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Volume 5 Number 6 June 1978 NliCleJC A c i d s Research Construction of recombinant plasmid carrying the X DNA fragment responsible for prophage integration N. Strizhov and L. Tikhomirova Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, 142292, USSR Received 4 April 1978 ABSTRACT The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of A DNA containing the genes responsible for prophage integration. The presence of these genes in recombinant plasmids was detected genetically. A int-gene was shown to be expressed in either orientation of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of A lysogens. The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with A phages. INTRODUCTION The stabilization of foreign DNA fragment in the recipient cells is one of the major problems of recombinant DNA research. The stabilization of foreign DNA sequences in the cell can be achieved by integration of these sequences into host chromosome. It may be carried out by means of A vectors containing the genes necessary for stable lysogenization. However, the maximal length of the fragment which can be cloned by the use of such phages as cloning vehicles is limited. Plasmid Col E1 and its derivatives are particularly convenient as cloning vehicles for the construction of recombinant DNA molecules. However, these plasmids can not integrate into host chromosome and are maintained in cells as independent replicons. In some cases it is important for succesful cloning that foreign DNA sequences are present in only one copy per cell otherwise the superproduction of the foreign gene product may be fatal for recipient cell. Taking into account the widespread application of plasmids as cloning vehicles for recombinant DNA research we investigat© Information Retrieval Limited 1 Falconberg Court London W1V5FG England 1737 Nucleic Acids Research ed the possibility of integration of plasmid DNA into the chromosome of E.coli. It was carried out using the in vitro constructed recombinant plasmid RSF2124 with A DNA fragment containing att-site and int-xis genes integrated into the EcoRI site. MATERIALS AND METHODS Containment classification; These experiments do not give rise to any recombinants which are thought to create novel biohazards. They were handled using normal sterile microbiological procedures. Bacteria and bacteriophage strains. The Escherichia coli K12 strains used and their origin were: C600 (from A.Kaiser), QD5OO3 (from L.Siminovitch), C600/RSF2124 from H.Boyers laboratory. C6OO( A. cI857) and C600 (A 1434) were-lysogenized in our laboratory. JC5088 rec A,-g (from A.Clark via K.Zlotnikov) 953 gal E~galK~trp~lac~su" (from Gellert) RW842 - the indicator strain for red plaque test from L.Enquist. The phages and their sources were: Agt- AC from G.Zavilgelsky, P1KC from K.Zlotnikov, A red 3 1 4 C I 8 5 7 and Aint4gam210CI857 from B.Malone via K.Zlotnikov, Ab2c, A + , ACI857, Ai434 from A.Kaiser and Aint am29 CI857 and Axisam6 from L.Enquist. Media. The standard rich media used were NB broth and NB broth agar. The NB broth medium contained (per liter of water): 5 g of Nutrient broth (Difco), 10 g of peptone (Spofa, CSR), 5 g of NaCl. It was solidified by the addition of Difco agar to a concentration of 1.3# for plates and 0.796 for top agar. EMBO-agar was prepared as described '. The minimal medium used was M9. For identification of gal + transductants 50 mg/1 2,3,5-triphenyltetrazolium chloride (TTC) was added. DNAs and enzymes. RSF2124 plasmid DNA was purified as described by Tanaka and Weisblum . The DNA of recombinant plasmids were purified according to Meyers et al. . Preparation of A g t - A C phage lysate was made by infection of exponentially growing culture of E.coli C600 in NB broth containing 10"**' M MgS0A« The phages were recovered by precipitation 1768 Nucleic Acids Research with polyethyleneglycol and purified by centrifugation through a CsCl gradient . The purified phage was dialysed against 10 mM tris-HCl pH 7,5, 50 mM NaCl, 1 mM MgSO^ and the DNA was extracted by gentle rolling with freshly distilled phenol 7 followed by dialysis against 10 mM tris-HCl pH 7,5, 50 mM NaCl. The EcoRI endonuclease was prepared by D.Vorozheikina from E.coli strain 1100/RI as described by Yoshimori 8 . The diQ gestion of DNAs was carried out as described . 