Analysis of a recessive plasmid copy number mutant: evidence for negative control of col E1 replication

Cell, Vol. 16. 267-275.

October

1979,

Copyright

0 1979

by MIT

Analysis of a Recessive Plasmid Copy Number Mutant: Evidence for Negative Control of Col El Replication H. Michael Shepard, David H. Gelfand and Barry Polisky Program in Molecular, Cell and Developmental Biology Department of Biology Indiana University Bloomington, Indiana 47405

Summary The Col El -derivative copy number mutant plasmid pOPlA6 has been used to investigate the control of plasmid replication. pOPlA6 normally exists at about 200 copies per chromosome, while the wildtype plasmid from which it was derived (pBGP120) exists at about 15 copies per chromosome. We have observed that in E. coli containing both pOP1 A6 and pBGP120, the copy number of pOP1 A6 is lowered to 4-6 copies per chromosome. Thus the mutation in pOPlA6 is recessive. The association between the two plasmids is stable in E. coli, indicating that incompatibility properties as well as replication control characteristics have been altered in pOPlA6. Coresidence of the unrelated plasmid pSC101 with pOPlA6 has no detectable effect on pOPlA6 copy number. These results suggest that a plasmid-specific, diffusible repressor may act negatively to control plasmid copy number, and that pOPlA6 produces a defective repressor or is altered in repressor synthesis. We have constructed in vitro a plasmid which is identical in size to pQPlA6 but contains a replication origin region derived from pBGPl20. Since this plasmid, pNOP1, exists stably (like pBGPl20) at lo-15 copies per chromosome, the high copy number of pOPlA6 is not related to its reduced size relative to pBGP120. To localize the mutation in pOPlA6 responsible for DNA overproduction, we have cloned fragments of pBGPl20 into pOPlA6 and selected for plasmids with wild-type copy number. We find that a 2.0 kh region of pBGP120 DNA surrounding the origin of plasmid DNA replication is capable of suppressing the DNA overproducer phenotype of pOPlA6. The 2.0 kb fragment is capable of independent selfreplication or can integrate into pOPlA6 in vivo to form a composite plasmid with two origins of replication. The overproducer phenotype of pOPlA6 is suppressed in either configuration.

The study of the control of DNA replication requires a simple and easily manipulated experimental system. Extrachrosomal elements of E. coli are useful for such studies because they have limited genetic complexity, are stably maintained at a constant intracellular copy

number and can be genetically manipulated in vivo and in vitro to generate replicons altered in replication control (Nordstrom, Ingram and Lundback, 1972; Kool and Nijkamp, 1974; Morris et al., 1974; Uhlin and Nordstrom, 1978; Andreoli et al., 1978; Gelfand et al., 1978). We have previously described the isolation and characterization of two plasmid copy number mutants of the 17.2 kb Col El derivative pBGP120 (Polisky, Bishop and Gelfand, 1976; Gelfand et al., 1978). The first of these Cop- mutants, designated pOP1, is identical in size and sequence organization to pBGPl20 but is present in exponentially growing cells at about 74 copies per chromosome, compared to about 15 copies for pBGP120. pOP1 is structurally unstable in certain genetic bckgrounds, breaking down to smaller plasmids which retain the overproducer phenotype. The best chracterized of these is pOP1 A6, a 6.9 kb plasmid which exists at 210 copies per chromosome (Figure 1). We have carried out experiments to probe the copy number control function altered in pOPlA6. In this paper we show that the overproducer phenotype is recessive; in E. coli containing both pBGP120 (Cop’) and pOPlA6 (Cop-). the copy number of pOPlA6 is reduced to levels equivalent to or less than that of pBGP120 alone. The reduction is plasmid-specific since it does not occur with the unrelated plasmid pSC101. Using in vitro recombination techniques, we have localized the Cop- mutation of pOP1 A6 to a 2.0 kb self-replicating DNA sequence surrounding the origin of replication of the plasmid. Results Cis-Trans Test of the pOPlA6 Mutation The high copy number of pOPlA6 (Table 2) could result from one of three classes of mutations in the plasmid genome: alterations in a plasmid-specified element positively required for plasmid replication (Jacob, Brenner and Cuzin, 1963); mutations in negatively acting elements of plasmid replication (Pritchard, Barth and Collins, 1969; Uhlin and Nordstrom, 1975, 1978); or mutations affecting the target(s) of positive or negative regulatory elements. To investigate how replication control of pOPlA6 was altered, strain DG75 was co-transformed with pOPlA6 and pBGPl20 DNAs. Transformation was carried out with a lo-fold molar excess of pOPlA6 DNA (ApRLacc; Table 1) relative to pBGP120 DNA (ApRLac+; Table 1). Transformants were selected on lac-minimal A plates containing 50 pg/ml ampicillin. Approximately 20% of the ApRLac’ colonies contained both pOP1 A6 and pBGP120. Both plasmids in cleared lysates from co-residents were Cop’ (Figure 2, lane 1; compare with lanes 2 and 3, which contain cleared lysates from DG75 carrying pBGP120 or

Cdl 268

12

14

16

17.2

I z

POPlh6 -

o@---poPl

----,---

[I

!

