JOURNAL OF BACTERIOLOGY, June 1979, p. 1033-1035 0021-9193/79/06-1033/03$02.00/0

Vol. 138, No. 3

Nature of Transforming Deoxyribonucleic Acid in CalciumTreated Escherichia coli PETER STRIKE,' * GWYNFOR 0. HUMPHREYS,2 AND R. JOHN ROBERTS' Department of Genetics' and Department of Microbiology,2 University of Liverpool, Liverpool L69 3BX,

England Received for publication 4 December 1978

A study of the reactivation of ultraviolet-irradiated plasmid and phage deoxyribonucleic acid molecules after transformation into Escherichia coli strains indicated that, when double-stranded deoxyribonucleic acid was used as the donor species, it was taken up without conversion to the single-stranded form.

The use of calcium treatment to render cells of Escherichia coli capable of uptake of exogenous DNA was first described by Mandel and Higa (12) for transfection with the DNA of bacteriophage lambda. The method has since been widely applied to transfection with several phages (1), to chromosomal transformation (13), and to transformation with plasmid DNA (4). Many of the recent techniques for the in vitro manipulation of DNA rely ultimately upon transformation of calcium-treated cells to determine the biological activity of the final product. However, despite the widespread use of the method and the improvements in the original procedure to give high efficiencies of transformation (8, 9), little is known about the mechanism of uptake of the transforming DNA. In the classical transformation systems of Streptococcus pneumoniae, Bacillus subtilis, and Haemophilus influenzae, a period of competence occurs naturally, during which a fraction of the cell population can take up exogenous DNA (10, 17). In the case of S. pneumoniae and B. subtilis, DNA is apparently taken up in the singlestranded state, whereas uptake in H. influenzae gives rise to at least partially single-stranded DNA (10, 11). There is no evidence that single strandedness results from uptake in calciumtreated E. coli, and this paper presents evidence that, at least in the case of one plasmid (NTP16) and one phage (4XtB), uptake of doublestranded DNA occurs. Previous studies with genetically marked heteroduplexes of bacteriophage lambda indicate that uptake of double-stranded DNA can occur, since some mixed bursts are detected (18). However, many centers produce pure phage bursts, indicating either single-strand uptake or repair of the mismatched bases before replication. The early work of Cohen et al. (4) shows that doublestranded covalently closed and open circular

plasmid DNAs are equally efficient substrates for transformation of calcium-treated cells, but they were unable to say anything about the nature of the DNA as it was taken up. One simple criterion which can be applied to transforming DNA to determine whether it is single or double stranded during uptake is that of susceptibility to excision repair. The repair of UV-damaged DNA by excision repair has been well characterized and is known to depend absolutely on the duplex nature of DNA (5). A single-strand break is introduced into the double-stranded structure, adjacent to the site of damage, by an ATP-dependent enzyme whose activity is govemed by the uvrA', B+, and C+ genes (14, 15). Excision of the damaged region follows, leading to the creation of a single-strand gap. This can be rapidly filled by DNA polymerase I, using the opposite strand as template, and the intact duplex is restored by polynucleotide ligase. If single-stranded DNA were taken up by calcium-treated cells, replication to the double-stranded form would be required before excision repair could function. Since UV-damaged DNA contains pyrimidine dimers, conversion to double-stranded DNA would require replication past such damage. There is only one known mechanism for replication past dimers, namely the error-prone type of DNA repair which is induced in UV-damaged cells (3). Induction of this process requires a functional recA+ gene product, and no such repair can occur in recA mutant strains (19). Thus, by transforming recA uvr' and recA uvr recipient strains with UV-damaged plasmid DNA and measuring the effects of excision repair on the survival of the plasmid, it should be possible to determine the nature of the transforming DNA. Figure 1 shows the results of such an experiment, in which covalently closed circular DNA of the plasmid NTP16 was used as the trans-

