Molec. gen. Genet. 169, 113-116 (1979) © by Springer-Verlag 1979
Short Communication
Construction in vitro of "Phage-Plasmid" Chimerae: a New Tool to Analyse the Mechanism of Plasmid Maintenance M. Couturier, J. Janssens, F. Bex, A. Desmyter, and I. Bonnevalle Laboratory of Genetics, Department of Molecular Biology, Universit6 libre de Bruxelles, rue des Chevaux, 67, B-1640 Rhode St Gen6se, Belgium
Summary. In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Ap ~ plasmid: pSC138 (Timmis et al., 1975). 2F recombinants were shown to be chimerae between the 2 and the F replicons. By genetical tests, we have demonstrated that both 2 and F replication mechanisms are functional: the 2F recombinant behaves as a non defective plaque forming phage on 2 sensitive bacteria and establishes itself as a stable plasmid on recA F - homoimmune bacteria. In the extra-chromosomal state, the 2F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed,
a
b
AWE
J ]
1.3_
Lac Z
bla ( ~
I
i.__2 Lac Z
c d
~tt redI NctqP
I
3 c17 n[n 5
Io
imm21 nm5
f5
e
..,,._ J. . . . . _~" bla f 5" f
QIS,,,R
/
L
cl7 nin5
cl7 nin5
f5
mm21
1. Construction of 2 F Recombinants
n5
f5
The maintenance of a defined plasmid in a bacterial strain involves several coordinated processes, including its controlled replication, its segregation and the expression of the incompatibility phenomenon. We intend to analyse the mechanism of plasmid maintenance in genetic and biochemical terms by cloning the genes implicated in these different processes in phage vectors. We thus cloned the EcoRI f5 fragment of the F' lac plasmid and an EcoRI fragment encoding ampicillin resistance (Ap r) in two lambdoid phage vectors. Both of these fragments exist in the mini F Apr plasmid pSC138 (Fig. 1, map d) (Timmis et al., 1975). It is known that the EcoRI f5 fragment of F carries two incompatibility loci incA and incB (Palchaudhuri and Maas, 1977; Manis and Kline, 1978) and all For offprints contact." M. Couturier
Fig. la-g. Represents the DNA maps of: b R631 (Murray et al., 1977); e 2imm21 816; d the miniF Apt: pSCI38 (Timmis et al., 1975); e the 2pSC138; f the 2631 f5 and g 2imm21 816 f5. Map a shows the position of some important markers on the 2 physical map. l indicates the position of an EcoRI target; -- the phage attachment site; gaps indicate the position of deletions and dotted lines represent substitutions, bla is the filactamase encoding gene of 2pSC138 which confers the ampicillin resistance phenotype
the genetic information necessary for the controlled autonomous replication of the F plasmid (Timmis et al., 1975; Lovett and Helinski, 1976). The vectors used are two replacement vectors, 2631 and 2imm21 816, both derived from 2 lac5 (Murray et al., 1977). They contain two EcoRI cleavage sites on both sides of their lac Z fragments (Fig. 1, maps b and c). EcoRI digested D N A mixtures of
0026-8925/79/0169/0113/$01.00
114
M. Couturier et al. : Construction in vitro of "Phage-Plasmid" Chimerae
pSC138 and 4631 DNA or pSC138 and )~imm21 816 DNA were ligated with T4 DNA ligase and used for transfection of Ca + + treated lac ZM15 bacteria (see Murray et al., 1977, for experimental procedures and references to the bacterial strain). The different 2F recombinants 2pSC138, 4631 f5 and 2imm21 816 f5 (Fig. 1, maps e f g) were identified among the Lac- plaque-forming phages by their ability to express, under 2 immunity, FI incompatibility and, in the case of 2pSC138, ampicillin resistance (see Sect. 3, iii).
2. Restriction Mapping of ApSC138, ~631 f5 and 2imm21 816 f5 As shown by the agarose slab gel electrophoretic profile (Fig. 2), the central lacZ fragment of 4631 was replaced either by the whole miniF Ap ~ plasmid (2pSC138 recombinant) or by the EcoRI f5 fragment of this plasmid (4631 f5 recombinant). In the 2imm21 816 f5 recombinant, the lacZ fragment of 2imm21 816 was replaced by the EcoRI f5 fragment.