1 pi of purified enzyme was enough for complete digestion of 1 ug of ADNA after 30 min at 37°C. The DNA ligase of phage T4 was kindly given us by A.Solonin. The hydroxylapatite fraction of the enzyme was used with the activity as high as 520 units/ml. Bam H1 restriction endonuclease was a generous gift from N.Kuzmin. The construction of recombinant plasmids. The central DNA fragment of phage Agt- AC was separated in a glycerol gradient 1 0 at 32.000 rev/min, 14 h, 15°C, ultracentrifuge L5-5O. The gradient solution was passed from the bottom of the tube through the Analabs u.v.monitor. The fractions of central fragment were collected and used in the ligase reaction. There was no noticeable contamination of the central fragment by the end fragments, as was shown by electrophoresis through an agarose gel. RSF2124 plasmid DNA was digested with EcoRI treated with phenol and after dialysis against 10 mM Tris-HCl pH 7,5, 50 mM NaCl was mixed with central fragment of Agt- Ac DNA (1,4 y.g and 1.7 ^g correspondingly) . Ligation and transformation were carried out as q described . Electrophoresis of DNAs was carried out on 1% agarose gels (Bio Rad) in 0.04 M Tris-acetate, pH 8.0, 0.02 M sodium acetate, 0.002 M EDTA at 2 v/cm for 20 h. Transduction experiments with P1 were carried out as described by Rhotman . Lft and Hft lysates of A phages were obtained by heat induction of A CI857 from bacteria containing parental or recombinant plasmids. All transducing phage lysates used were sterile. 1769 Nucleic Acids Research The assay of the Lft and Hft lysates was carried out in the preformed gradient of CsCl in SW 50.1 at 30.000 rev/min for 5 h. The fractions were collected in 2 ml of Nutrient broth. Colicine E1 was isolated from E.coli JC411/Col E1 (from D.Helinski) by the method of Schwartz and Helinski . Electron microscopic observation of plasmid DNA was carried out by micro-method of Lang and Mitani . RESULTS AND DISCUSSION The construction of hybrid plasmid. The mixture of central Agt- Ac DNA fragment and EcoRI endonuclease digested DNA of plasmid RSF2124 was treated with ligase and used to transform CaCl2-treated QD5OO3 cells. The results of transformation experiment are presented in table 1. Table 1. The appearance of ampicillin resistant (Apr) clones in the transformation experiments. DNA Transformants/ }ig RSF2124 DNA RSF2124,native RSF2124, E.coRI RSF2124, EcoRI+ligase RSF2124,EcoRI+ ADNA fragment+ligase 3«10 5 2,3«1O2 6,2«103 8,6«103 The efficiency of transformation increased 27 times when mixture of EcoRI-digested DNA of plasmid RSF2124 and central fragment of Agt- Ac DNA was treated with T4 ligase. 100 Apresistant transformants were assayed for the ability to produce colicine E1. It was found that 9 of 100 assayed clones were immune to colicine E1, but not able to synthesize it. Plasmid DNA was purified from these clones. The electrophoresis in agarose gels of EcoRI digestion products of purified plasmids shows two fragments - one corresponding to linear form of RSF2124 DNA and other corresponding to central EcoRI fragment of A g t - A C (fig.1). The insertion of DNA fragment into plasmid RSF2124 was confirmed by electron microscopy of parental and recombinant plasmid DNAs. The measuring of the contour lengths of plas1770 Nucleic Acids Research a b e d Fig.1. Agarose gel electrophoresis of DNA digested with EcoRI endonuclease: