Proc. Nati. Acad. Sci. USA
Vol. 75, No. 5, pp. 2200-2204, May 1978
Biochemistry
Construction of a novel plasmid-phage hybrid: Use of the hybrid to demonstrate ColE 1 DNA replication in vivo in the absence of a ColEl-specified protein (bacteriophage P4/gene cloning/chloramphenicol/plasmid incompatibility)
MICHAEL KAHN AND DONALD R. HELINSKI Department of Biology, University of California, San Diego, La Jolla, California 92093
Communicated by E. Peter Geiduschek, February 21, 1978
ABSTRACT
A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid CoWEl. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColEl replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColEl DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColEl plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that CoEl does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.
Plasmid DNA replication in bacteria is of particular interest in view of the diversity of replication modes and the variety of control systems that may be involved in the replication of different plasmids. Biochemical approaches to the in vivo study of plasmid replication, incompatibility, and expression, however, have been limited to cells in which plasmids have previously been established. Because there has been no efficient method of introducing plasmids into cells synchronously and quantitatively, many techniques used successfully in the study of other DNA replication systems have not been available for the study of plasmids. This difficulty could conceivably be overcome by reconstructing the plasmid to allow it to be packaged into bacteriophage particles. Infection of bacterial cells with these phage particles would then result in the injection of the plasmid DNA carried by the particles. In this way, methods successfully used to study bacteriophage DNA replication in a bacterial cell could be applied to plasmid DNA
replication. Bacteriophage P4 has several properties that can be utilized to facilitate the packaging of a plasmid. In Escherichia coli, P4 lytic growth is dependent on the presence of a helper bacteriophage, P2 (1), and P4 has no lytic functions when grown in the absence of P2. In this respect, P4 resembles a defective virus except that it can efficiently activate a P2 prophage and produce P4 phage free of P2 helper contamination. Souza et al. (2) have shown in vivo that the 8-kilobase (kb) fragment produced by digestion of P4 with the restriction enzyme EcoRI is sufficient for lytic growth if the size of the phage DNA is maintained near 11 kb by the substitution of about 3 kb of foreign DNA. In this paper we report the construction by in vitro techThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
niques of a hybrid DNA molecule made up of a segment of the P4 genome and a low molecular weight derivative of plasmid ColEl. This ColEl-P4 recombinant molecule can exist in E. coli as a plasmid and can be packaged into infective bacteriophage particles. In this study, E. coli was infected with the hybrid, and ColEl-directed replication of the hybrid DNA was analyzed in the presence of the protein synthesis inhibitor chloramphenicol. Evidence is presented that replication of ColEl in vivo does not require a ColEl-specified protein. MATERIALS AND METHODS Reagents. Sources for reagents were: Sigma (lysozyme, chloramphenicol, puromycin, kanamycin, streptomycin, and rifampicin); New England Nuclear [[methyl-3H]thymine (>10 Ci/mmol), [2-'4C]thymine (>50 mCi/mmol)]; Baker (polyethylene glycol 6000); New England Biolabs (Pst I, Sma I, Hae II, HindIII, Sal I, and Hpa I restriction enzymes); Miles Laboratories (T4 DNA ligase). EcoRI was prepared according to Greene et al. (3). All antibiotics used in inhibition studies were added as powders except rifampicin which was used as a 10 mg/ml solution in dimethyl sulfoxide. LB and TPG media have been described (4, 5). Bacterial Strains. HF4704 (5) and AB2497 (6) have been described. C-2110 (polAl his rha) was obtained from M. Sunshine and a Thy- derivative, C-2110thy, was selected. N2076T (rnc+ thyA deo thi purI argH nadB lacY gal maIA xyl ara mtl str tonA supE44) and N2077T (N2076 rnc thyA deo) were made by using a trimethoprim selection (7) from the thymine prototrophs described by Apirion and Watson (8) and obtained from D. Apirion. C-2326 (r-m- supD argstrp tonB P21g) was obtained from R. Calendar. An r-mK+trp + derivative of this strain, CK2326, was constructed by conjugal mating using HF958 (HfrH r-mK+ leu thy) (9). Plasmids. The construction of pMK20 and pMKl will be described elsewhere. pMK1 contains a 1.3-kb replication region of ColEl and codes for tetracycline resistance. Plasmid pCR1 was isolated by covey et al. (10) and has been described by Armstrong et al. (11). Bacteriophages. P4 vir, was obtained from R. Calendar. Phage stocks were grown on CK2326 and purified by using polyethylene glycol as described by Barrett et al. (4). P420, the P4-ColEl hybrid constructed in this study, was found to be unstable in CsCl. Therefore, all phage stocks were precipitated with polyethylene glycol, resuspended in 100 mM Tris/10 mM MgCl2, pH 8, and stored at 1012 plaque-forming units (PFU)/ ml. Clone Analysis. Plasmid DNA corresponding to individual transformants or transfectants was prepared from cells that had Abbreviations: kb, kilobase(s); PFU, plaque-forming unit.
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Proc. Natl. Acad. Sc. USA 75 (1978)
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_~~~~~~~~~~ FIG. 1. Agarose gel analysis of P420 hybrid DNA. The following DNA samples were examined by electrop'horesis in 1% agarose gels: EcoRI digests of DNA purified from P4 yinl infected cells (a), plasmid pMK2O DNA (b), P420 DNA isolated from plasmid containing cells (c), P420ODNA purified from infected cells (d), and P420 DNA isolated from infected, choapeio-treated cells (e); Pst I digests of DNA from cells infected with phage P4 yir1 (f) and phage P420 (g); and undigested DNA from cells infected by P4 vir1 (h) and P420 (i).
been treated with chloramphenicol (250,Mg/mi) to enrich for resident plasmids (12) or from P2 nonlysogens that had been infected for 2 hr with phage derived from individual plaques. Cells were lysed by using the lysozyme/Triton X-100 procedure of Katz eat al. (13). All extracts were phenol-extracted and in certain cases the DNA was further purified by using CsCl/ ethidium, bromide gradients. The yield of covalently closed DNA from these procedures was typically 0.5-1 1tg of DNA per ml of culture. Analysis of DNA. Restriction, ligation, and gel electrophoresis conditions are described elsewhere (14). CK2326 was transfected by wsing the procedure of Lederberg and Cohen (15). All recombinant DNA procedures were carried out in accordance with the National Institutes of Health "Guidelines on Recombinant DNA Research" (16). RESULTS Construction of the P4-ColEl Hybrid. A low molecular weight derivative of ColEl was isolated by cutting ColEl and pCR1 DNA with the restriction enzyme Hae II and joining the fragments together with DNA ligase. AB2497 was transformed with the mixture and kanamycin-resistant transformants were selected. The resulting plasmid, pMK2O, contains both the Hae II A and E fragments of ColEl1 (17) and a 1. 1-kb Hae II fragment from pCR1 that specifies kanamycin resistance. pMK2O is 3.6 kb long and has a single EcoRI site. EcoRI digestion of closed circles of bacteriophage P4 yields three fragments designated AD, B, and C (18) (Fig. 1, lane a). P4 vir, DNA and
Sma
FIG. 2. Restriction map of P420. The restriction sites in P4 are from Kahn and Hopkins (19); restriction sites in pMK20 were derived from unpublished data. The site of the P4 cohesive ends (P4 cos) is defined as the left end of the linear map. Restriction enzymes used were HindIll (Ill), EcoRI (RI), Pst I (Pst), Sma I (Sma), Hpa I (Hpa), Sal I (Sal), and Hae II.