3. General Properties of ~pSC138 (i) Establishment of 2pSC138 as a Plasmid As shown on Fig. 2, 2pSC138 is a chimera between two repticons, the 2 replicon and the miniF Ap ~ replicon.
Repression of the 2 replicon by 2 immunity and selection of ampicillin resistance allowed us to analyse the replication of the chimeric 2pSC138 under the control of the cloned F genes. If 2pSC138 is able to establish itself as a plasmid (under conditions where the 2 replicon is repressed by 2 immunity), ampicillin resistant clones should be found among the 2 lysognic recA F - bacteria infected with 2pSC138. As shown in table 1, the number of ampicillin resistant clones formed on plates supplemented with ampicillin after infection of recA F-(2) strain with 2pSC138 represents 50 to 150% of the number of phage plaques formed on the corresponding 2 sensitive strain. These Apr clones cannot result from the integration of 2pSC138 in the E. coli chromosome or in the prophage since )~pSC138 carries an att-red deletion, the prophage the red3 mutation and the bacterial strain recA mutation. The extrachromosomal state of ,~pSC138 in these Ap r clones has been definitively proved by the facts that 2pSC138 can be cured by acridine orange and removed by incompatibility curing (see Sect. 3, iii). This experiment therefore demonstrates that 2pSC138 is able to establish itself as a plasmid at a frequency of almost 100% under 2 immunity: the F replication mechanism in 2pSC138 is thus perfectly functional.
(ii) Control of the Copy Number and Stability of 2pSCI38 as a Plasmid
Fig. 2. Agarose slab gel electrophoretic profiles of DNA from lambdoid vectors, miniF plasmid and 2F recombinants digested or not by EcoRI. Electrophoresis was performed in a 0.7% agarose slab gel in Tris 40 mM, acetate 20 raM, EDTA 2 mM buffer at pH 7.7 and photographed after staining with ethidium bromide. Slots 1 to 7 contained EcoRI digested DNA of (1) 2; (2) 2631 ; (3) miniF Apt: pSC138; (4) 2631 f5; (5) 2pSC138; (6) 2imm21 816f5 and (7) 2imm21 816. Slots 8, 9, 10 are unrestricted DNA; (8) 2pSC138; (9) 2631 f5; (10))~irnm21 816 f5. The two largest EcoRI fragments in slots 2 and 7 correspond to fragments 1 and 3 of 2631 and 2imm2l 816 (see Fig. 1). The smallest EcoRI fragment in slot 2 and 7 is the lacZ fragment of both vectors. The largest EcoRI fragment in slot 3 (EcoRI digested pSC138 DNA) corresponds to EcoRI f5 fragment of F while the smallest fragment is the EcoRI fragment encoding ampicillin resistance
We observed that 15 to 35 gg/ml of ampicillin was the maximum ampicillin concentration at which 100% of the AB2463(2)/2pSC138 cells were able to form clones. In view of the direct relationship that has been observed between the level of ampicillin resistance expressed by a given cell and the number of [3 lactamase encoding genes present (Uhlin and Nordstr6m, 1975), the level of ampicillin resistance conferred by 2pSC138 to the AB2463(2) bacterial strain suggests a copy number of one or two plasmids per bacterium. This copy number is similar to that of the F plasmid. 2pSC138 is maintained stably as a plasmid. When the AB2463()0/2pSC138 was allowed to grow for 10 generations on non selective medium and was then tested for expression of the ampicillin resistance marker, only 1 in 500 clones were Ap s. The same
M. Couturier et al. : Construction in vitro of "Phage-Plasmid" Chimerae level of maintenance was observed for the F'lac plasmid.