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FIG. 1. Survival of UV-irradiated DNA of the plasmid NTP16 on repair-proficient and -deficient recipient cells. Symbols: 0, AB1157 urv' rec'; 0, B W41 uvr' recA13; U, ABI885 uvrB5 rec'; El, AB2480 uvrA6 recA13. All four strains are essentially isogenic. Data is shown for strain AB1885 uvrB5 rec'k;

data obtained with strain AB1886 uvrA6 rec' revealed no significant difference between these strains with respect to plasmid survival. Both uvrA and uvrB mutations are known to affect the same step in excision repair (see text). Transformation was carried out by a minor modification of the procedure of Humphreys et al. (8). Overnight cultures of the strains to be transformed were diluted 1:20 in fr-esh nutrient broth and grown with aeration for 90 to 1(X) min. Cells were harvested and washed once in 0.5 volume of 10 mM CaC12-10 mM morpholinopropane sulfonic acid, pH 6.8, at O'C. The washed cells were resuspended in 0.05 volume of 75 mM CaCl2-10 mM morpholinopropane sulfonic acid, pH 6.8, at 00C. and 0.1 ml of these reci]pient cells was transformed directly in 75 mM CaC12 in a final volume of 0.25 ml with an almost saturating concentration of DNA (1 pg/ml) for 45 min at O'C. The transformation mixture was then transferred to 420C for 10 min, an equal volume of nutrient broth was added, and the culture was incubated at 37 C for 2 h with shaking. Samples were plated onto selectiveplates containing amnpieillin and kanamycin to determine the number oftransformants and onto nutrient agar plates to determine total cell numbers. Absolute transformation fr-equencies (i.e., fr-action of cells transformed) were: for AB1157, 1.3 X 10-3; for AB1885., 1.5 x 10-3; for B W41, 1.0 x 10-3; and for AB2480, 1.8 X 10-3. plaSMid DNA wats amPlified by growing for 3 h in the presence of chlor-

forming species. This plasmid has a molecular weight of 5.8 x 106 and codes for resistance to ampicillin and kanamycin (8). It is apparent that excision repair is effective on this substrate and that the recA+ gene product is not essential for excision repair in this system. Plasmid survival is consistently slightly lower in the recA background, which may reflect a minor pathway of repair controlled by this gene product or simply the fact that degradation of UV-damaged plasmid DNA is more likely to occur in these strains. Excision repair also proved efficient in increasing survival when the UV-irradiated open circular forn of NTP16 was used as the transforming DNA (data not shown). The amount of reactivation observed is consistent with the hypothesis that the DNA taken up when double-stranded DNA is used as substrate remains in its double-stranded form. At a dose of UV light which reduces plasmid survival in the uvr strain to 37% (i.e., an average of one lethal hit per molecule), more than 98% of the plasmid survives in the uvr' strain. At this dose, therefore, more than 96% of the DNA molecules which have received at least one lethal hit and are taken up by the recipient cell remain susceptible to excision repair. The small fraction of plasmid molecules which do not survive this dose may represent those which receive adjacent multiple hits and thus become refractory to excision repair. In contrast, if single-stranded DNA was used as the transforming species, the inability of excision repair to act on this substrate became apparent. Figure 2 shows the survival of singleand double-stranded (RFI [covalently closed circular replicative form]) DNA of phage 4XtB (2) on excision-proficient and -deficient recipient strains. Both bacterial strains are rec+, and it is apparent that there is no effective rescue of damaged single-stranded DNA, despite the possibility of error-prone repair. Similar results have been reported for other purified phage DNAs and other transfecting systems (7, 16). One theoretical alternative consistent with these results and yet involving uptake of singlestranded DNA would be if the plasmid duplex were taken up as two separate complementary strands which reannealed to give doublestranded DNA inside the cell. Such a complex procedure seems unlikely, especially as dose-response curves indicate that uptake of a single molecule is sufficient to allow subsequent plasmid survival (8). This possibility is being explored further, however. It is clear, therefore, at least in the case of amphenicol (200 pg/ml) and extracted by a minor modification of the procedure of Guerry et al. (6).

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FIG. 2. Survival of UV-irradiated oXtB DNA on excision repair-proficient (AB1157 uvrr, closed symbols) and -deficient (AB1886 uvrA6, open symbols) calcium-treated cells. Circles indicate survival of double-stranded RFI DNA, and squares indicate single-strandedphage DNA. Isolation and transfection of RFI DNA was carried out as described forplasmid DNA in the legend to Fig. 1. Single-stranded DNA was isolated by phenol treatment of purified phage particles and was transfected as described for RFI. Plating procedures were as described previously (2), using strain AB1886 as the indicator bacterium. The efficiencies of transfection (fraction of cells transfected) of the recipient strains were: for AB1157, 1.9 X 10-3 (double stranded) and 1.2 x 10-3 (single stranded); and for AB1886, 2.1 x 10-3 (double stranded) and 3.1 x 10-3 (single stranded). p.f.u., Plaque-forming units.