pMK20 DNA were digested with EcoRI, ligated together, and used to transfect the P4 indicator strain CK2326. Cells at the margins of the resultant plaques were tested for kanamycin resistance (50 ,ug/ml). Many plaques yielded stable kanamycin-resistant cells that were found to contain supercoiled plasmid DNA. These cells spontaneously released phage that gave clear plaques on CK2326 that also had kanamycin-resistant bacteria at their margins. The P420 hybrid phage made a slightly larger plaque than did P4 vir1 and could be grown lytically in good yield (1010-o10l1 PFU/ml). Restriction analysis of the plasmid DNA isolated from kanamycin-resistant cells demonstrated that the plasmids they contained were recombinants between P4 and pMK20. An analysis by gel electrophoresis of one of these recombinant plasmids, P420, is shown in Fig. 1. Digestion of the DNA with EcoRI (Fig. 1, lane c) showed that the molecule is composed of the large EcoRI AD fragment of P4 (Fig. 1, lane a) and the entire chromosome of pMK20 (Fig. 1, lane b). The structure of P420 deduced from restriction enzyme analysis is shown in Fig. 2. A recombinant molecule that had the other orientation of pMK20 relative to P4 was also isolated. The DNA in both P4infected cells (Fig. 1, lane h) and P420-infected cells (Fig. 1, lane i) was present as covalently closed circles. The single band produced after digestion of P420 DNA with Pst I (Fig. 1, lane g) is slightly larger than the band from a similar digest of P4 virl DNA (Fig. 1, lane f) because the pMK20 insert (3.6 kb) is slightly larger than the deleted B and C fragments of P4 (total size, 3.2 kb). This additional DNA may account for the greater instability of P420 phage in CsCl solution compared with P4 virl. These results indicate that the hybrid molecule can exist either as a plasmid or as an infectious bacteriophage. P420 DNA can be obtained in three ways: (i) from the bacteriophage particles; (U) as covalently closed molecules from infected cells that are either lysogenic or nonlysogenic for the P2 helper phage; or (iii) after chloramphenicol amplification of cells carrying the hybrid as a stable plasmid. P420 DNA obtained from all three procedures was identical as judged by its physical
Biochemistry: Kahn and Helinski
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Proc. Nati. Acad. Sci. USA 75 (1978) 10 -
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FIG. 3. DNA synthesis in P420-infected cells. HF4704 was grown overnight at 370 in TPG medium supplemented with thymine at 25 ,ug/ml, 0.5% Casamino Acids, 0.2% glucose, 0.02% rhamnose, and thiamine and niacin (2 yg/ml) and then diluted 1:50 into the same medium containing 4 jug/ml of [14C]thymine (4 ;g/ml; 1 MCi/ml) to prelabel cells. At a density of 3 X 108 cells per ml, the culture was pelleted by centrifugation and resuspended in an equal volume of unlabeled medium. Chloramphenicol (250 gg/ml) was added 15 min later. After 5 min the culture was divided into quarters and to two portions were added P4 (&) and P420 (-) separately at a multiplicity of infection of 10. After an additional 2.5 hr, P420 was added to a third portion (0) and [3H]thymine (50 MCi/ml) was added to all cultures, including the uninfected control (-). Samples (0.2 ml) were removed and added to a solution containing 0.2 M NaOH/1% Sarkosyl at the times indicated. Salmon sperm DNA (50 Mg/ml) was added to each sample and the samples were precipitated with 5% trichloroacetic acid, filtered through glass fiber filters, and assayed for radioactivity. Plotted are the raw ratios of 3H to 14C. For the experiments in this and other figures, 14C was typically 3000-5000 cpm per sample. Arrow, actual time of phage addition; [3H]thymine was then added and the first samples were taken at time zero.
characteristics (Fig. 1, lanes c, d, and e) and its biological activity, except that DNA derived from phage particles is linear. P420 DNA Replication in the Absence of Protein Synthesis. HF4704 was infected with P420 in the presence of chloramphenicol at 250 ,g/ml. Under the conditions used in this study, protein synthesis is less than 3% of that found in the absence of the inhibitor, and this residual incorporation is probably into small peptides (20). In the absence of protein synthesis, chromosomal DNA replication stops within 2 hr (21). As shown in Fig. 3, uninfected cells incorporated only a very small amount of [3H]thymine under these conditions. P4 DNA replication requires a phage-coded protein, and it is not surprising that, because this protein is not made in the presence of chloramphenicol (22), P4-infected cells show no additional incorporation of label. However, cells infected with P420 did synthesize DNA under these conditions. This synthesis was linear for at least 3 hr and the rate of synthesis was the same whether the P420 multiplicity of infection was 5 or 50 PFU per cell. There was no lag in the initiation of P420 DNA synthesis. Cells infected with P420 2.5 hr prior to the addition of [3H]thymine showed the same kinetics of label incorporation as cells infected only 10 min before label addition. The rate of P420 DNA synthesis under these conditions was comparable to that found for a resident GoIEl plasmid in the presence of chloramphenicol.
FIG. 4. Analysis of DNA made after P420 infection. HF4704 was grown as described in Fig. 3 and infected with P420 2.5 hr prior to the addition of [3H]thymine. Four hours after the label was added, a sample was taken, the cells were lysed with lysozyme and Sarkosyl, and the lysate was analyzed by CsCl/ethidium bromide centrifugation (13). The majority of 3H-labeled DNA represents DNA synthesized 2.5-6.5 hr after infection with P420; "4C-labeled DNA represents chromosomal DNA made prior to the addition of chloramphenicol.