(iii) 2pSC138 Expresses and is Sensitive to FI Incompatibility In Table 1, we compare quantitatively the efficiency of plating of 2pSC138 on F - , F + and Hfr 2-sensitive (i.e. non-lysogenic) strains with the efficiency of establishment of 2pSC138 as a plasmid in F - , F + and Hfr strains insensitive to 2 (i.e. 2 lysogens). Little variation was observed for the efficiency of plating on the three different sensitive strains. As pointed out before, we observed a one to one relationship between the efficiency of establishment of ,~pSC138 as a plasmid in lysogenic F - strains and the number of plaque-forming particles used in the infection. On an F + lysogenic strain, however, the efficiency of establishment of 2pSC138 as a plasmid was reduced at least 5 fold and on a Hfr Iysogenic strain, it was reduced by a factor of 2,000, showing that, as a plasmid, 2pSC138 is sensitive to incompatibility. The following experiments show that 2pSC138 also expresses incompatibility. When a recA F'lac strain lysogenic for 2 was infected by 2pSC138, less than 1% of the Apr clones formed on medium supplemented with ampicillin still exhibited a Lac ÷ phenotype. Under these conditions, the establishment of ),pSC138 as a plasmid apparently caused curing of the unselected F'lac plasmid. Moreover we have analyzed the incompatibility curing of 2pSCl38 established as a plasmid by an incoming F'lac plasmid. For this purpose, Str 5 recA F - ( 2 ) strain with and without resident 2pSC138 were mated with a Str s F'lac donor strain and the resulting Lac ÷ Sm r excortju-
115
gants (isolated on MacConkey lac agar plates supplemented with SM, 100 gg/ml) were analyzed. As expected, the Lac + phenotype acquired by the F strain, which did not contain the 2pSC138, was perfectly stable and perpetuated generation after generation. Conversely, most of the Lac + exconjugants resulting from the mating with the 2pSC138 containing strain were unstable and segregated Lac-clones. When these unstable Lac + exconjugants were subcultured in non selective conditions for at least 20 generations, most of the resulting clones exhibited either a stable L a c + A p s phenotype or a stable Lac Apr phenotype. These results clearly show that 2pSC138 as a plasmid expresses and is sensitive to FI incompatibility.
Discussion
We have constructed )~F chimerae and demonstrated that they can behave as a phage, as a plasmid and possibly as a prophage (result not shown) depending on the presence or absence of FI incompatibility and/ or 2 immunity. In the extrachromosomal state, 2F recombinants retain the controlled autonomous replication and FI incompatibility characteristics of the F plasmid. These chimeric phages should facilitate the genetic and biochemical analysis of plasmid replication, incompatibility and segregation mechanisms, since these processes may now be studied by phage experimental procedures including mutagenesis, mutants selection, complementation tests, recombination mapping and isolation of F gene products resulting from the F gene amplification by 2 replication. Some m u -
Table 1. Efficiencyof plating and plasmid estabiishment of the chimeric 2pSC138 Part A
Part B
Bacteria
Sex
Efficiency of plating of 2pSC138
Bacteria
Sex
Apr transducing efficiency
AB2463 AB2463/F'lacpro KL 1699
FF+ Hfr
i 1.4 1.5
AB2463().) AB2463(2)/F'lacpro KL 1699(2)
FF+ Hfr
0.5-1.5 0.02~.2 0.00014).0005
Overnight cultures of the sensitive and lysogenic strains were resuspended into 10- ~'M MgSO4 and infected by 2pSC138 at a multiplicity less than I. After 20 min adsorption, soft agar was added and the mixture poured onto tryptone agar plates (part A), or onto tryptone agar plates supplemented with ampicillin (20 ~tg/ml) (part B). AB2463 (Howard-Flanders, I966) and HfrKL 1699 (Low, 1968) are recA strains and the 2 prophage carries the red3 mutation. The efficiency of plating was calculated relative to phage titer on AB2463. A p r transducing efficiency was determined as the ratio of the number of ampicillin resistant clones formed on ampicillin plates relative to the number of infecting phages (titered on AB2463). Apr transducing efficiency was taken as a measure of the establishment of the chimeric 2pSC138 as a plasmid
116
M. Couturier et al. : Construction in vitro of ~'Phage-Plasmid" Chimerae
tants which phenotypically express a modified copy number control, sensitivity to incompatibility and ability to express incompatibility, have been selected. They are currently being analyzed. I n d e p e n d e n t l y o f t h i s w o r k , H a y a k a w a , Y. a n d Matsubara, K. have construted a derivative of the p l a s m i d F w h i c h is p a c k a g e a b t e i n ,~ c o a t .