--I 4 -4 4

pOP1 A6, respectively). A co-resident clone, designated PSOOl , was studied further. Quantitative determination of total plasmid copy number in PSOOl confirmed the qualitative conclusion from cleared lysates that both plasmids were Cop+. pOPlA6 and pBGPl20 together comprise 1.5% of the total intracellular DNA in PSOOl (Table 2). Densitometric analysis of ethidium bromide-stained bands in agarose gels of cleared lysates prepared from PSOOl indicated that copy numbers were 5 and 2 for pOP1 A6 and pBGPl20, respectively. These results indicate that the Cop- phenotype of pOPlA6 is suppressed when it co-resides with pBGP120. Since related plasmids are usually incompatible, it was surprising to observe apparent compatibility between pOPlA6 and pBGPl20. To determine the stability of the co-residence, we took advantage of the observation that pBGP120 confers a Lac+ phenotype of DG75 while pOPlA6 does not (Table 1; Gelfand et al., 1978). Segregation of pBGPl20 from pOPlA6 can be quantitated easily on lactose indicator plates, since loss of pBGP120 results in a Lac- phenotype for PSOOl . Over a period of 100 generations in nonselective medium (minimal medium E), the segregation frequency of pBGPl20 from pOP1 A6 was t2 x 1 Oe4 per cell (data not shown). These results indicate a high degree of stability in the association and suggest that pOPlA6 may be altered in incompatibility properties as well as in replication control. It was conceivable, although a remote possibility, that the 6.9 kb plasmid in PSOOl was a Cop+ revertant of pOPlA6. To investigate this, supercoiled DNA was isolated from PSOOl and the 17.2 and 6.9 kb plasmid DNA species were separated by neutral sucrose gradient centrifugation as described in Experimental Procedures. The plasmid DNA content of the gradient fractions was monitored by agarose gel electrophoresis. Peak fractions containing either the 17.2 kb species (pBGP120, designated pool A) or the 6.9 kb species (pOPlA6, designated pool B) were used to transform DG75. Transformants were plated on lacminimal A plates containing 50 pg/ml ampicillin. Each

Figure tives

1. Structure

of pBGPl20

and Deriva-

Plasmid designations and Cop phenotype of each plasmid are shown on the left. Dotted lines indicate sequences in common between the deletion mutant and the parent plasmid. W indicates a 140 kbp inverted repeat which flanks the Tn3 transposon (Heffron et al.. 1977). (X1 indicates a portion of the 82 region originating in X plac 5 (Polisky et al., 1976). P. 0. and 2 indicate the promoter, operator and structural gene for fl-galactosidase of E. coli. All Hae II and Hae Ill sites are shown only for pNOPl0. Cop phenotype was determined as described in Experimental Procedures. The prefix OP designates a Cop- phenotype; an NOP prefix designates a Cop+ phenotype.

of the ApRLac+ transformants examined from pool A DNA contained only a 17.2 kb Cop+ plasmid species (Figure 2, lanes 4 and 5). Similarly, the ApRLactransformants from pool B DNA contained only a Cop6.9 kb plasmid species (Figure 2, lanes 6 and 7). These results rule out the possibility that reversion was responsible for the Cop+ phenotype of pOPlA6 when pOPlA6 co-resided with pBGPl20 in DG75. To investigate whether suppression of pOPlA6 in trans was specific for related plasmids, DG75 was cotransformed with pOPlA6 and the unrelated plasmid pSClO1 which confers resistance to tetracycline (TcR). Cleared lysates from ApRTcR transformants were examined by agarose gel electrophoresis. Figure 2, lane 8 shows a representative lysate, demonstrating that pSClO1 has no detectable effect on the copy number of pOP1 A6. These results support the conclusion that pOPlA6 carries a mutation in a repliconspecific, negative control function of replication, and that the mutation is complemented by the wild-type gene product in trans. Localization of Replication Control Sequences on pOPlA6 and pBGPl20 We used in vitro recombination techniques to map the cop mutation in pOP1 A6. Both pBGP120 and pOP1 A6 contain single recognition sites for Eco RI and Barn HI restriction endonucleases (Gelfand et al., 1978; Figure 1). Double digestion of pOPlA6 generates two fragments of 5.2 and 1.7 kb; similar treatment of pBGPl20 generates 12.0 and 5.2 kb fragments. In both plasmids, the 5.2 kb fragment contains the Col El-derived origin of replication and part of the Tn3 transposon (Figure 1). The 1.7 kb fragment of pOP1 A6 contains Tn3 sequences encoding /?-lactamase and a small segment of the E. coli /?-galactosidase structural gene. The 1.7 kb fragment is not capable of self-replication (our unpublished observations). The 5.2 kb Eco RI-Barn HI fragments of pOPiA6 and pBGP120 and the 1.7 kb Eco RI-Barn HI fragment of pOPlA6 were isolated separately by preparative agarose gel electrophoresis. The 5.2 kb fragments

Control 269

Table

of Col El Replication

1. Characteristics

of Col El, pBGPl20

and Its Derivatives

Colicin Plasmid

kb

Apa = Productior?

Immunity’

Lac’

Copd +

6.3

-

+

+

-

pBGPl20

17.2

+

-

+

+

i

POP1

17.2

+

-

+

+

-

-

-

Col El

POP1 A6

6.9

+

-

+

pNOP1 pNOPl0

6.9

+

-

+

-

+

2.0

-

-

+

-

+ + +

pNOPl1

6.9

+

-

+

-

pNOPl2

8.9

+

-

+

-

a Resistance to 50 pg/ml ampicillin in plates containing minimal E agar b As shown by Polisky et al. (1976). ’ As described in Experimental Procedures. d Cop’ denotes wild-type copy number and Cop- denotes an overproducer plasmid.

were then ligated separately to the 1.7 kb fragment in vitro using T4 DNA ligase. The ligation mixtures were used to transform DG75, and ApR transformants were selected. Ligation of the 5.2 and 1.7 kb fragments of pOP1 A6 is expected to recreate a 6.9 kb Cop- plasmid identical to pOPlA6. If the cop mutation were located in the 5.2 kb fragment of pOP1 A6, analogous ligation of the 5.2 kb pBGP120 fragment would be expected to generate a 6.9 Cop’ plasmid. Figure 3A (lanes B-F) shows DNA present in cleared lysates from five ApR colonies derived from transformation with the p6GPl20 5.2 kb fragment ligated to the 1.7 kb fragment. Each clone contained a 6.9 kb Cop’ plasmid. The plasmid from one clone was designated pNOP1. Figure 38 (lanes B-F) shows DNA present in clones derived from the pOPlA6 5.2 kb fragment ligated to the 1.7 kb fragment. Each clone contained a Cop- plasmid identical to pOPlA6. A total of 50 transformants from the two ligation mixtures were examined for plasmid DNA copy number phenotype, and the results were entirely consistent with those presented in Figure 3. The copy number of pNOP1 in DG75 was determined by the saturation hybridization method (Gelfand et al., 1978) and found to be 15 per chromosome equivalent, identical to that of pBGPl20 (Table 1). From these results it is clear that the cop mutation is located in the 5.2 kb fragment of pOPlA6. To localize the mutation more precisely, fragments of pBGP120 DNA were inserted into the Eco RI site of pOPlA6. Selection was for a pBGPl20 DNA fragment which would lower the copy number of pOPlA6 in vivo. This experiment required a simple method for distinguishing between cells containing Cop+ and Cop- plasmids. As shown in Figure 4, DG75 carrying pOPlA6 can grow on plates containing 10 mg/ml ampicillin, while DG75 carrying pBGP120 cannot. If a