a - RSF2124 b - RSF2124 - AC <?c - Agt - AC d - RSF2124 - AC + Agt - AC mids showed that recombinant plasraid differs from parental RSF2124 by length corresponding to Ac fragment (fig.2). Expression of A genes integrated into plasmid RSF2124. Expression of the int-gene of A inserted into RSF2124 DNA molecule by an in vitro recombination was studied by assaying the ability of recombinant plasmids to complement lnt~ mutation of A phage . The int-mutants of A are not able to produce stable lysogens. Abortive lysogens picked from the turbid plaques of integration-negative mutants may be distinguished from stable lysogens picked from the plaques of wild type phage by their response on a test plate to a continuous challenge to their immunity. On a EMBO-test plate spread with Ab2c large pink colonies appear after incubation at 33 C for 20 h if the A lysogens transferred to the plate are 14 stable . Abortive lysogens appear as a small dark colonies. The stable lysogenization of hybrid plasmid carrying cells by A int mutants is possible if the plasmids provides the int product defficient in A int mutants. Other possibility for stable lysogenization by int" phages may occur from the recombination of int" phages with plasmid DNA in homologous regions producing phages of int+ genotype. The EMBO-test allow us to differenciate stable lysogens from abortive 1771 Nucleic Acids Research B 0,2? Fig.2. Electron micrographs of RSF2124 - Ac (A) and RSF2124 (B) plasmid DNA. ones. When bacteria containing RSF2124-AC plasmids were infected with int-mutants of A stable lysogens appeared with high efficiency. They did not appear at all when int-mutants infected the strain containing parental plasmid RSF2124. High frequency of stable lysogenization in the first case indicates that rather the complementation than the recombination may be a reason for lysogenization by int~ phages. It was supported by the fact that phages obtained from lysogenic bacteria containing recombinant plasmids were of int~ genotype. Expression of int gene carried out by the RSF2124-AC plasmid was confirmed by red plaque test developed by L.Enquist and R.Weisberg . According to this test the expression of int and xis genes of A can be detected by their ability to promote the int and xis dependent excision of a cryptic prophage inserted within gal T gene of E.coli. Excision of a cryptic 1772 Nucleic Acids Research prophage results in gal + reversion of the cells that survived infection. For this purpose parental and recombinant plasmid DNAs were transformed to the strain RW842 containing the cryptic prophage. When int~ xis + phages were plated on this strain red plaques similar to ones of wild type A were observed if the strain carried the plasmid RSF2124- AC. If the strain RW842/RSF2124 was infected with Aint~xis + phages there appeared plaques with turbid but completely colourless centers. Excision of prophage requires both int and xis gene products . The int gene is expressed from its own promoter AC. A*"? * therefore its expression does not depend on orientation of A DNA fragment in plasmid. Expression of xis gene within the plasmid probably must be regulated by transcription starting from a promoter for colicin E1 protein synthesis. The red plaque test can give a semiquantitative estimate of int and xis functions . The expression of xis gene inserted into the plasmid in both orientation (it was shown by endonuclease EcoRI and Bam H1 restriction analysis of recombinant plasmids; data not shown) was very low. When gal~ strain RW842 was transformed with parental plasmid RSF2124 the red gal + clones were not observed. RW842 does not revert spontaneously to gal + . In the case of transformation by RSF2124- AC plasmid the red gal + clones appeared but with low frequency. In contrast to the plating of int~xis+ phages the effect of complementation was not observed