Acknowledgements. M.C. is grateful to Marc Van Montagu, Jeff Schell, Pierre Pohl, Norren and Ken Murray, Geoff Wilson and Ken Timmis for introducing her in the plasmid and the in vitro recombination worlds and for gift of phages, plasmid DNA and restriction enzymes. We want to thank all our friends from the laboratory and Wener Maas, Bruce Kline, Kenichi Matsubara and Ren~ Thomas for stimulating discussions. We are grateful to Jeff Schell, Marc Van Montagu and Oliver Doubleday for critical reading of the manuscript. This work was carried out under a contract with the "Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (I.R.S.I.A.) ", an agreement between the Belgian Government and the Universit6 libre de Bruxelles "Action de Recherche Concert~e", and with the support from the Fonds National de la Recherche Scientifique. M.C. is "chercheur qualifi+" in the Fonds National de la Recherche Scientifique Belge. J.J. is "attach6" and F.B. is "assistant" from the I.R.S.I.A. A.D. is boursi6re I.R.S.I.A.
References Howard-Flanders, P. : Mutants of Escherichia coil K-12 defective in DNA repair and in genetic recombination. Genet. 53, 1137-1150 (1966) Lovett, M., Helinski, D. : Method for the isolation of the replication region of a bacterial replicon: construction of a Mini-F' km plasmid. J. Bacteriol. 127, 982-987 (1976) Low, B.: Formation of merodiptoids in matings with a class of Rec- recipient strains ofEscherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 60, 160-167 (1968) Mants, J., Kline, B.: F plasmid incompatibility and copy number genes. Their map locations and interactions. Plasmid 1, (in press) (1978) Murray, N., Brammar, W., Murray, K.: Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150, 53-61 (1977) Palchaudhuri, S., Maas, W. : Physical mapping of a DNA sequence common to plasmids of incompatibility group F1. Proc. Natl. Acad. Sci. U.S.A. 74, 1190-1194 (1977) Timmis, K., Cabello, F., Cohen, S. : Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc. Natl. Acad. Sci. U.S.A. 72, 2242-2246 (1975) Uhlin, B., Nordstr6m : Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12. J. Bacteriol. 124, 641-649 (1975)
Communicated
b y J. S c h e l l
Received October 24, 1978
Short Communication
Construction in vitro of "Phage-Plasmid" Chimerae: a New Tool to Analyse the Mechanism of Plasmid Maintenance M. Couturier, J. Janssens, F. Bex, A. Desmyter, and I. Bonnevalle Laboratory of Genetics, Department of Molecular Biology, Universit6 libre de Bruxelles, rue des Chevaux, 67, B-1640 Rhode St Gen6se, Belgium
Summary. In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Ap ~ plasmid: pSC138 (Timmis et al., 1975). 2F recombinants were shown to be chimerae between the 2 and the F replicons. By genetical tests, we have demonstrated that both 2 and F replication mechanisms are functional: the 2F recombinant behaves as a non defective plaque forming phage on 2 sensitive bacteria and establishes itself as a stable plasmid on recA F - homoimmune bacteria. In the extra-chromosomal state, the 2F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed,
a
b
AWE
J ]
1.3_
Lac Z
bla ( ~
I
i.__2 Lac Z
c d
~tt redI NctqP
I
3 c17 n[n 5
Io
imm21 nm5
f5
e
..,,._ J. . . . . _~" bla f 5" f
QIS,,,R
/
L
cl7 nin5
cl7 nin5
f5
mm21
1. Construction of 2 F Recombinants
n5
f5
The maintenance of a defined plasmid in a bacterial strain involves several coordinated processes, including its controlled replication, its segregation and the expression of the incompatibility phenomenon. We intend to analyse the mechanism of plasmid maintenance in genetic and biochemical terms by cloning the genes implicated in these different processes in phage vectors. We thus cloned the EcoRI f5 fragment of the F' lac plasmid and an EcoRI fragment encoding ampicillin resistance (Ap r) in two lambdoid phage vectors. Both of these fragments exist in the mini F Apr plasmid pSC138 (Fig. 1, map d) (Timmis et al., 1975). It is known that the EcoRI f5 fragment of F carries two incompatibility loci incA and incB (Palchaudhuri and Maas, 1977; Manis and Kline, 1978) and all For offprints contact." M. Couturier