Puromycin-treated cells (1 ug/ml) showed the same rate of DNA synthesis after P420 infection as did the chloramphenicol-treated cells. Streptomycin-treated cells (100 Mg/ml) had a lower rate of P420-induced DNA replication but, when either puromycin or chloramphenicol also was added, the full rate of phage-dependent synthesis was observed. Most of the DNA synthesized in P420-infected cells (80%) was in the form of covalently closed circles, as determined by ethidium bromide/CsCl equilibrium centrifugation (Fig. 4). These circular DNA molecules also showed the sedimentation characteristics of P420 in neutral sucrose gradients and yielded the expected fragments after digestion with EcoRI (Fig. 1, lane e), Sma I, Hae II, Hpa I, Pst I, and HindIII. Several lines of evidence indicate that the DNA synthesis observed in P420-infected cells in the presence of chloramphenicol is dependent on the ColEl replicon. No synthesis was seen in cells infected with P4 virn (Fig. 3). In addition, P420infected cells that contained the polAl mutation did not have a rate of DNA synthesis greater than that found in uninfected cells (Fig. 5 left). ColEl DNA synthesis is dependent on DNA polymerase I (23) but P4 phage DNA replicates normally in polAl mutants (unpublished data). Phage adsorption was not inhibited in C-2110thy because viable cells of this strain and of its P2 lysogen were efficiently killed by the phage. ColEl DNA synthesis is known to require concomitant RNA synthesis that is sensitive to rifampicin (24, 25). On the other hand, P4 DNA synthesis requires a phage-coded RNA polymerase that is rifampicin resistant (22). As shown in Fig. 5 right, P420 DNA replication was inhibited by rifampicin (100,ug/ml) even when the antibiotic was added to a culture actively synthesizing P420 DNA.
P420 Replication in Cells Containing CoIEl. In an attempt to determine if P420 could be used to probe the mechanism of ColEl incompatibility, chloramphenicol-treated cells that had a resident ColEl plasmid were infected with P420 phage. In cells that contained pMK1, a 3.2-kb derivative of ColEl that specifies tetracycline resistance, the level of DNA synthesis in the presence of chloramphenicol was the same with or without infection with P420 (Fig. 6). Because considerably more DNA synthesis occurred in P420-infected cells lacking any plasmid than in cells containing pMK1, it is clear that the presence of pMK1 interferes with P420 DNA replication. The level of DNA synthesis in the presence of chloramphenicol in cells carrying
Proc. Natl. Acad. Sci. USA 75 (1978)-
Biochemistry: Kahn and Helinski
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FIG. 5. (Left) Requirement of DNA polymerase I for P420 DNA replication in the presence of chloramphenicol. P420 was used to infect C-2110thy (polAl) and HF4704 (polA+) 2.5 hr prior to the addition of [3H]thymine as described in Fig. 3. Samples were taken at the times indicated and radioactivity was determined as in Fig.3. A, P420-infected C-2110thy; A, uninfected C-2110thy; 0, P420-infected HF4704; 0, uninfected HF4704. (Right) Rifampicin-sensitivity of P420 DNA synthesis. HF4704 cells were infected with P420 2.5 hr prior to the addition of [3H~thymine as described in Fig. 3. At the time [3H]thymine was added (A) or 2 hr later (A), rifampicin (100 Atg/ml) was added. One culture was not infected with phage (0) and another was infected but not treated with the drug (-). Because rifampicin was added as a solution in dimethyl sulfoxide, an equal volume of this solvent was added to the control cultures.
the ColEl derivative pMK20 or pCRl also was unchanged by infection with P420. In these cases, however, the basal level of DNA synthesis is already as high as it is in cells not containing the plasmid and infected with P420. Sucrose gradient analysis of the products of DNA synthesis in pMK1- and pMK20-containing cells infected with P420 showed that essentially all of the label was incorporated into DNA of the length of the resident plasmid. We also found that an amber mutant of P420 will grow lytically on various nonsuppressing strains unless these strains carry pMK1, pMK20, or pCR1 as a resident plasmid (unpublished data). DISCUSSION A ColEl derivative, pMK20, has been joined to bacteriophage P4 to yield a hybrid with both plasmid and phage properties. This phasmid,* P420, was used to establish the ability of ColEl to replicate in the absence of a phage-coded protein. P420 can be easily interconverted between the phage and plasmid states. The existence of a stable plasmid form of the hybrid, especially in P2 lysogens, was unexpected. P4 vir associations with nonlysogens have been described (5), but they are temperature-sensitive for growth and lose P4 readily (unpublished data). Cells carrying the P420 phasmid lack these properties, possibly due to deletion of the P4 EcoRI B and C fragments or because of some moderating influence of the ColEl DNA. The phage component of the phasmid used in this work, bacteriophage P4, has several properties that should be useful in further hybrid constructions. The P4 EcoRI A-D fragment *
This word, from the Greek word phasm meaning apparition, is appropriate to describe the changeable properties of the phage-plasmid combinations. The possible use of this term for phage-plasmid combinations was brought to our attention by Dr. Sidney Brenner.
0
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FIG. 6. P420 DNA synthesis in ColEl-containing cells. Cells were grown and infected with P420, as described in Fig. 5 left: *, P420infected HF4704; 0, uninfected HF4704; A, P420-infected HF4704 (pMK1); A, uninfected HF4704 (pMK1). Samples were taken and radioactivity was determined as described in Fig. 3.