wild-type repressor structural gene were inserted into pOPlA6 and properly expressed, these results and those of the co-residence experiment predict that the resulting pOPlA6 recombinant should grow on agar containing 50 pg/ml ampicillin, but not on agar containing 10 mg/ml ampicillin. To obtain a broad size range of DNA fragments, pBGPl20 DNA was partially digested with Hae Ill. Synthetic Eco RI decamers were ligated onto the flush-ended Hae Ill-generated fragments and the products were ligated into the Eco RI site of pOP1 A6. To eliminate vector self-ligation, Eco RI-cleaved pOPlA6 was treated with alkaline phosphatase (UIIrich et al., 1977). The ligation mixture was used to transform DG75. Transformants were plated on LBagar containing 50 pg/ml ampicillin and were subsequently tested for ability to grow on agar containing 10 mg/ml ampicilin. One clone, designated PSO03, was unable to grow on 10 mg/ml ampicillin plates. Plasmid DNA in a cleared lysate of this clone was analyzed by agarose gel electrophoresis. Surprisingly, the lysate contained two Cop+ plasmids with molecular sizes of 6.9 and 2.0 kb (Figure 5, lane 2; compare with lane 1). The 6.9 kb plasmid was tentatively identified as suppressed pOP1 A6, while the 2.0 kb plasmid was designated pNOP10. To determine the Cop phenotype of each plasmid in PSO03 and the sequence relationship between pNOP10 and Col El DNA, it was necessary to separate pNOP10 from its 6.9 kb plasmid co-resident. Preliminary analysis indicated that pNOP10 contained a single Eco RI site (Figure 5, lane 9) but lacked Pst I and Barn HI sites (data not shown). This information was used to purify pNOPl0 by restriction endonuclease digestion of PSO03 plasmid DNA and subsequent dye-buoyant density centrifugation. Purified pNOPl0 DNA was digested separately with Hae II and Hae Ill (Figure 6A, lanes 2 and 3). Col El DNA was digested in a similar manner (Figure 6A, lanes 1 and 4). This analysis and double digestion with Eco RI and Hae II or Hae III (data not shown) showed that pNOP10 contained fragments of identical electrophoretic mobility as Col El Hae II fragments A2 and E, and Col El Hae Ill fragments B2, F, H, N, E and L. Thus pNOP10 is a subset of the pBGPl20 sequences extending from the pBGP120 Eco RI site through the origin of replication to the border of the Tn3 transposon. These results are summarized schematically in Figure 1. This conclusion was confirmed by analysis of heteroduplex molecules composed of Eco RI-cleaved pNCPl0 and Barn HI-cleaved pNOP (Figure 6s). The heteroduplex consists of a 2.0 + 0.3 kb double-strand region flanked by single-strand regions of 1.8 + 0.2 and 3.0 f 0.3 kb. Since the Eco RI site of pNOP1 is about 1.7 kb from the Barn H’I site, the heteroduplex structure demonstrates that pNOPl0 is a contiguous 2.0 kb sequence originating at the Eco RI site of

Cell 270

1234567

pOP1 A6+ pSClO1. (CCC) represents covalently closed DNA. 0.8% agarose gel electrophoresis was performed were prepared from 5 ml cultures grown to ODsso of 3.0.

a

Figure 2. Effect of pBGPl20 Co-residents on pOP1 A6 Copy

and pSClO1 Number

Agarose gel analysis of plasmid DNA in cleared lysates of DG7WpOPl A6 + pBGPl20 (PSOOl ; lane I), DG75/pBGPl20 (lane 2) and DG75/pOPlA6 (lane 3). Also shown is a similar analysis of plasmid DNA -OC+ linear content of Ap’Lac+ transformants (lanes 4 and pOPlA6 5) and ApsLactransformants (lanes 6 and 7) obtained after transformation with plasmid -ccc pSClOl DNA from the DG75/pOPlA6 + pBGPl20 -CCC pOPlO co-resident as described in the text. Lane 6 shows DNA in a cleared lysate from DG75/ circular DNA and (OC) is open circular DNA. The upper band in each lane is chromosomal as described in Experimental Procedures. Cleared lysates in this and subsequent figures

pBGPl20 and spanning the origin of replication (see Figure 1). Furthermore, the conclusion that the other end of Eco RI-cleaved pNOPl0 is in the Hae II C fragment of pBGP120 is also confirmed by the heteroduplex structure. Given the previously observed behavior of pOP1 A6 in co-residence with pBGP120, it seemed plausible that pNOPl0 negatively controlled the copy number of the 6.9 kb plasmid in PSO03 in trans. The equivalence of the 6.9 kb plasmid and the Cop- pOPlA6 species was demonstrated by the transformation of DG75 with total plasmid DNA isolated from PSO03. Transformants were selected for ApR. Nine of ten ApR transformants examined contained a single 6.9 kb Cop- plasmid species indistinguishable from pOP1 A6 (Figure 5, lane 4). Since pNOPl0 does not contain sequences coding for ,L?-lactamase (Figure l), no ApR colonies containing pNOPl0 alone were obtained. An unexpected result of this experiment was that one ApR transformant contained a single Cop+ plasmid of 8.9 kb (Figure 5, lane 5). This plasmid, designated pNOP12, contained two Eco RI sites. Cleavage of pNOP12 with Eco RI generated fragments of 6.9 and 2.0 kb (Figure 5, lane 6). Double digestion of pNOPl2 with Hae II and Barn HI (data not shown) indicated that pNOP10 was integrated into the Eco RI site of pOPlA6 as a direct repeat of pOP1 A6 sequences spanning the region from the Eco RI site to the Hae II C fragment (see Figure 1). These events are shown schematically in Figure 7. Thus pNOP12 apparently arose as a consequence of integration of the independently replicating 2.0 kb species, pNOPl0, into pOPlA6 in vivo to form an 8.9 kb composite plasmid.