Fig. la-g. Represents the DNA maps of: b R631 (Murray et al., 1977); e 2imm21 816; d the miniF Apt: pSCI38 (Timmis et al., 1975); e the 2pSC138; f the 2631 f5 and g 2imm21 816 f5. Map a shows the position of some important markers on the 2 physical map. l indicates the position of an EcoRI target; -- the phage attachment site; gaps indicate the position of deletions and dotted lines represent substitutions, bla is the filactamase encoding gene of 2pSC138 which confers the ampicillin resistance phenotype
the genetic information necessary for the controlled autonomous replication of the F plasmid (Timmis et al., 1975; Lovett and Helinski, 1976). The vectors used are two replacement vectors, 2631 and 2imm21 816, both derived from 2 lac5 (Murray et al., 1977). They contain two EcoRI cleavage sites on both sides of their lac Z fragments (Fig. 1, maps b and c). EcoRI digested D N A mixtures of
0026-8925/79/0169/0113/$01.00
114
M. Couturier et al. : Construction in vitro of "Phage-Plasmid" Chimerae
pSC138 and 4631 DNA or pSC138 and )~imm21 816 DNA were ligated with T4 DNA ligase and used for transfection of Ca + + treated lac ZM15 bacteria (see Murray et al., 1977, for experimental procedures and references to the bacterial strain). The different 2F recombinants 2pSC138, 4631 f5 and 2imm21 816 f5 (Fig. 1, maps e f g) were identified among the Lac- plaque-forming phages by their ability to express, under 2 immunity, FI incompatibility and, in the case of 2pSC138, ampicillin resistance (see Sect. 3, iii).
2. Restriction Mapping of ApSC138, ~631 f5 and 2imm21 816 f5 As shown by the agarose slab gel electrophoretic profile (Fig. 2), the central lacZ fragment of 4631 was replaced either by the whole miniF Ap ~ plasmid (2pSC138 recombinant) or by the EcoRI f5 fragment of this plasmid (4631 f5 recombinant). In the 2imm21 816 f5 recombinant, the lacZ fragment of 2imm21 816 was replaced by the EcoRI f5 fragment.
3. General Properties of ~pSC138 (i) Establishment of 2pSC138 as a Plasmid As shown on Fig. 2, 2pSC138 is a chimera between two repticons, the 2 replicon and the miniF Ap ~ replicon.
Repression of the 2 replicon by 2 immunity and selection of ampicillin resistance allowed us to analyse the replication of the chimeric 2pSC138 under the control of the cloned F genes. If 2pSC138 is able to establish itself as a plasmid (under conditions where the 2 replicon is repressed by 2 immunity), ampicillin resistant clones should be found among the 2 lysognic recA F - bacteria infected with 2pSC138. As shown in table 1, the number of ampicillin resistant clones formed on plates supplemented with ampicillin after infection of recA F-(2) strain with 2pSC138 represents 50 to 150% of the number of phage plaques formed on the corresponding 2 sensitive strain. These Apr clones cannot result from the integration of 2pSC138 in the E. coli chromosome or in the prophage since )~pSC138 carries an att-red deletion, the prophage the red3 mutation and the bacterial strain recA mutation. The extrachromosomal state of ,~pSC138 in these Ap r clones has been definitively proved by the facts that 2pSC138 can be cured by acridine orange and removed by incompatibility curing (see Sect. 3, iii). This experiment therefore demonstrates that 2pSC138 is able to establish itself as a plasmid at a frequency of almost 100% under 2 immunity: the F replication mechanism in 2pSC138 is thus perfectly functional.