when int+xis~ phages were plated on the RW842 carrying recombinant plasmid. About 2/3 of the int+xis~ phage plaques were completely colourless and 1/3 contained one to four red cured clones. We cannot exclude that cured clones may be the int + xis + recombinant phages, formed as a result of recombination of infecting int+xis~ phages with RSF2124-AC plasmid. Insertion of recombinant plasmid into chromosome. Having convinced that inserted DNA fragment contained functional int gene of A the possibility of integration of plasmid RSF2124-AC in the chromosome of bacteria was assayed. The P1 mediated cotransduction of chromosome gal marker and Ap r Col10111 plasmid markers must be observed if the recombinant plasmid is able to integrate in the attachment site for A. However, we could not observe cotransduction of these 1773 Nucleic Acids Research Table 2. P1 mediated transduction. Donor s t r a i n gal + gal + An1* C6OO/RSF2124 C6OO/RSF2124- AC C6OO(AcI857)/RSF2124 C600(AcI857)/RSF2124- Ac JC5088(Ared 314)/RSF2124 JC5088(Ared 314)/RSF2124- AC 225 735 411 258 467 596 Ap Col i m m 0 0 0 % of cotransduction 0 0 0 17 6,6 0 0 0,8 5 markers if the transduction was carried out by P1 phage obtained from the gal + strain C600/RSF2124- AC. The results of transduction experiments are shown in the table 2. It is known that besides the int gene expression the function of 18 19 other genes are necessary for stable lysogenization by A ' . Cotransduction of gal + Ap r Col markers to gal" strain 953 was observed when P1 transducing phages were obtained from A-lysogenic E.coli. Frequency of cotransduction for gal + Ap r Colm m markers in these experiments was 6£>% (table 2 ) . It is not clear how prophage can promote the site specific recombination between plasmid. and chromosome. It is not excluded that recombinant plasmid may be inserted into the chromosome of bacteria by means of general recombination in the homologic region of plasmid and prophage. In order to eliminate the possibility of recombination between prophage and plasmid DNA the recombinant and parental plasmids were transferred into strain defective for general recombination system and lysogenic for recombination defective A prophage JC5088 recAcg ( A red 314). In this strain insertion of plasmid RSF2124— AC into chromosome may occur only by means of site specific recombination responsible for prophage integration. The frequency of cotransduction of gal + A p r Colm m markers reduced when P1 lysate was obtained from rec A~ (A red) strain, however, it was sufficiently high to allow us to suppose that integration should be carried out by site-specific recombination. It is known that 1774 Nucleic Acids Research Table 3. Specialized transduction by A . Donor strain Lft C600(AcI857)/RSF2124 _n_ _ti_ _n — Hft C600(AcI857)/RSF2124- Ac C600/RSF2124 C600/RSF2124 - AC C600(AcI857)AprColimm Preparation of phage lysate Recipient strain Ap r Col inun transducing phage frequency heat induction C600 1«10~ 9 C6OO C600 C6OO C6OO 5*10~6 5«10~ 9 4'10~7 1.10"4 1.10"5 — n_ infection _n_ heat induction c6oo(/V) rec~ mutations have no apparent effect on the ability of the cells to maintain the Col E1 factor but can prevent the spontaneous and induced production of colicine. These mutations do not affect the expression of immunity of Col fac20 tor . If the expression of some function from colicine gene promoter is necessary the frequency of plasmid insertion in rec A~ strain may be reduced. Other confirmation for recombinant plasmid insertion into chromosome of bacteria was obtained from specialized transduction experiments. A transducing phages were prepared from appropriate lysogens by heat induction. Lft lysates were used to infect E.coli C600 and Ap r C 6 1 i m m transductants were counted on the plates with ampicillin and colicin E1. The frequency of transducing phages in Lft lysates