Table 2. Plasmid

Copy

Numbers

% Intracellular Host/Plasmid

-CAM

DG7WpBGPl20

6

DG7WpOPlA6 DG75/pNOPl DG75/pBGP120 POP1 A6

DNA

+CAM’ 45

Plasmid Copy Numbers -CAM 15

+CAM 185

27

36

210

300

3

40

15

350

1.5

16

2/5’

+ 20/60c

a Plasmid copy number was calculated from the weight ratio of plasmid and chromosomal DNA. The plasmid molecular sizes are given in Table I. The molecular weight of the DG75 chromosome is 3.75 x 1 O6 bp. The percentage of intracellular DNA as plasmid is the average of three plateau values for each determination. Copy number is expressed per chromosome mass equivalent. b Cultures were treated for 17-I 8 hr with 250 pg/ml chloramphenicol (CAM). ’ pBGPl2O/pOPl A6. Relative copy numbers were estimated by integrating scans of ethidium bromide-stained gels of cleared lysate DNA.

pNOP10 DNA (Figure 5, lane 3). This result supports the conclusions obtained from the restriction endonuclease mapping of pNOPl0 and confirms the location of the colicin immunity gene in Col El. Taken together, these observations indicate that a 2.0 kb sequence of pBGP120 DNA spanning the origin of plasmid replication is capable of not only independent, regulated replication, but also controlling the replication of a related Cop- plasmid in the cis or trans configurations. Discussion

pNOPl0 Encodes Colicin El Immunity From studies of a variety of Col El insertion and deletion mutants, lnselburg (1977) has argued that the colicin immunity gene maps between the Col El Eco RI site and the origin of plasmid replication. Since this region is present in pNOPl0, we transformed DG75 with pNOPl0 DNA and plated transformants on nutrient agar containing colicin El. Cleared lysates colicin-immune transformants from contained

Two major conclusions can be drawn from the data presented here: first, that the DNA overproducer phenotype of the Cop- plasmid pOPlA6 can be suppressed by the presence of the related Cop+ plasmid pBGPl20 in the same cell; and second, that the region of the pBGP120 (and Col El ) genome responsible for suppression of the pOPlA6 Cop- phenotype is at or near the origin of plasmid replication. The results

Control 271

A.

of Col El Replication

ABCDEF

C

-CCC pNOP1

R

“-

A

B

C

D

E

F

I

I

I

I

I

I

2

4

6

8

10

mg/ml Figure

4. Ampicillin

Resistance

Ap

of Plasmid-Containing

Strains

Dilutions of fresh 5 ml overnight cultures of DG75/pBGP120 or DG75/pOPlA6 grown in L broth were plated onto L plates containing various amounts of ampicillin. Plates were incubated at 37°C for 24 hr. and colonies appearing on the plates were counted. (0) DG75/ pOPl A6; (0) DG75/pBGP120.

.ccc Figure

3. Cop Phenotype

pOPlA6

of in Vitro Recombinants

Agarose gel analysis of cleared lysate DNA from Apa transformants. (A) (lane A) DG75/pBGPlZO; (lanes B-F) separate clones derived from ligation of pBGPl20 5.2 kb Eco RI-Barn HI fragment ligated to pOP1 A6 1.7 kb Eco RI-Barn I fragment. (B) (lane A) DG75/pBGP120: (lanes 6-F) separate clones derived from ligation of pOPlA6 5.2 kb fragment to pOP1 A6 1.7 kb fragment. Cleared lysates were prepared as described in Experimental Procedures. Transformants were selected on L plates containing 50 pg/ml ampicillin.

confirm and extend our conclusion that these Col El derivatives contain genetic information involved in the control of plasmid copy number (Gelfand et al., 1978). pOP1 A6 exists stably at about 210 copies per chromosome in DG75 (Table 2; Gelfand et al., 1978). In cells containing both pOP1 A6 and the parent plasmid pBGP120, however, the copy number of pOPlA6 drops to five copies per chromosome equivalent. No effect is observed when the unrelated plasmid pSC101 co-resides with pOPlA6. This behavior is most simply interpreted by postulating a negative control of replication mediated by a plasmid-specific re-

pressor (Pritchard et al., 1969). In this model, pOP1 A6 is understood to contain a mutation which alters the production of repressor or produces an inactive repressor. The observation that pOPlA6 copy number is lowered in the presence of active repressor produced by pBGP120 indicates that pOP1 A6 retains a functional recognition site for repressor. Several features of plasmid replication have recently been observed which are consistent with the major tenets of the negative control model of replication control put forward by Pritchard and his colleagues (1969, 1978). Genetic evidence for the existence of a repressor function in the replication of the R plasmid, Rldrd-19, has been established by the isolation of nonsense and temperature-sensitive plasmid copy number mutants (Gustafsson and Nordstrom, 1978; Uhlin and Nordstrom, 1978). The amber copy number mutant, pKN303, has a copy number identical to that of the wild-type plasmid in the presence of a strong amber suppressor. In the absence of suppression, the copy number of pKN303 increases !&fold over that of wild-type (Gustafsson and Nordstrom, 1978). Another mutant of Rldrd-19, pKN401, replicates without control when the host is grown at