(ii) Control of the Copy Number and Stability of 2pSCI38 as a Plasmid
Fig. 2. Agarose slab gel electrophoretic profiles of DNA from lambdoid vectors, miniF plasmid and 2F recombinants digested or not by EcoRI. Electrophoresis was performed in a 0.7% agarose slab gel in Tris 40 mM, acetate 20 raM, EDTA 2 mM buffer at pH 7.7 and photographed after staining with ethidium bromide. Slots 1 to 7 contained EcoRI digested DNA of (1) 2; (2) 2631 ; (3) miniF Apt: pSC138; (4) 2631 f5; (5) 2pSC138; (6) 2imm21 816f5 and (7) 2imm21 816. Slots 8, 9, 10 are unrestricted DNA; (8) 2pSC138; (9) 2631 f5; (10))~irnm21 816 f5. The two largest EcoRI fragments in slots 2 and 7 correspond to fragments 1 and 3 of 2631 and 2imm2l 816 (see Fig. 1). The smallest EcoRI fragment in slot 2 and 7 is the lacZ fragment of both vectors. The largest EcoRI fragment in slot 3 (EcoRI digested pSC138 DNA) corresponds to EcoRI f5 fragment of F while the smallest fragment is the EcoRI fragment encoding ampicillin resistance
We observed that 15 to 35 gg/ml of ampicillin was the maximum ampicillin concentration at which 100% of the AB2463(2)/2pSC138 cells were able to form clones. In view of the direct relationship that has been observed between the level of ampicillin resistance expressed by a given cell and the number of [3 lactamase encoding genes present (Uhlin and Nordstr6m, 1975), the level of ampicillin resistance conferred by 2pSC138 to the AB2463(2) bacterial strain suggests a copy number of one or two plasmids per bacterium. This copy number is similar to that of the F plasmid. 2pSC138 is maintained stably as a plasmid. When the AB2463()0/2pSC138 was allowed to grow for 10 generations on non selective medium and was then tested for expression of the ampicillin resistance marker, only 1 in 500 clones were Ap s. The same
M. Couturier et al. : Construction in vitro of "Phage-Plasmid" Chimerae level of maintenance was observed for the F'lac plasmid.
(iii) 2pSC138 Expresses and is Sensitive to FI Incompatibility In Table 1, we compare quantitatively the efficiency of plating of 2pSC138 on F - , F + and Hfr 2-sensitive (i.e. non-lysogenic) strains with the efficiency of establishment of 2pSC138 as a plasmid in F - , F + and Hfr strains insensitive to 2 (i.e. 2 lysogens). Little variation was observed for the efficiency of plating on the three different sensitive strains. As pointed out before, we observed a one to one relationship between the efficiency of establishment of ,~pSC138 as a plasmid in lysogenic F - strains and the number of plaque-forming particles used in the infection. On an F + lysogenic strain, however, the efficiency of establishment of 2pSC138 as a plasmid was reduced at least 5 fold and on a Hfr Iysogenic strain, it was reduced by a factor of 2,000, showing that, as a plasmid, 2pSC138 is sensitive to incompatibility. The following experiments show that 2pSC138 also expresses incompatibility. When a recA F'lac strain lysogenic for 2 was infected by 2pSC138, less than 1% of the Apr clones formed on medium supplemented with ampicillin still exhibited a Lac ÷ phenotype. Under these conditions, the establishment of ),pSC138 as a plasmid apparently caused curing of the unselected F'lac plasmid. Moreover we have analyzed the incompatibility curing of 2pSCl38 established as a plasmid by an incoming F'lac plasmid. For this purpose, Str 5 recA F - ( 2 ) strain with and without resident 2pSC138 were mated with a Str s F'lac donor strain and the resulting Lac ÷ Sm r excortju-
115
gants (isolated on MacConkey lac agar plates supplemented with SM, 100 gg/ml) were analyzed. As expected, the Lac + phenotype acquired by the F strain, which did not contain the 2pSC138, was perfectly stable and perpetuated generation after generation. Conversely, most of the Lac + exconjugants resulting from the mating with the 2pSC138 containing strain were unstable and segregated Lac-clones. When these unstable Lac + exconjugants were subcultured in non selective conditions for at least 20 generations, most of the resulting clones exhibited either a stable L a c + A p s phenotype or a stable Lac Apr phenotype. These results clearly show that 2pSC138 as a plasmid expresses and is sensitive to FI incompatibility.
