic charactersof host by bacteriophageP 1. Virology 1, I90206.

J., AND MOLIN, S. (1982). The sites of action of the two copy number control functions of plasmid R I.

LIGHT,

Mol. Gen. Genet. 187, 486-493.

LIGHT,J., NORDSTROM, M., ANDNORDSTROM, K. ( 198I). Identification and characterization of new copy mutants of plasmid RI and identification of a polypeptide involved in copy number control. ilfol. Gen. Genef. 181, 123-130. MIKI, T., EASTON,A. M., AND ROWND, R. H. (1980). Cloning of replication, incompatibility, and stability functions of R plasmid NRI. J. Bacreriol. 141, 87-99. MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MOLIN, S., AND NORDSTROM,K. (1980). Control of plasmid RI replication. Functions involved in replication, copy number control, incompatibility and switch-off of replication. J. Bacterial. 141, I I I-120. MOLIN, S., STOUGAARD,P., UHLIN, B. E., GUSTAFSSON, P., AND NORDSTROM,K. ( 1979). Clustering of genes involved in replication. copy number control, incompatibility, and stable maintenance of the resistance plasmid Rldrd19. J. Bacterial. 138, 70-79. MOLIN, S., STOUGAARD,P., OGURA,T., MIKI, T., AND MIRAGA, S. (1980). Copy number mutants of the plasmid carrying the replication origin of the Escherichiu coli chromosome: Evidence for a control region of replication. Proc. Narl. Acad. Sci. USA 77, 3993-3991. REED, R. R.. YOUNG, R. A., STEITZ,J. A., GRINDLEY, N. D. F., AND GUYER, M. S. (1979). Transposition of the Escherichia co/i insertion element yd generates a five-base-pair repeat. Proc. Natl. .&ad. Sci. USA 76,

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tional characterization of a copy number control gene (copB) of plasmid Rl. J. Bacferiol. 151, 1136-I 145. ROSEN,J., RYDER,T., INOKUCHI,H., OHTSUBO, H., AND OHTSUBO, E. (1980). Genes and sites involved in replications and incompatibility of an RIO0 plasmid derivative based on nucleotide sequence analysis. Mol. Gen. Genet. 179, 527-537.

RYDER,T. B., DAVISON,D. B., ROSEN,J. I., OHTSLJEQ E., AND OHTSUBO,H. (1982). Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids of Escherichiu coli. Gene 17,229-3 IO. SHARP,P. A., COHEN,S. N., AND DAVIDSON,N. (1973). Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. II. Structure of drug resistance(R) facton and F factors. J. Mol. Biol. 75, 235-255.

STOUGAARD,P.. MOLIN, S., NORDSTROM,K., AND HANSEN, F. G. (198 la). The nucleotide sequence of the replication control region of the resistance plasmid Rldrd19. Mol. Gen. Genet. 181, 116-122. STOUGAARD, P., MOLIN, S., AND NORDSTROM, K. ( 198I b). RNAs involved in copy-number control and incompatibility of plasmid RI. Proc. N&l. Acad. Sci. USA 78, 6008-60 12. TAYLOR,D. P., AND COHEN,S. N. (1979). Structural and functional analysis of cloned DNA segmentscontaining the replication and incompatibility regions of a miniplasmids derived from a copy number mutant of NR I. J. Bacterial. 137, 92-104. TIMMIS, K., CAPBELLO,F., AND COHEN, S. N. (1975). Cloning, isolation and characterization of replication regionsof complex genomes.Proc. Natl. .4cad Sci. USA 72,2242-2246.

UHLIN, B. I!., AND NORDSTROM,K. (1977). R plasmid gene dosageeffectsin Escherichiu co/i K- 12: copy mutants of the R plasmid Rldrd-19. Plasmid 1, 1-7. WOMBLE,D. D., TAYLOR, D. P., AND ROUND, R. H. ( 1977).Method for obtaining more accurate covalently 4882-4886. closed circular plasmid to chromosome ratios from RIISE, E., STOUGAARD,P., BINDSLEV.B., NORDSTROM, bacterial lysatesby dye-bouyant density centrifugation. K., AND MOLIN, S. ( 1982).Molecular cloningand funcJ. Bacterial. 130, 148-153.