Cell 272

6789

12345

OC+lineor pOPl A6 CCC pOPlA6

-ccc

pNOPl2

Figure 5. Control of pOPlA6 Trans and in Cis by pNOPl0

.Qg RI - digested pOPIn

.Eco RIpNOPl0

CCC pNOPl0

temperatures above 35”C, and eventually inhibits host cell growth. Finally, copy number behavior of composite plasmids of Col El and pSClO1 is also consistent with plasmid-specific negative control of replication (Cabello, Timmis and Cohen, 1976). In addition to these data indicting negative control, a strong case can be argued that Col El replication is nof under positive control by plasmid-specified effectors. The most compelling evidence for this theory is the observation that Col El -bacteriophage hybrids can utilize the Col El origin of replication in the absence of protein synthesis (Donoghue and Sharp, 1976; Kahn and Helinski, 1976). Loci of Control of Plasmid Replication What parameters are sensed in the decision to replicate? We have previously shown that pBGP120 and Col El reside at approximately the same copy number, 15 per chromosome equivalent, despite their substantial difference in size. We have shown in this paper that pNOP1, a plasmid containing the origin of pBGPl20 and identical in size to pOP1 A6 , also exists at about 16 copies per chromosome. These results indicate that the various manipulations involved in the construction of these Col El derivatives [for example, insertion of the Tn3 transposon (So, Gill and Falkow, 19751, X plac 5 and E. coli DNAs (Polisky et al., 197611 had no significant effect on copy number control. Hence, regardless of size, the number of plasmid origins is maintained at a constant level. A different pattern emerges, however, in the Cop- plasmid pOP1 and its deletion derivative pOPlA6. These plasmids exist at different copy numbers but comprise the same total amount of cell DNA (25-30%; Gelfand et al., 1976). Thus when plasmid-specific regulation is eliminated or diminished by mutation, control is no longer mediated by origin concentration, but perhaps by limitation of host replication factors. Nordstram, Engberg and Nordstrijm (1974) have noted competition between the host chromosome and R factors for DNA polymerase Ill under some conditions of cell growth. Similar competition may limit replication of pOPlA6 and pOP1.

digested

Replication

in

Agarose gel analysis of plasmid DNA in cleared lysates of DG75/pOPlA6 (lane 1). DG75/pOPlA6 + pNOPl0 (PSOO3; lane 2). DG75/pNOPlO (lane 3). DG75/pOPlA6 (recovered from clone PSOO3; lane 4) and DG7WpNOPi 2 (lane 5). Lanes 6 and 7 contain Eco RI-digested pNOPl2 and plasmid DNA from PSOO3. respectively. Eco Rl-digested pOPlA6 (6.9 kb) is shown in lane 8 and Eco RI-digested DNOP~O (2.0 kb) is shown in lane 9. Symbols are as in Figure 2. The upper band in lanes l-5 is chromosomal DNA.

Hershfield et al. (1976) observed that certain deletion derivatives of Col El have higher copy numbers than Col El. They proposed that plasmid mass might be an important parameter in replication control. It is possible, however, that these derivatives were in fact altered in replication control functions since they may express altered incompatibility properties (Warren and Sherratt, 1978). Altered incompatibility has also been observed in Cop- mutants of Rl drd-19 (Uhlin and Nordstrijm, 19751, mini-F plasmids (Manis and Kline, 1976) and in the present study for Col El Copmutants. Localization of the Cop- Mutation in pOPlA6 The altered control function in pOPlA6 was mapped by in vitro recombination. Initial mapping efforts localized the mutation to the 5.2 kb Barn HI-Eco RI fragment containing the plasmid origin of replication as well as Tn3 sequences. Subsequent experiments involved the cloning of pBGP120 fragments into pOPlA6 in an attempt to suppress the overproducer phenotype. These experiments implicated a 2.0 kb fragment spanning the origin of replication. Unexpectedly, we found that this 2.0 kb sequence was able to suppress the Cop- phenotype of pOPlA6 in trans as a co-resident (pNOPlO), or in cis after integration into the Eco RI site of pOP1 A6. Integration of pNOPl0 into pOPlA6 generated the 8.9 kb Cop’ plasmid pNOP12. Integration of pNOP10 at the Eco RI site of pOPlA6 was not observed as expected after transformation with the initial ligation mixture, but only after transformation of DG75 with total supercoiled DNA from clone PSO03. This observation indicated that integration of pNOPl0 occurred in vivo, most plausibly by a recA-mediated recombination event between homologous regions of pNOP10 and pOP1 A6 DNAs. Regardless of the mechanism giving rise to pNOP12, the data clearly indicate that the region of pBGPl20 which suppresses mutation(s) responsible for altered copy number control in pOP1 A6 is located at or near the origin of plasmid replication. This type of organization has also been observed in the F plasmid (Manis and Kline, 19781, R6K (Figurski et al.,

Control 273

of Col El Replication

A. ii\-CD-

E-

-J

Figure 7. pNOPl2 pNOPl0 Replicons

Is a Composite

Plasmid

Containing

pOP1 A6 and

The Hae II (1). Eco RI (A) and Barn HI (A) sites of pNOPl0 pOP1 A6 used to establish the orientation of pNOPl0 in pNOPl2 shown. Lengths of the fragments are shown in Figure 1.

and are

1978) and Rsc derivatives of Rl drd-19 (Kollek, Oertel and Goebel. 1978). Although the nature of the repressor and its mode of action are unknown, the existence of temperaturesensitive and nonsense mutants indicates that the repressor is probably a polypeptide. Fralick (1978) has recently shown that the rate-limiting step in the replication of the E. coli N167 chromosome (dnaAts) is not formation of the initiation complex, but rather the association of a negative control element with the replication initiation complex. This element is postulated to prevent progression of the fully formed initiation complex into the elongation phase of DNA replication. Our results are consistent with a similar mode of Col El plasmid replication control. Experimental

Figure 6. (A) Comparison of the Structures of pNOPl0 and Col El by Restriction Endonuclease Analysis and (B) Heteroduplex Molecules between Eco RI-Cleaved pNOPl0 and Barn HI-Cleaved pNOP1 (A) Hae II digests of Col El and pNOPl0 are shown in lanes 1 and 2, respectively. Lanes 3 and 4 compare the fragments obtained from Hae Ill digestions of pNOPl0 and Col El, respectively. Fragment designations and lengths in kilobases shown are from Oka and Takanami (1976). The smallest fragments (Hae II F; Hae Ill K. L. M and N) are not visible in the photograph. Fragments were separated in a 5-20% (w/v) exponential polyacrylamide gradient gel as described in Experimental Procedures. Hae II Col El fragment sizes (kb) are (A) 2.33; (B) 1.73; (C) 1 .13: (D) 0.69; (E) 0.34. Hae Ill Col El fragment sizes are (A) 1 .lO; (B) 1.05; (C) 0.90; (Cl) 0.88; (E) 0.44; (F) 0.42; (G and H) 0.41; (I) 0.25; (J) 0.17. (B) Heteroduplexes were formed as described in Experimental Procedures. Open circle pBR 322 (4.3 kb) was used as a length standard for double-strand regions.