was 5*10 (table 3). When Lft lysate was obtained from the strain containing parental plasmid RSF2124 Ap r Col i m m transductants were not observed. Hft lysates transduced corresponding markers to recipient strain with frequency 1.10 . The recombination in homology regions is not excluded when propagation of phages occur in recombinant plasmid carrying strains. In order to estimate this the bacteria carrying parental or recombinant plasmids were infected with phage AcI857 at multiplicity of infection equal to 0.1 and lysates obtained were assayed for the presence of transducing phages. It was shown that recombinant Ap r Col m m transducing 1775 Nucleic Acids Research TO* A I A o 1 6 i I I.1B0 Air /o2 i; i f.375 1.370 5 10 •"— bottom \5 25 30 3S 40 10 (5 20 2* 30 FRACTIONS Fig.3. Density gradient analysis of Lft (A) and. Hft (B) lysates. Gradient fractions were titrated for total phage (closed circles) and for transducing phage (open circles). phages arise with 10 times lower frequency as compeared with prophage induction (table 3 ) . Additional information was obtained about structure of transducing phages by assaying Lft and Hft lysates in the CsCl gradient. The fractions of the gradients were plated on C600 (A ) and C600 ( A i434) and the peak fractions of marker phage A i43^, phages from Lft and Hft lysates were identified. The presence of transducing phages in the fractions was determined by incubation of fraction aliquots with starved E.coli C600 cells and subsequent plating on the medium containing ampicillin and colicin E1. The results of these experiments are presented in figure 3. The sedimentogram of Lft lysate shows wide distribution of Ap r transducing phages in the gradient fractions. Transducing phages from Hft lysate were localized as a sharp peak . The frequency of transducing phages increased 10 times if the presence of transducing phages in the gradient fractions was assayed on the A lysogenic strain. Experiments, are in progress to elucidate the structure of transducing phages and the mechanism of recomblnant plasmid integration. 1776 Nucleic Acids Research ACKNOWLEDGEMENTS We are indebted to Dr. N.Matvienko for helpful advice and discussions. We thank Dr. I.Fodor for his critical reading of the manuscript. We are also grateful to Dr. L.Enquist for making the red plaque test available for us and L.Matrosova for technical help. REFERENCES •\. Enquist.L.W. and Weisberg.R.A. (1976) Virology, 72, 147-153 2. Thomas,M., Cameron,J.R. and Davis,R.W. (1974) Proc.Nat. Acad.Sci.USA, 71, 4579-4583. 3. Miller,J.H. (1972) Experiments in Molecular genetics. Cold Spring Harbor Laboratory. 4. Tanaka,T. and Weisblum.B. (1975) J.Bact., 121, 354-362. 5. Meyers,J.A., Sanchez,D., Elwell,L.P. and Falkow.S. (1976) J.Bact. 127, 1529-1537. 6. Thomas,M. and Davis,R.W. (1975) J.Mol.Biol., 91, 315-328. 7. Kaiser,A. and Hogness.D. (1960) J.Mol.Biol., 2, 392-399. 8. Yoshimori.R.W. (1971) Ph.D.Thesis. University of California, San Francisco Medical Centre. 9. Tikhomirova,L.P., Solonin,A.S., Ksenzenko,V.N. and Matvienko,N.I. (1976) Nucl.Acids Res. 3, 2485-2490. 10. Ihler,G. and Kawai.Y. (1971) J.Mol.Biol. 61, 311-328. 11. Rothman.J.L. (1965) J.Mol.Biol. 12, 892-912. 12. Schwartz,S. and Helinski.D. (1971) J.Biol.Chem. 246, 63186327. 13. Lang,D. and Mitani,M. (1970) Biopolymers, 9, 373-379. 14. Gottesman.M. and Yarmolinsky,M. (1968) J.Mol.Biol., 31, 487-505. 15. Guarnerous.G. and Echols,H. (1970) J.Mol.Biol., 47, 565574. 16. Shimada,K. and Campbell,A. (1974) Proc.Nat.Acad. Sci.USA, 71, 237-241. 17. Oppenheim,A.B. (1976) Nature, 261, 615-616. 18. Katzir,N., Oppenheim.A.• Belfort,M. and Oppenheim,A.B. (1976) Virology, 74, 324-331. 19. Echols,H., Chung,H. and Green,L. (1974) Mechanisms in Recombination. Ed. by Grell,Rh.F., 69-77. 20. Helinski.D.R. and Herichman,H.R. (1967) J.Bact., 94, 700-706. 1777 Nucleic Acids Research 1778