will grow lytically only if it is joined to some additional DNA (ref. 2; unpublished data). The minimal insert that will permit growth is somewhat less than 2 kb and the maximum is about 3.6 kb. In this respect, P4 resembles the X gt cloning system of Thomas et al. (26). The P4 system does offer certain unique advantages. Because P4 can activate bacteriophage P2 without inducing it, P4 phage can be purified relatively free of helper contamination. P4 DNA also can be packaged into the larger P2-sized heads (27). P4 could carry an insert of 13 to 30 kb if it were packaged into the larger P2 phage capsid. Preliminary experiments have shown that at least 25 kb of DNA can be packaged in vivo into P2 particles when the DNA is coupled to the P4 cohesive end site (unpublished data). In vitro packaging of recombinant DNA by extracts or P4- and P2-infected cells (27) could improve the efficiency of recombinant DNA recovery. This method has been used successfully with X (28). In addition to our observation that P420 will replicate in vio in the presence of an inhibitor of protein synthesis, several other observations support the conclusion that ColEl replication does not require a positive-acting protein: (i) a plasmid-specified protein is not required for ColEl DNA synthesis in vitro (29), (ii) attempts to identify a trans-acting protein that permits the origin region of ColEl to replicate in vivo have been unsuccessful (unpublished data), (iii) an amber mutant defective in replication has not been isolated despite an extensive search (K. Armstrong, personal communication), and (iv) studies with a ColEl-bacteriophage X hybrid have led to a similar conclusion that no plasmid-coded protein is required for ColEl DNA replication (30). However, these observations do not exclude the possible existence of a negative control protein specified by the plasmid, which regulates plasmid copy number. It clearly is difficult to establish experimentally that there is absolutely no synthesis of a plasmid-coded protein. It could be argued that chloramphenicol is ineffective in inhibiting synthesis of this hypothetical protein. However, because DNA synthesis begins immediately after infection (Fig. 3), the protein would have to be synthesized rapidly. This-is unlikely in the presence of high concentrations of chloramphenicol because this inhibitor slows ribosome movement (20). Other inhibitors that have different modes of action (puromycin and streptomycin) also fail to block DNA synthesis. We also consider it
2204
Biochemistry: Kahn and Helinski
unlikely that a ColEl-specified protein that is required for replication is packaged by P420 and introduced into the cell during infection. There is no evidence for packaging of cellular proteins by bacteriophage particles of the P4 type (31). The finding of a high level of P420 DNA synthesis at a low multiplicity of infection would demand'an efficient mechanism for the packaging of a replication protein. The possibility that tightly bound proteins in the form of a relaxation complex are packaged is unlikely in view of our failure to detect the presence of the parent plasmid pMK20 as a relaxation complex (unpublished data). Plasmid incompatibility, the inability of two similar plasmids to coexist stably in a cell, has been difficult to study experimentally. Many plasmids have been assigned to groups on the basis of their compatibility relationships, but in no case has a mechanism for incompatibility been demonstrated. Use of a phasmid may permit new insight into incompatibility. It was found that a resident plasmid inhibits the replication of entering P420 (Fig. 6). This inhibition by plasmid pMK1 may be due to direct competition for replication components, in which case plasmid pMK1 has an advantage either by virtue of its high copy number relative to the phasmid or because as a resident plasmid it may sequester these components before P420 enters. Alternatively, pMK1 may specify a protein that specifically inhibits ColEl replication. The finding that ColEl DNA replication does not require a plasmid-encoded protein makes it unlikely that ColEl replication is controlled by a positive-acting regulatory protein as proposed in the model of Jacob et al. (32). It is conceivable that the sequence or structure of DNA at the origin of replication, or of an RNA transcript of this DNA, provides sufficient information for cellular enzymes to recognize it as an origin of replication. The procedures demonstrated here for manipulating a plasmid as a phage should be very useful in investigating various aspects of plasmid biology. Plasmid mutants may be easier to find with the phasmid system because the efficient injection of mutagenized plasmid into cells by the phasmid particles will permit a larger number of potential mutants to be screened. Complementation analysis of plasmid replication and incompatibility mutants should be facilitated by conversion of the plasmids into infective phage elements. It is also possible that new classes of mutants will be found that cannot be stably maintained in cells but can be propagated as phage. Furthermore, by using techniques similar to those used here, the events that lead to plasmid establishment and the biochemical basis of host mutations that block plasmid maintenance can be dissected. Finally, the unique properties of the P4 phage as a vehicle for cloning DNA in E. coli may prove useful in situations in which present plasmid and phage vehicles are unsuitable. We thank Richard Calendar for helpful discussions about P4. This investigation was supported by grants from the National Institute of
Proc. Natl. Acad. Sci. USA 75 (1978) Allergy and Infectious Diseases (AI-07194) and the National Science Foundation (PCM77-0653). M.K. was supported by a postdoctoral fellowship from the SmithKline Corporation. 1. Six, E. W. & Klug, C. (1973) Virology 51,327-344. 2. Souza, L., Geisselsoder, J., Hopkins, A. & Calendar, R. (1978) Virology, in press. 3. Greene, P. J., Betlach, M. C., Goodman, H. M. & Boyer, H. W. (1974) in Methods in Molecular Biology, ed. Wickner, R. B. (Marcel Dekker, New York), Vol. 7, pp. 87-111. 4. Barrett, K. J., Marsh, M. L. & Calendar, R. (1976) J. Mol. Biol. 106,683-707. 5. Lindqvist, B. H. & Six, E. W. (1971) Virology 43, 1-7. 6. Bachmann, B. J. (1972) Bacteriol. Rev. 36,525-557. 7. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, NY), p. 220. 8. Apirion, D. & Watson, N. (1975) J. Bacteriol. 124,317-324. 9. Wood, W. B. (1966) J. Mol. Biol. 16, 118-133. 10. Covey, C., Richardson, D. & Carbon, J. (1976) Mol. Gen. Genet. 145, 155-158. 11. Armstrong, K. A., Hershfield, V. & Helinski, D. R. (1977) Science 196, 172-174. 12. Clewell, D. B. & Helinski, D. R. (1972) J. Bacteriol. 110, 1135-1146. 13. Katz, L., Kingsbury, D. T. & Helinski, D. R. (1973) J. Bacteriol. 114,577-591. 14. Meyer, R., Figurski, D. & Helinski, D. R. (1977) Mol. Gen. Genet. 152, 129-135. 15. Lederberg, E. M. & Cohen, S. N. (1974) J. Bacteriol. 119, 1072-1074. 16. National Institutes of Health (1976) Fed. Reg. 41, 769-775. 17. Oka, A. & Takanami, M. (1976) Nature 264, 193-194. 18. Goldstein, L., Thomas, M. & Davis, R. W. (1975) Virology 66, 420-427. 19. Kahn, M. & Hopkins, A. (1978) Virology, in press. 20. Pestka, S. (1971) Annu. Rev. Microbiol. 25,487-562. 21. Maaloe, 0. & Hanawalt, P. C. (1961) J. Mol. Biol. 3,144-155. 22. Barrett, K. J., Gibbs, W. & Calendar, R. (1972) Proc. Natl. Acad. Sci. USA 69,2986-2990. 23. Kingsbury, D. T. & Helinski, D. R. (1973) J. Bacteriol. 114, 1116-1124. 24. Sakakibara, Y. & Tomizawa, J.-I. (1974) Proc. Natl. Acad. Sci.
USA 71,802-806. 25. Clewell, D. B., Evenchik, B. & Cranston, J. W. (1972) Nature New Biol. 237, 29-31. 26. Thomas, M., Cameron, J. R. & Davis, R. W. (1974) Proc. Natl. Acad. Sci. USA 71, 4579-4583. 27. Pruss, G., Goldstein, R. & Calendar, R. (1974) Proc. Natl. Acad. Sci. USA 71, 2367-2371. 28. Hohn, B. & Murray, K. (1977) Proc. Natl. Acad. Sci. USA 74, 3259-3263. 29. Tomizawa, J., Sakakibara, Y. & Kakefuda, T. (1975) Proc. Natl. Acad. Sci. USA 72,1050-1054. 30. Donoghue, D. J. & Sharp, P. A. (1978) J. Bacteriol. 133, 12871294. 31. Casiens, S. & King, J. (1975) Annu. Rev. Biochem. 44, 555611. 32. Jacob, F., Brenner, S. & Cuzin, F. (1963) Cold Spring Harbor Symp. Quant. Biol. 28,329-348.