Procedures

Materials, Strains and Plasmids Barn HI. E. coli DNA polymerase I. calf intestine alkaline phosphatase (Grade 1). T4-polynucleotide kinase and deoxyribonucleotide triphosphates were purchased from Boehringer-Mannheim. Pst I, Hae II and Hae Ill were obtained from BRL. Eco RI was prepared as described by Greene, Betlach and Bayer (1974). T4 DNA ligase was purchased from BRL or isolated through the hydroxyapatite step as described by Higgins et al. (1977). y-32P-ATP (2.8 x lo3 Ci/mole) was supplied by NEN and 3H-dTTP (96 Ci/mmole) was supplied by ICN. Eco RI decamers were purchased from Collaborative Research and unlabeled ATP from PL Biochemicals. SeaKem agarose was obtained from Marine Colloids Division (Rockland. Maine) and hydroxyapatite was DNA-grade from BioRad. Media used were 2X VT (Miller, 1972). minimal medium E (Vogel and Bonner. 1956). lactose minimal medium A (lac-minimal A) (Pardee. Jacob and Monod. 1959) and lactose minimal medium B (lacminimal B). Lac-minimal A was supplemented with 0.2% casamino acids; lac-minimal B lacked casamino acids. Lac+ and Lac colonies grow overnight on plates containing lac-minimal A. but Lac’ cells form much larger colonies. Lace cells will not grow on plates containing lac-minimal B. MacConkey indicator agar (Difco) was used to distinguish Lac’ and Lac colonies in some experiments. PlateS containing Luria broth (LB) or nutrient broth agar were prepared as described by Miller (1972). Plates were supplemented with 50 pg/ml

Cdl 274

or 10 mg/ml sodium ampicillin (Polycillin N; Bristol Laboratories) as indicated in the text and figure legends. The E. coli derivative DG75 (O’Farrell, Polisky and Gslfand, 1978) was used in this study. Table 1 and Figure 1 summarize the biological and structural properties of the derivaNves of pBGPl20 used here. Recombinant DNA experiments ware carried out under PI conditions in accordance with the NIH Guidelines for recombinant DNA research. Plasmid Transformation, Isolation and Copy Number Determinations Transformations with plasmid DNA prepared from cesium chlorideethidium bromide centrifugation of cleared lysates (Gelfand et al. 1978) were carried out according to the method of Cohen, Chang and Hsu (1972). 5 x lo6 ampicillin-resistant transformants were usually obtained with 100 ng of input DNA. In co-transformation experiments. pBGPl20 and pOPlA6 were mixed at a molar ratio of 1 :lO, respectively. Transformants were selected on lac-minimal A agar containing 50 pg/ml ampicillin, and were subsequently analyzed for plasmid content by agarose gel electrophoresis of cleared lysate DNA as described by Polisky et al. (1976). The separation of the co-residing plasmids pBGPl20 and pOP1 A6 was accomplished in 5-20% (w/w) exponential sucrose gradients formed in 50 mM Tris-HCI (pH 7.5), 0.1 M sodium acetate and 10 mM EDTA. Gradients were centrifuged for 15 hr at 39,000 rpm and 22’C in an SW41 rotor. Fractions (0.2 ml) were pumped from the bottom of the centrifuge tube and analyzed for plasmid content by electrophoresis through 0.8% agarose gels. Fractions were pooled as described in the text, precipitated with ethanol and subsequently used for transformation. Estimates of plasmid copy number can be made by comparison of samples of known and unknown copy number after agarose gel electrophoresis of purified cleared lysate DNA (Gelfand et al.. 1978) or by measurement of plasmid-encoded /3-lactamase (Uhlin and Nordstrdm. 1978). We have previously described a more accurate method for the determination of plasmid copy number by saturation hybridization (Gelfand et al., 1978). In this procedure, total DNA from plasmid-containing cells is isolated and labeled in vitro (Galau et al., 1976). The DNA is then used as tracer in hybridization reactions driven by purified plasmid DNA. The resulting hybrids are assayed on hydroxyapatite. In these reactions plasmid-specific tracer is rapidly driven into duplex form because of its low complexity compared to the E. coli chromosome. The percentage of total radioactivity bound to hydroxyapatite at the end of the reaction after correction for tracer reactivity (70-l 00%) is equal to the percentage of total cellular DNA which is plasmid-specific, Restriction Endonuclease Treatment of Plasmid DNA Restriction endonuclease digestions were with Barn HI [20 mM TrisHCI (pH 7.5). 7 mM MgC12. 2 mM P-mercaptoethanol]. Eco RI [lo0 mM Tris-HCI (pH 7.5). 50 mM NaCI. 5 mM MgCl& Hae II [6 mM TrisHCI (pH 7.5), 6 mM MgCI,. 6 mM 2-mercaptoethanol], Hae III (same as Hae II except 6 mM NaCI) and Pst I [20 mM Tris-HCI (pH 7.5), 10 mM MgCl*. 50 mM (NH,), SO,]. Double digestions were performed sequentially in the buffer described for each enzyme, except in the case of Hae II-Barn HI digestions which are performed simultaneously in Hae II buffer. All reactions contained 100 pg/ml autoclaved gelatin and were performed at 37”C, except the Pst I digestions which took place at 30°C. Analysis of restriction fragments by acrylamide gel electrophoresis was according to the method of Jeppesen (1974), except that exponential 5-20% (w/v) acrylamide gels were used,