Discussion
We have constructed )~F chimerae and demonstrated that they can behave as a phage, as a plasmid and possibly as a prophage (result not shown) depending on the presence or absence of FI incompatibility and/ or 2 immunity. In the extrachromosomal state, 2F recombinants retain the controlled autonomous replication and FI incompatibility characteristics of the F plasmid. These chimeric phages should facilitate the genetic and biochemical analysis of plasmid replication, incompatibility and segregation mechanisms, since these processes may now be studied by phage experimental procedures including mutagenesis, mutants selection, complementation tests, recombination mapping and isolation of F gene products resulting from the F gene amplification by 2 replication. Some m u -
Table 1. Efficiencyof plating and plasmid estabiishment of the chimeric 2pSC138 Part A
Part B
Bacteria
Sex
Efficiency of plating of 2pSC138
Bacteria
Sex
Apr transducing efficiency
AB2463 AB2463/F'lacpro KL 1699
FF+ Hfr
i 1.4 1.5
AB2463().) AB2463(2)/F'lacpro KL 1699(2)
FF+ Hfr
0.5-1.5 0.02~.2 0.00014).0005
Overnight cultures of the sensitive and lysogenic strains were resuspended into 10- ~'M MgSO4 and infected by 2pSC138 at a multiplicity less than I. After 20 min adsorption, soft agar was added and the mixture poured onto tryptone agar plates (part A), or onto tryptone agar plates supplemented with ampicillin (20 ~tg/ml) (part B). AB2463 (Howard-Flanders, I966) and HfrKL 1699 (Low, 1968) are recA strains and the 2 prophage carries the red3 mutation. The efficiency of plating was calculated relative to phage titer on AB2463. A p r transducing efficiency was determined as the ratio of the number of ampicillin resistant clones formed on ampicillin plates relative to the number of infecting phages (titered on AB2463). Apr transducing efficiency was taken as a measure of the establishment of the chimeric 2pSC138 as a plasmid
116
M. Couturier et al. : Construction in vitro of ~'Phage-Plasmid" Chimerae
tants which phenotypically express a modified copy number control, sensitivity to incompatibility and ability to express incompatibility, have been selected. They are currently being analyzed. I n d e p e n d e n t l y o f t h i s w o r k , H a y a k a w a , Y. a n d Matsubara, K. have construted a derivative of the p l a s m i d F w h i c h is p a c k a g e a b t e i n ,~ c o a t .
Acknowledgements. M.C. is grateful to Marc Van Montagu, Jeff Schell, Pierre Pohl, Norren and Ken Murray, Geoff Wilson and Ken Timmis for introducing her in the plasmid and the in vitro recombination worlds and for gift of phages, plasmid DNA and restriction enzymes. We want to thank all our friends from the laboratory and Wener Maas, Bruce Kline, Kenichi Matsubara and Ren~ Thomas for stimulating discussions. We are grateful to Jeff Schell, Marc Van Montagu and Oliver Doubleday for critical reading of the manuscript. This work was carried out under a contract with the "Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (I.R.S.I.A.) ", an agreement between the Belgian Government and the Universit6 libre de Bruxelles "Action de Recherche Concert~e", and with the support from the Fonds National de la Recherche Scientifique. M.C. is "chercheur qualifi+" in the Fonds National de la Recherche Scientifique Belge. J.J. is "attach6" and F.B. is "assistant" from the I.R.S.I.A. A.D. is boursi6re I.R.S.I.A.
References Howard-Flanders, P. : Mutants of Escherichia coil K-12 defective in DNA repair and in genetic recombination. Genet. 53, 1137-1150 (1966) Lovett, M., Helinski, D. : Method for the isolation of the replication region of a bacterial replicon: construction of a Mini-F' km plasmid. J. Bacteriol. 127, 982-987 (1976) Low, B.: Formation of merodiptoids in matings with a class of Rec- recipient strains ofEscherichia coli K-12. Proc. Natl. Acad. Sci. U.S.A. 60, 160-167 (1968) Mants, J., Kline, B.: F plasmid incompatibility and copy number genes. Their map locations and interactions. Plasmid 1, (in press) (1978) Murray, N., Brammar, W., Murray, K.: Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150, 53-61 (1977) Palchaudhuri, S., Maas, W. : Physical mapping of a DNA sequence common to plasmids of incompatibility group F1. Proc. Natl. Acad. Sci. U.S.A. 74, 1190-1194 (1977) Timmis, K., Cabello, F., Cohen, S. : Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc. Natl. Acad. Sci. U.S.A. 72, 2242-2246 (1975) Uhlin, B., Nordstr6m : Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12. J. Bacteriol. 124, 641-649 (1975)
Communicated
b y J. S c h e l l
Received October 24, 1978