Vol. 75, No. 5, pp. 2200-2204, May 1978
Biochemistry
Construction of a novel plasmid-phage hybrid: Use of the hybrid to demonstrate ColE 1 DNA replication in vivo in the absence of a ColEl-specified protein (bacteriophage P4/gene cloning/chloramphenicol/plasmid incompatibility)
MICHAEL KAHN AND DONALD R. HELINSKI Department of Biology, University of California, San Diego, La Jolla, California 92093
Communicated by E. Peter Geiduschek, February 21, 1978
ABSTRACT
A hybrid bacteriophage, P420, was constructed in vitro; it contains part of bacteriophage P4 and a 3.6-kilobase derivative of plasmid CoWEl. In Escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. Replication of P420, directed by the ColEl replicon, was found to occur after P420 phage infection of E. coli cells that had been incubated in the presence of chloramphenicol. Replication began without a lag period and resulted in the synthesis of covalently closed circles of P420 DNA. Like ColEl DNA replication but unlike that of P4, replication was dependent on DNA polymerase I and was sensitive to rifampicin. The presence of a resident ColEl plasmid in the infected cells resulted in an inhibition of the replication of the incoming P420 DNA. These results indicate that CoEl does not require a plasmid-coded protein to replicate its DNA in vivo and demonstrate the utility of P4 bacteriophage for coupling bacteriophage properties to a plasmid replicon.
Plasmid DNA replication in bacteria is of particular interest in view of the diversity of replication modes and the variety of control systems that may be involved in the replication of different plasmids. Biochemical approaches to the in vivo study of plasmid replication, incompatibility, and expression, however, have been limited to cells in which plasmids have previously been established. Because there has been no efficient method of introducing plasmids into cells synchronously and quantitatively, many techniques used successfully in the study of other DNA replication systems have not been available for the study of plasmids. This difficulty could conceivably be overcome by reconstructing the plasmid to allow it to be packaged into bacteriophage particles. Infection of bacterial cells with these phage particles would then result in the injection of the plasmid DNA carried by the particles. In this way, methods successfully used to study bacteriophage DNA replication in a bacterial cell could be applied to plasmid DNA
replication. Bacteriophage P4 has several properties that can be utilized to facilitate the packaging of a plasmid. In Escherichia coli, P4 lytic growth is dependent on the presence of a helper bacteriophage, P2 (1), and P4 has no lytic functions when grown in the absence of P2. In this respect, P4 resembles a defective virus except that it can efficiently activate a P2 prophage and produce P4 phage free of P2 helper contamination. Souza et al. (2) have shown in vivo that the 8-kilobase (kb) fragment produced by digestion of P4 with the restriction enzyme EcoRI is sufficient for lytic growth if the size of the phage DNA is maintained near 11 kb by the substitution of about 3 kb of foreign DNA. In this paper we report the construction by in vitro techThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
niques of a hybrid DNA molecule made up of a segment of the P4 genome and a low molecular weight derivative of plasmid ColEl. This ColEl-P4 recombinant molecule can exist in E. coli as a plasmid and can be packaged into infective bacteriophage particles. In this study, E. coli was infected with the hybrid, and ColEl-directed replication of the hybrid DNA was analyzed in the presence of the protein synthesis inhibitor chloramphenicol. Evidence is presented that replication of ColEl in vivo does not require a ColEl-specified protein. MATERIALS AND METHODS Reagents. Sources for reagents were: Sigma (lysozyme, chloramphenicol, puromycin, kanamycin, streptomycin, and rifampicin); New England Nuclear [[methyl-3H]thymine (>10 Ci/mmol), [2-'4C]thymine (>50 mCi/mmol)]; Baker (polyethylene glycol 6000); New England Biolabs (Pst I, Sma I, Hae II, HindIII, Sal I, and Hpa I restriction enzymes); Miles Laboratories (T4 DNA ligase). EcoRI was prepared according to Greene et al. (3). All antibiotics used in inhibition studies were added as powders except rifampicin which was used as a 10 mg/ml solution in dimethyl sulfoxide. LB and TPG media have been described (4, 5). Bacterial Strains. HF4704 (5) and AB2497 (6) have been described. C-2110 (polAl his rha) was obtained from M. Sunshine and a Thy- derivative, C-2110thy, was selected. N2076T (rnc+ thyA deo thi purI argH nadB lacY gal maIA xyl ara mtl str tonA supE44) and N2077T (N2076 rnc thyA deo) were made by using a trimethoprim selection (7) from the thymine prototrophs described by Apirion and Watson (8) and obtained from D. Apirion. C-2326 (r-m- supD argstrp tonB P21g) was obtained from R. Calendar. An r-mK+trp + derivative of this strain, CK2326, was constructed by conjugal mating using HF958 (HfrH r-mK+ leu thy) (9). Plasmids. The construction of pMK20 and pMKl will be described elsewhere. pMK1 contains a 1.3-kb replication region of ColEl and codes for tetracycline resistance. Plasmid pCR1 was isolated by covey et al. (10) and has been described by Armstrong et al. (11). Bacteriophages. P4 vir, was obtained from R. Calendar. Phage stocks were grown on CK2326 and purified by using polyethylene glycol as described by Barrett et al. (4). P420, the P4-ColEl hybrid constructed in this study, was found to be unstable in CsCl. Therefore, all phage stocks were precipitated with polyethylene glycol, resuspended in 100 mM Tris/10 mM MgCl2, pH 8, and stored at 1012 plaque-forming units (PFU)/ ml. Clone Analysis. Plasmid DNA corresponding to individual transformants or transfectants was prepared from cells that had Abbreviations: kb, kilobase(s); PFU, plaque-forming unit.
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Proc. Natl. Acad. Sc. USA 75 (1978)
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_~~~~~~~~~~ FIG. 1. Agarose gel analysis of P420 hybrid DNA. The following DNA samples were examined by electrop'horesis in 1% agarose gels: EcoRI digests of DNA purified from P4 yinl infected cells (a), plasmid pMK2O DNA (b), P420 DNA isolated from plasmid containing cells (c), P420ODNA purified from infected cells (d), and P420 DNA isolated from infected, choapeio-treated cells (e); Pst I digests of DNA from cells infected with phage P4 yir1 (f) and phage P420 (g); and undigested DNA from cells infected by P4 vir1 (h) and P420 (i).