Isolation of the pBGPl20 Replication Control Region For cloning wild-type replication control functions from pBGPl20 into the Eco RI site of pOPlA6. blunt-ended DNA fragments of pBGPl20 were generated by partial digestion with Hae Ill. Eco RI decamers were ligated onto the blunt-ended fragments as described by E. Craig (personal communication). The reaction mix contained kinase-treated Eco RI decamers and pBGPl20 DNA fragments in a 25:l molar ratio. The pBGPl20 DNA concentration in the reaction was 100 pg/ml and contained the concentrations of salts and buffers described above, except that 100 U/ml of T4 DNA ligase were used and incubation was for 3 hr at 17’C. After digestion of the reaction products with Eco RI. 0.1 vol of 3 M sodium acetate was added and the DNA was precipitated overnight with 2 vol of ethanol. This step removed most of the unreacted Y-~‘P-ATP in the reaction mixture. Following centrifugation. the DNA pellet was resuspended in 100 pl of 10 mM TrisHCI (pH 8.0). 0.1 mM EDTA (TE). and the Hae Ill-Eco RI DNA fragments were separated from decamers by passage through a column (1 .l cm X 29 cm) of Sephadex G75 equilibrated in 10 mM Tris-HCI (pH 8.0). 1 mM EDTA, 0.1 M sodium acetate. The excluded volume contained the 3ZP-labeled Hae Ill-Eco RI DNA fragments and was ethanol-precipitated. Vector recircularization was prevented in the subsequent ligation reaction by treatment of Eco RI-digested pOPlA8 DNA with calf intestine alkaline phosphatase (0.2 U/pg of vector DNA at 65°C for 30 min). For ligation of the pBGPl20 DNA into pOP1 A6, DNAs were mixed in a molar ratio of 1 :l at a total DNA concentration of 4 pg/ml in a volume of 100 pl. The concentration of T4 DNA ligase in this reaction was 2.5 U/ml and incubation was at 12.5”C for 17 hr. The reaction mixture was used directly to transform DG75 and the transformants were spread onto LB plates containing 50 gg/ml ampicillin. 2.5 x IO3 ampicillin-resistant transformants were obtained. The iransformants were subsequently tested for growth on LB plates containing 10 mg/ml ampicillin. Isolation of pNOPl0 When present as co-residents, the 6.9 (pOPlAG) and 2.0 kb (pNOP10) plasmids were difficult to separate in large amounts by agarose gel electrophoresis or sucrose gradient centrifugation. Because pNOPl0 lacked Barn HI sites. digestion of co-resident plasmid DNA linearized only the pOP1 A6 DNA (Figure 1). leaving pNOPl0 as supercoiled DNA. A combination of this digestion with subsequent cesium chloride-ethidium bromide centrifugation was used in our initial purification of pNOP10. After two cycles of digestion and centrifugation. only pNOPl0 could be observed when the supercoiled DNA (1 ag) was analyzed by agarose gel electrophoresis. Col El Selection for DG75/pNOPlO Transformants A crude Col El preparation was prepared from JC411 (Co1 El) as described by Tanaka and Weisblum (1975). 200-400 81 of this lysate, when spread onto nutrient agar plates, reduced the background of non-immune cells sufficiently to allow selection of cells harboring plasmids conferring colicin immunity. Transformation with 0.1 pg of pNOPl0 DNA gve rise to 1 X 1 O5 colicin El -resistant clones. Heteroduplex Analysis Heteroduplexes were prepared by the formamide procedure of Davis, Simon and Davidson (1971) and examined in a Phillips 300 electron microscope. Length measurements were made with a Numonics graphics calculator. Acknowledgments

In Vitro Recombination of the pBGPl20 and pOPlA6 Origins of Plasmid Replication DNA fragments were purified by electroelution from preparative agarose gels (McDonell, Simon and Studier, 1977). Ligation mixtures (50 gl) contained approximately equimolar amounts of fragments at a total DNA concentration of 2 pg/ml. 2 U/ml of T4 DNA ligase were added and the mixtures were incubated overnight at 12.5”C. The ligation reactions were then used directly to transform DG75 and transformants were selected on minimal E plates containing 50 pg/ml ampicillin.

We thank Tom Blumenthal and S. R. Jaskunas for comments on the manuscript, John Kaumeyer for DNA ligase and E. Craig for advice on blunt-end ligation. H.M.S. was supported by a Damon RunyonWalter Winchell Cancer Research Fund postdoctoral fellowship. This work was supported by an NIH grant to B.P. and a grant from the Indiana University Biomedical Research Support Committee. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

Control 275

of Col El Replication

marked “advertisement” in accordance solely to indicate this fact. Received

April 30. 1979;

revised

with 18 U.S.C.

Section

1734

July 2. 1979

Kool. A. J. and Nijkamp, H. J. J. (1974). Isolation and characterization of a copy mutant of the bacteriocinogenic plasmid Clo DF13. Bacterial. 7 20, 569-578.

References Andreoli, P. M.. Brandsma, R. A., Veltkamp. E. and Nijkamp. H. J. J. (1978). Isolation and characterization of a Clo DF13::TngOl plasmid mutant with thermosensitive control of DNA replication. J. Bacterial. 135. 612-621. Cabello, P., Timmis, K. and Cohen, in a composite plasmic constructed replicons. Nature 259, 285-290.

terization of the minimal fragment required for autonomous replication (“basic replicon”) of a COPY mutant fpKN102) of the antibiotic resistance factor RI. Mol. Gen. Genet. 762, 51-58.

S. N. (1976). Replication control by in vitro linkage of two distinct

Cohen, S. N.. Chang. A. C. Y. and Hsu. L. (1972). Non-chromosomal antibiotic resistance in bacteria: genetic transformation of E. coli by R factor DNA. Proc. Nat. Acad. Sci. USA 69, 21 lo-21 14.

J.

McDonell. M. W., Simon, M. N. and Studier, F. W. (1977). Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 7 70. 119-146. Manis. J. J. and Kline, B. C. (1978). F Plasmid incompatibility and copy number genes: their map locations and interactions. Plasmid 7, 492407. Miller, J. H. (1972). Cold Spring Harbor

Experiments Laboratory).

in Molecular

Genetics,

(New

York:

Morris, C. F., Hashimoto. H.. Mickel. S. and Rownd. R. (1974). Round of replication mutant of a drug resistance factor. J. Bacterial. 7 78. 855-866.