plasmid NTP16 and oXtB RFI DNA, that covalently closed circular DNA is taken up in the double-stranded form by calcium-treated E. coli cells. Uptake of single-stranded DNA can and does occur, however, when a substrate such as 4XtB viral DNA is used as the transfecting species. Susceptibility to excision repair appears to be a useful criterion for determining whether a particular DNA species is single or double stranded in vivo, and the interpretation of the data does not appear to be complicated by the possibility of error-prone repair. Given the wellunderstood molecular details of many repair processes, it should be possible to exploit other aspects of these mechanisms to investigate further the nature of incoming transfecting DNA. Such experiments are currently in progress.

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This work was supported by a Medical Research Council project grant to P.S., and a Science Research Council postgraduate studentship to R.J.R. We thank C. E. Dowell for the kind gift of phage oXtB and Carole Alston for her excellent technical assistance. LITERATURE CITED 1. Benzinger, R. 1978. Transfection of Enterobacteriaceae and its applications. Microbiol. Rev. 42:194-236. 2. Bone, D. R., and C. E. Dowell. 1973. A mutant of bacteriophage OX174 which infects E. coli K12 strains. I. Isolation and partial characterization of 4XtB. Virology 52:319-329. 3. Caillet-Fauquet, P., M. Defais, and M. Radman. 1977. Molecular mechanisms of induced mutagenesis. Replication in vivo of bacteriophage OX174 single stranded, ultraviolet light-irradiated DNA in intact and irradiated host cells. J. Mol. Biol. 117:95-112. 4. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Nonchromosomal antibiotic resistance in bacteria-genetic transformation of E. coli by R factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2214. 5. Grossman, L, A. Braun, R. Feldberg, and I. Mahler. 1975. Enzymatic repair of DNA. Annu. Rev. Biochem. 44:19-43. 6. Guerry, P., D. J. Leblanc, and S. Falkow. 1973. General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116:1064-1066. 7. Heijneker, H. L. 1975. Physico-chemical and biological study of excision-repair of UV-irradiated 4X174 RF DNA in vitro. Nucleic Acids Res. 2:2147-2161. 8. Humphreys, G. O., A. Weston, M. G. M. Brown, and J. R. Saunders. 1979. Plasmid transformation of Escherichia coli, p. 254-279. In S. W. Glover and L. 0. Butler (ed.), Transformation 1978. Cotswold Press, Oxford. 9. Inoue, M., and R. Curtiss m. 1978. Transformation procedure to E. coli X1776 strain, p. 248. In W. A. Scott and R. Werner (ed.), Molecular cloning of recombinant DNA. Academic Press Inc., New York. 10. Lacks, S. 1977. Binding and entry of DNA in bacterial transformation, p. 179-232. In J. L. Reissig (ed.), Microbial interactions. Chapman and Hall, London. 11. Leclerc, J., and J. Setlow. 1974. Transformation in Haemophilus influenzae., p. 187-207. In R. F. Grell (ed.), Mechanisms in recombination. Plenum Press, New York. 12. Mandel, M., and A. Higa. 1970. Calcium dependent bacteriophage DNA infection. J. Mol. Biol. 53:159-162. 13. Oishi, M., and R. M. Irbe. 1977. Circular chromosomes and genetic transformation in E. coli, p. 121-134. In A. Portoles, R. Lopes, and M. Espinosa (ed.), Modern trends in bacterial transformation and transfection. North-Holland Publishing Co., New York. 14. Seeberg, E., J. Nissen-Meyer, and P. Strike. 1976. Incision of ultraviolet irradiated DNA by extracts of E. coli requires three different gene products. Nature (London) 263:524-526. 15. Seeberg, E., and P. Strike. 1976. Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli. J. Bacteriol. 125:787795. 16. Taketo, A., S. Yasuda, and M. Sekiguchi. 1972. Initial step of excision repair in Escherichia coli: replacement of defective function of uvr mutants by T4 endonuclease V. J. Mol. Biol. 70:1-14. 17. Tomasz, A. 1969. Some aspects of the competent state in genetic transformation. Annu. Rev. Genet. 3:217-232. 18. Wagner, R., Jr., and M. Meselson. 1976. Repair tracts in mismatched DNA heteroduplexes. Proc. Natl. Acad. Sci. U.S.A. 73:4135-4139. 19. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40: 869-907.