been treated with chloramphenicol (250,Mg/mi) to enrich for resident plasmids (12) or from P2 nonlysogens that had been infected for 2 hr with phage derived from individual plaques. Cells were lysed by using the lysozyme/Triton X-100 procedure of Katz eat al. (13). All extracts were phenol-extracted and in certain cases the DNA was further purified by using CsCl/ ethidium, bromide gradients. The yield of covalently closed DNA from these procedures was typically 0.5-1 1tg of DNA per ml of culture. Analysis of DNA. Restriction, ligation, and gel electrophoresis conditions are described elsewhere (14). CK2326 was transfected by wsing the procedure of Lederberg and Cohen (15). All recombinant DNA procedures were carried out in accordance with the National Institutes of Health "Guidelines on Recombinant DNA Research" (16). RESULTS Construction of the P4-ColEl Hybrid. A low molecular weight derivative of ColEl was isolated by cutting ColEl and pCR1 DNA with the restriction enzyme Hae II and joining the fragments together with DNA ligase. AB2497 was transformed with the mixture and kanamycin-resistant transformants were selected. The resulting plasmid, pMK2O, contains both the Hae II A and E fragments of ColEl1 (17) and a 1. 1-kb Hae II fragment from pCR1 that specifies kanamycin resistance. pMK2O is 3.6 kb long and has a single EcoRI site. EcoRI digestion of closed circles of bacteriophage P4 yields three fragments designated AD, B, and C (18) (Fig. 1, lane a). P4 vir, DNA and
Sma
FIG. 2. Restriction map of P420. The restriction sites in P4 are from Kahn and Hopkins (19); restriction sites in pMK20 were derived from unpublished data. The site of the P4 cohesive ends (P4 cos) is defined as the left end of the linear map. Restriction enzymes used were HindIll (Ill), EcoRI (RI), Pst I (Pst), Sma I (Sma), Hpa I (Hpa), Sal I (Sal), and Hae II.
pMK20 DNA were digested with EcoRI, ligated together, and used to transfect the P4 indicator strain CK2326. Cells at the margins of the resultant plaques were tested for kanamycin resistance (50 ,ug/ml). Many plaques yielded stable kanamycin-resistant cells that were found to contain supercoiled plasmid DNA. These cells spontaneously released phage that gave clear plaques on CK2326 that also had kanamycin-resistant bacteria at their margins. The P420 hybrid phage made a slightly larger plaque than did P4 vir1 and could be grown lytically in good yield (1010-o10l1 PFU/ml). Restriction analysis of the plasmid DNA isolated from kanamycin-resistant cells demonstrated that the plasmids they contained were recombinants between P4 and pMK20. An analysis by gel electrophoresis of one of these recombinant plasmids, P420, is shown in Fig. 1. Digestion of the DNA with EcoRI (Fig. 1, lane c) showed that the molecule is composed of the large EcoRI AD fragment of P4 (Fig. 1, lane a) and the entire chromosome of pMK20 (Fig. 1, lane b). The structure of P420 deduced from restriction enzyme analysis is shown in Fig. 2. A recombinant molecule that had the other orientation of pMK20 relative to P4 was also isolated. The DNA in both P4infected cells (Fig. 1, lane h) and P420-infected cells (Fig. 1, lane i) was present as covalently closed circles. The single band produced after digestion of P420 DNA with Pst I (Fig. 1, lane g) is slightly larger than the band from a similar digest of P4 virl DNA (Fig. 1, lane f) because the pMK20 insert (3.6 kb) is slightly larger than the deleted B and C fragments of P4 (total size, 3.2 kb). This additional DNA may account for the greater instability of P420 phage in CsCl solution compared with P4 virl. These results indicate that the hybrid molecule can exist either as a plasmid or as an infectious bacteriophage. P420 DNA can be obtained in three ways: (i) from the bacteriophage particles; (U) as covalently closed molecules from infected cells that are either lysogenic or nonlysogenic for the P2 helper phage; or (iii) after chloramphenicol amplification of cells carrying the hybrid as a stable plasmid. P420 DNA obtained from all three procedures was identical as judged by its physical
Biochemistry: Kahn and Helinski
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Proc. Nati. Acad. Sci. USA 75 (1978) 10 -
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FIG. 3. DNA synthesis in P420-infected cells. HF4704 was grown overnight at 370 in TPG medium supplemented with thymine at 25 ,ug/ml, 0.5% Casamino Acids, 0.2% glucose, 0.02% rhamnose, and thiamine and niacin (2 yg/ml) and then diluted 1:50 into the same medium containing 4 jug/ml of [14C]thymine (4 ;g/ml; 1 MCi/ml) to prelabel cells. At a density of 3 X 108 cells per ml, the culture was pelleted by centrifugation and resuspended in an equal volume of unlabeled medium. Chloramphenicol (250 gg/ml) was added 15 min later. After 5 min the culture was divided into quarters and to two portions were added P4 (&) and P420 (-) separately at a multiplicity of infection of 10. After an additional 2.5 hr, P420 was added to a third portion (0) and [3H]thymine (50 MCi/ml) was added to all cultures, including the uninfected control (-). Samples (0.2 ml) were removed and added to a solution containing 0.2 M NaOH/1% Sarkosyl at the times indicated. Salmon sperm DNA (50 Mg/ml) was added to each sample and the samples were precipitated with 5% trichloroacetic acid, filtered through glass fiber filters, and assayed for radioactivity. Plotted are the raw ratios of 3H to 14C. For the experiments in this and other figures, 14C was typically 3000-5000 cpm per sample. Arrow, actual time of phage addition; [3H]thymine was then added and the first samples were taken at time zero.
characteristics (Fig. 1, lanes c, d, and e) and its biological activity, except that DNA derived from phage particles is linear. P420 DNA Replication in the Absence of Protein Synthesis. HF4704 was infected with P420 in the presence of chloramphenicol at 250 ,g/ml. Under the conditions used in this study, protein synthesis is less than 3% of that found in the absence of the inhibitor, and this residual incorporation is probably into small peptides (20). In the absence of protein synthesis, chromosomal DNA replication stops within 2 hr (21). As shown in Fig. 3, uninfected cells incorporated only a very small amount of [3H]thymine under these conditions. P4 DNA replication requires a phage-coded protein, and it is not surprising that, because this protein is not made in the presence of chloramphenicol (22), P4-infected cells show no additional incorporation of label. However, cells infected with P420 did synthesize DNA under these conditions. This synthesis was linear for at least 3 hr and the rate of synthesis was the same whether the P420 multiplicity of infection was 5 or 50 PFU per cell. There was no lag in the initiation of P420 DNA synthesis. Cells infected with P420 2.5 hr prior to the addition of [3H]thymine showed the same kinetics of label incorporation as cells infected only 10 min before label addition. The rate of P420 DNA synthesis under these conditions was comparable to that found for a resident GoIEl plasmid in the presence of chloramphenicol.
FIG. 4. Analysis of DNA made after P420 infection. HF4704 was grown as described in Fig. 3 and infected with P420 2.5 hr prior to the addition of [3H]thymine. Four hours after the label was added, a sample was taken, the cells were lysed with lysozyme and Sarkosyl, and the lysate was analyzed by CsCl/ethidium bromide centrifugation (13). The majority of 3H-labeled DNA represents DNA synthesized 2.5-6.5 hr after infection with P420; "4C-labeled DNA represents chromosomal DNA made prior to the addition of chloramphenicol.