Davis, R. W.. Simon, M. and Davidson, N. (1971). Electron microscope hetero-duplex methods for mapping regions of base sequence homology in nucleic acids. In Methods in Enzymology, 21, D. L. Grossman and K. Moldave. eds. (New York: Academic Press), pp. 413-428.

Nordstrom, K.. Ingram, L. C. and Lundback. A. (1972). Mutations in R-factors in E. co/i causing an increased number of R-factor copies per chromosome. J. Bacterial. 7 70, 562-569.

Donoghue. D. J. and Sharp, plasmid DNA in vivo requires teriol. 133, 1287-l 294.

Nordstrom, U. M.. Engberg, B. and Nordstrom, K. (1974). Competition for DNA polymerase Ill between the chromosome and the R-factor RI. Mol. Gen. Genet. 735. 185-l 90.

P. A. (1978). Replication of colicin El no plasmid-encoded proteins. J. Bac-

Figurski. D.. Kolter. R.. Meyer, R., Kahn, M.. Eichenlaub. R. and Helinski. D. R. (1978). Replication regions of plasmids ColEl , F, R6K and RK2. In Microbiology 1978, D. Schlessinger, ed. (Washington: American Society for Microbiology), pp. 105-l 09. Fralick. J. A. (1978). Studies on the regulation of initiation of chromosome replication in Escherichia CO/I. J. Mol. Biol. 122. 271-286. Gala”. G. A., Klein, W. H.. Davis, M. M.. Wold, B. J.. Briten. R. J. and Davidson, E. H. (1976). Structural gene sets active in embryos and adult tissues of the sea urchin. Cell 7. 487-505. Gelfand, D. H.. Shepard, H. M.. O’Farrell, P. H. and Polisky. B. (1978). Isolation and characterization of a ColEl-derived plasmid copy number mutant. Proc. Nat. Acad. Sci. USA 75, 5869-5873. Greene, P. J., Betlach, restriction endonuclease. Wickner. ed. (New York:

M. and Boyer, H. W. (1974). The Eco RI In Methods in Molecular Biology, 7, R. B. Marcel Dekker). pp. 87-l 11.

Gustafsson, P. and Nordstrom, K. (1978). Temperature-dependent and amber copy mutants of plasmid Rldrd-79 in Escherichia Plasmid 7, 134-144.

co/!.

Heffron, F., Bedinger, P.. Champoux. J. J. and Falkow. S. (1977). Deletions affecting the transposition of an antibiotic resistance gene. Proc. Nat. Acad. Sci. USA 74, 702-706. Hershfield. V.. Boyer. H. W.. Chow, L. and Helinski. D. R. (1976). Characterization of a mini-ColEl plasmid. J. Bacterial. 126. 447453. Higgins, N. P.. Geballe. A. P.. Snopeck. T. J., Sugino. A. and Cozzarelli. N. (1977). Bacteriophage T4 RNA ligase: preparation of a physically homogeneous, nuclease-free enzyme from hyperproducing cells. Nucl. Acids Res. 4, 3175-3186. Inselburg, J. (1977). Studies of colicin El plasmid functions by analysis of deletions and TnA insertions of the plasmid. J. Bacterial. 7 32, 332-340. Jacob, F.. Brenner, S. and Cuzin. F. (1963). On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. &ant. Biol. 28. 329-348. Jeppesen, P. G. N. (1974). A method by electrophoresis in polyacrylamide gels. Anal. Biochem. 58. 195-207. Kahn, M. and phage hybrid: in viva in the Sci. USA 75, Kollek.

for separating concentration

DNA fragments gradient slab

Helinski. D. R. (1978). Construction of a novel plasmiduse of the hybrid to demonstrate ColEl DNA replication absence of a ColEl-specified protein. Proc. Nat. Acad. 2200-2204.

R.. Oertel.

W. and Goebel.

W. (1978).

Isolation

and charac-

O’Farrell, P. H.. Polisky. B. and Gelfand. D. H. (1978). Regulated expression by readthrough translation from a plasmid-encoded pgalactosidase. J. Bacterial. 734, 645-654. Oka. A. and Takanami. M. (1976). plasmid. Nature 264, 193-l 96.

Cleavage

map

of colicin

Pardee. A. B.. Jacob, F. and Monod. J. (1979). The genetic and cytoplasmic expression of inducibility in the synthesis galactosidase of E. coli. J. Mol. Biol. 7. 165-178.

El

control of (c-

Polisky. B., Bishop, A. J. and Gelfand. D. H. (1976). A plasmid cloning vehicle allowing regulated expression of eukaryotic DNA in bacteria. Proc. Nat. Acad. Sci. USA 73, 3900-3904. Pritchard, R. H. (1978). Control of DNA replication in bacteria. In DNA Synthesis, I. Molineux and M. Kohiyama. eds. (Plenum Press: New York and London). pp. l-26 Pritchard, synthesis

R, H.. Barth, P. T. and Collins, J. (1969). Control of DNA in bacteria. Symp. Sot. Gen. Microbial. 79, 263-298.

So, M., Gill, R. and Falkow. S. (1975). The generation of a ColEl-Aps cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet. 742, 239-249. Tanaka, T. and Weisblum. B. (1975). Construction of a colicin El-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bacterial. 72 7, 354-262. Uhlin. B. E. and Nordstrdm. K. (1975). Plasmid incompatibility and control of replication: copy mutants of the R-factor RI in E. co/i K-l 2. J. Bacterial. 724, 641-649. Uhlin. 8. E. and Nordstrom, K. (1978). A runaway-replication of plasmid Rl drd-79: temperature dependent loss of copy control. Mol. Gen. Genet. 765, 167-179.

mutant number

Ullrich. A., Shine, J.. Chirgwin, J.. Pictet. R.. Tischer, E.. Rutter. W. J. and Goodman, H. M. (1977). Rat insulin genes: construction of plasmids containing the coding sequences. Science 796, 13131319. Vogel, H. J. and Banner, D. M. (1956). Acetylornithinease of Escherichia co/i: partial purification and some properties. J. Biol. Chem. 278, 97-102. Warren, G. and Sherratt, D. (1978). Incompatibility and transforming efficiency of ColEl and related plasmids. Mol. Gen. Genet. 767, 3948.