Puromycin-treated cells (1 ug/ml) showed the same rate of DNA synthesis after P420 infection as did the chloramphenicol-treated cells. Streptomycin-treated cells (100 Mg/ml) had a lower rate of P420-induced DNA replication but, when either puromycin or chloramphenicol also was added, the full rate of phage-dependent synthesis was observed. Most of the DNA synthesized in P420-infected cells (80%) was in the form of covalently closed circles, as determined by ethidium bromide/CsCl equilibrium centrifugation (Fig. 4). These circular DNA molecules also showed the sedimentation characteristics of P420 in neutral sucrose gradients and yielded the expected fragments after digestion with EcoRI (Fig. 1, lane e), Sma I, Hae II, Hpa I, Pst I, and HindIII. Several lines of evidence indicate that the DNA synthesis observed in P420-infected cells in the presence of chloramphenicol is dependent on the ColEl replicon. No synthesis was seen in cells infected with P4 virn (Fig. 3). In addition, P420infected cells that contained the polAl mutation did not have a rate of DNA synthesis greater than that found in uninfected cells (Fig. 5 left). ColEl DNA synthesis is dependent on DNA polymerase I (23) but P4 phage DNA replicates normally in polAl mutants (unpublished data). Phage adsorption was not inhibited in C-2110thy because viable cells of this strain and of its P2 lysogen were efficiently killed by the phage. ColEl DNA synthesis is known to require concomitant RNA synthesis that is sensitive to rifampicin (24, 25). On the other hand, P4 DNA synthesis requires a phage-coded RNA polymerase that is rifampicin resistant (22). As shown in Fig. 5 right, P420 DNA replication was inhibited by rifampicin (100,ug/ml) even when the antibiotic was added to a culture actively synthesizing P420 DNA.
P420 Replication in Cells Containing CoIEl. In an attempt to determine if P420 could be used to probe the mechanism of ColEl incompatibility, chloramphenicol-treated cells that had a resident ColEl plasmid were infected with P420 phage. In cells that contained pMK1, a 3.2-kb derivative of ColEl that specifies tetracycline resistance, the level of DNA synthesis in the presence of chloramphenicol was the same with or without infection with P420 (Fig. 6). Because considerably more DNA synthesis occurred in P420-infected cells lacking any plasmid than in cells containing pMK1, it is clear that the presence of pMK1 interferes with P420 DNA replication. The level of DNA synthesis in the presence of chloramphenicol in cells carrying
Proc. Natl. Acad. Sci. USA 75 (1978)-
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FIG. 5. (Left) Requirement of DNA polymerase I for P420 DNA replication in the presence of chloramphenicol. P420 was used to infect C-2110thy (polAl) and HF4704 (polA+) 2.5 hr prior to the addition of [3H]thymine as described in Fig. 3. Samples were taken at the times indicated and radioactivity was determined as in Fig.3. A, P420-infected C-2110thy; A, uninfected C-2110thy; 0, P420-infected HF4704; 0, uninfected HF4704. (Right) Rifampicin-sensitivity of P420 DNA synthesis. HF4704 cells were infected with P420 2.5 hr prior to the addition of [3H~thymine as described in Fig. 3. At the time [3H]thymine was added (A) or 2 hr later (A), rifampicin (100 Atg/ml) was added. One culture was not infected with phage (0) and another was infected but not treated with the drug (-). Because rifampicin was added as a solution in dimethyl sulfoxide, an equal volume of this solvent was added to the control cultures.
the ColEl derivative pMK20 or pCRl also was unchanged by infection with P420. In these cases, however, the basal level of DNA synthesis is already as high as it is in cells not containing the plasmid and infected with P420. Sucrose gradient analysis of the products of DNA synthesis in pMK1- and pMK20-containing cells infected with P420 showed that essentially all of the label was incorporated into DNA of the length of the resident plasmid. We also found that an amber mutant of P420 will grow lytically on various nonsuppressing strains unless these strains carry pMK1, pMK20, or pCR1 as a resident plasmid (unpublished data). DISCUSSION A ColEl derivative, pMK20, has been joined to bacteriophage P4 to yield a hybrid with both plasmid and phage properties. This phasmid,* P420, was used to establish the ability of ColEl to replicate in the absence of a phage-coded protein. P420 can be easily interconverted between the phage and plasmid states. The existence of a stable plasmid form of the hybrid, especially in P2 lysogens, was unexpected. P4 vir associations with nonlysogens have been described (5), but they are temperature-sensitive for growth and lose P4 readily (unpublished data). Cells carrying the P420 phasmid lack these properties, possibly due to deletion of the P4 EcoRI B and C fragments or because of some moderating influence of the ColEl DNA. The phage component of the phasmid used in this work, bacteriophage P4, has several properties that should be useful in further hybrid constructions. The P4 EcoRI A-D fragment *
This word, from the Greek word phasm meaning apparition, is appropriate to describe the changeable properties of the phage-plasmid combinations. The possible use of this term for phage-plasmid combinations was brought to our attention by Dr. Sidney Brenner.
0
1
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FIG. 6. P420 DNA synthesis in ColEl-containing cells. Cells were grown and infected with P420, as described in Fig. 5 left: *, P420infected HF4704; 0, uninfected HF4704; A, P420-infected HF4704 (pMK1); A, uninfected HF4704 (pMK1). Samples were taken and radioactivity was determined as described in Fig. 3.
will grow lytically only if it is joined to some additional DNA (ref. 2; unpublished data). The minimal insert that will permit growth is somewhat less than 2 kb and the maximum is about 3.6 kb. In this respect, P4 resembles the X gt cloning system of Thomas et al. (26). The P4 system does offer certain unique advantages. Because P4 can activate bacteriophage P2 without inducing it, P4 phage can be purified relatively free of helper contamination. P4 DNA also can be packaged into the larger P2-sized heads (27). P4 could carry an insert of 13 to 30 kb if it were packaged into the larger P2 phage capsid. Preliminary experiments have shown that at least 25 kb of DNA can be packaged in vivo into P2 particles when the DNA is coupled to the P4 cohesive end site (unpublished data). In vitro packaging of recombinant DNA by extracts or P4- and P2-infected cells (27) could improve the efficiency of recombinant DNA recovery. This method has been used successfully with X (28). In addition to our observation that P420 will replicate in vio in the presence of an inhibitor of protein synthesis, several other observations support the conclusion that ColEl replication does not require a positive-acting protein: (i) a plasmid-specified protein is not required for ColEl DNA synthesis in vitro (29), (ii) attempts to identify a trans-acting protein that permits the origin region of ColEl to replicate in vivo have been unsuccessful (unpublished data), (iii) an amber mutant defective in replication has not been isolated despite an extensive search (K. Armstrong, personal communication), and (iv) studies with a ColEl-bacteriophage X hybrid have led to a similar conclusion that no plasmid-coded protein is required for ColEl DNA replication (30). However, these observations do not exclude the possible existence of a negative control protein specified by the plasmid, which regulates plasmid copy number. It clearly is difficult to establish experimentally that there is absolutely no synthesis of a plasmid-coded protein. It could be argued that chloramphenicol is ineffective in inhibiting synthesis of this hypothetical protein. However, because DNA synthesis begins immediately after infection (Fig. 3), the protein would have to be synthesized rapidly. This-is unlikely in the presence of high concentrations of chloramphenicol because this inhibitor slows ribosome movement (20). Other inhibitors that have different modes of action (puromycin and streptomycin) also fail to block DNA synthesis. We also consider it
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unlikely that a ColEl-specified protein that is required for replication is packaged by P420 and introduced into the cell during infection. There is no evidence for packaging of cellular proteins by bacteriophage particles of the P4 type (31). The finding of a high level of P420 DNA synthesis at a low multiplicity of infection would demand'an efficient mechanism for the packaging of a replication protein. The possibility that tightly bound proteins in the form of a relaxation complex are packaged is unlikely in view of our failure to detect the presence of the parent plasmid pMK20 as a relaxation complex (unpublished data). Plasmid incompatibility, the inability of two similar plasmids to coexist stably in a cell, has been difficult to study experimentally. Many plasmids have been assigned to groups on the basis of their compatibility relationships, but in no case has a mechanism for incompatibility been demonstrated. Use of a phasmid may permit new insight into incompatibility. It was found that a resident plasmid inhibits the replication of entering P420 (Fig. 6). This inhibition by plasmid pMK1 may be due to direct competition for replication components, in which case plasmid pMK1 has an advantage either by virtue of its high copy number relative to the phasmid or because as a resident plasmid it may sequester these components before P420 enters. Alternatively, pMK1 may specify a protein that specifically inhibits ColEl replication. The finding that ColEl DNA replication does not require a plasmid-encoded protein makes it unlikely that ColEl replication is controlled by a positive-acting regulatory protein as proposed in the model of Jacob et al. (32). It is conceivable that the sequence or structure of DNA at the origin of replication, or of an RNA transcript of this DNA, provides sufficient information for cellular enzymes to recognize it as an origin of replication. The procedures demonstrated here for manipulating a plasmid as a phage should be very useful in investigating various aspects of plasmid biology. Plasmid mutants may be easier to find with the phasmid system because the efficient injection of mutagenized plasmid into cells by the phasmid particles will permit a larger number of potential mutants to be screened. Complementation analysis of plasmid replication and incompatibility mutants should be facilitated by conversion of the plasmids into infective phage elements. It is also possible that new classes of mutants will be found that cannot be stably maintained in cells but can be propagated as phage. Furthermore, by using techniques similar to those used here, the events that lead to plasmid establishment and the biochemical basis of host mutations that block plasmid maintenance can be dissected. Finally, the unique properties of the P4 phage as a vehicle for cloning DNA in E. coli may prove useful in situations in which present plasmid and phage vehicles are unsuitable. We thank Richard Calendar for helpful discussions about P4. This investigation was supported by grants from the National Institute of
Proc. Natl. Acad. Sci. USA 75 (1978) Allergy and Infectious Diseases (AI-07194) and the National Science Foundation (PCM77-0653). M.K. was supported by a postdoctoral fellowship from the SmithKline Corporation. 1. Six, E. W. & Klug, C. (1973) Virology 51,327-344. 2. Souza, L., Geisselsoder, J., Hopkins, A. & Calendar, R. (1978) Virology, in press. 3. Greene, P. J., Betlach, M. C., Goodman, H. M. & Boyer, H. W. (1974) in Methods in Molecular Biology, ed. Wickner, R. B. (Marcel Dekker, New York), Vol. 7, pp. 87-111. 4. Barrett, K. J., Marsh, M. L. & Calendar, R. (1976) J. Mol. Biol. 106,683-707. 5. Lindqvist, B. H. & Six, E. W. (1971) Virology 43, 1-7. 6. Bachmann, B. J. (1972) Bacteriol. Rev. 36,525-557. 7. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, NY), p. 220. 8. Apirion, D. & Watson, N. (1975) J. Bacteriol. 124,317-324. 9. Wood, W. B. (1966) J. Mol. Biol. 16, 118-133. 10. Covey, C., Richardson, D. & Carbon, J. (1976) Mol. Gen. Genet. 145, 155-158. 11. Armstrong, K. A., Hershfield, V. & Helinski, D. R. (1977) Science 196, 172-174. 12. Clewell, D. B. & Helinski, D. R. (1972) J. Bacteriol. 110, 1135-1146. 13. Katz, L., Kingsbury, D. T. & Helinski, D. R. (1973) J. Bacteriol. 114,577-591. 14. Meyer, R., Figurski, D. & Helinski, D. R. (1977) Mol. Gen. Genet. 152, 129-135. 15. Lederberg, E. M. & Cohen, S. N. (1974) J. Bacteriol. 119, 1072-1074. 16. National Institutes of Health (1976) Fed. Reg. 41, 769-775. 17. Oka, A. & Takanami, M. (1976) Nature 264, 193-194. 18. Goldstein, L., Thomas, M. & Davis, R. W. (1975) Virology 66, 420-427. 19. Kahn, M. & Hopkins, A. (1978) Virology, in press. 20. Pestka, S. (1971) Annu. Rev. Microbiol. 25,487-562. 21. Maaloe, 0. & Hanawalt, P. C. (1961) J. Mol. Biol. 3,144-155. 22. Barrett, K. J., Gibbs, W. & Calendar, R. (1972) Proc. Natl. Acad. Sci. USA 69,2986-2990. 23. Kingsbury, D. T. & Helinski, D. R. (1973) J. Bacteriol. 114, 1116-1124. 24. Sakakibara, Y. & Tomizawa, J.-I. (1974) Proc. Natl. Acad. Sci.
USA 71,802-806. 25. Clewell, D. B., Evenchik, B. & Cranston, J. W. (1972) Nature New Biol. 237, 29-31. 26. Thomas, M., Cameron, J. R. & Davis, R. W. (1974) Proc. Natl. Acad. Sci. USA 71, 4579-4583. 27. Pruss, G., Goldstein, R. & Calendar, R. (1974) Proc. Natl. Acad. Sci. USA 71, 2367-2371. 28. Hohn, B. & Murray, K. (1977) Proc. Natl. Acad. Sci. USA 74, 3259-3263. 29. Tomizawa, J., Sakakibara, Y. & Kakefuda, T. (1975) Proc. Natl. Acad. Sci. USA 72,1050-1054. 30. Donoghue, D. J. & Sharp, P. A. (1978) J. Bacteriol. 133, 12871294. 31. Casiens, S. & King, J. (1975) Annu. Rev. Biochem. 44, 555611. 32. Jacob, F., Brenner, S. & Cuzin, F. (1963) Cold Spring Harbor Symp. Quant. Biol. 28,329-348.
