Vol. 138, No. 3
JOURNAL OF BACTERIOLOGY, June 1979, p. 779-782
0021-9193/79/06-0779/04$02.00/0
Penicillin Selection of Escherichia coli Deoxyribonucleic Acid Repair Mutants AZIZ SANCARt* AND CLAUD S. RUPERT
University of Texas at Dallas, Programs in Biology, Richardson, Texas 75080 Received for publication 22 March 1979
A rapid method has been developed for isolation ofultraviolet-sensitive mutants of Escherichia coli, by inducing delay in the growth and/or division of repairdeficient cells with low fluences of far-ultraviolet radiation, and killing with penicillin the repair-proficient cells, which continue to grow and divide. With this technique, we have achieved about a 3,000-fold enrichment for photoreactivationless (phr) cells and have isolated and characterized three phr mutants.
In seeking photoreactivationless (phr) mutants of Escherichia coli K-12 for genetic analysis, random screening was too slow to provide the desired numbers, whereas positive selection for repair deficiency by the "suicidal-phage reactivation" method (8) was cumbersome, because of the need to photoreactivate phage-bacterium complexes. Other cell properties correlated with repair deficiencies give no help in selecting phr mutants. We consequently adapted the penicillin selection technique (3, 16), commonly used to isolate auxotrophs, for selection of repair mutants. UV (254 nm) irradiation will cause growth, and/or division delay at nonlethal doses (12; see also 11 and 20), and the UV fluences required for this effect are somewhat lower in the absence of DNA repair (4, 19). Since penicillin acts only on growing and/or dividing cells, it was possible that repair-proficient cells could be preferentially killed after suitable, low UV fluences. Development of this technique has allowed isolation of several phr mutants from E. coli K-12. The method is also applicable to isolation of other UV-sensitive mutants as well. MATERIALS AND METHODS Bacterial strains, plasmid, and phage. The E. coli K-12 strains AB1157 (uvrA +) and AB1886 (uvrA6) are described elsewhere (7). DNA from plasmnid R6K (amp+ str+; see 14) was supplied by R. C. Clowes, and the mutant phage T4vl (5) was supplied by W. Harm. Media. LB medium consists of the following (per liter): tryptone (Difco), 10 g; yeast extract (Difco), 5 g; NaCl, 10 g. K medium (17) is M9 medium plus decolorized vitamin-free amino acids (Difco), and 1 jg of thiamine per ml. Ml medium contains (per liter): Na2HPO4, 7.5 g; KH2PO4, 3.75 g; NaCl, 0.6 g; and sucrose, 120 g. M2 medium contains (per liter): decolorized vitamin-free Casamino Acids, 50 g; glucose, 30 t Present address: Yale University School of Medicine, Radiobiology Laboratories, New Haven, CT 06510.
g; sucrose, 120 g; thiamine, 5 mg; and MgSO4, 12.5 g. KMS medium is a mixture of Ml and M2 in a ratio of 4 to 1 (giving essentially K medium plus 0.01 M MgSO4 and 12% sucrose). UV irradiation and photoreactivation. The UV radiation was from a low-pressure mercury lamp (Osram HNS 12) emitting mostly at 254 nm. UV fluence rates were measured with a Jagger meter (10), calibrated against a standard lamp (U.S. Bureau of Standards) and thermopile. Photoreactivation was carried out in a glass-bottomed water bath with three 15-W Westinghouse Daylight fluorescent lamps at 2-cm distance. Unless otherwise stated, all experiments involving UV irradiation were done under yellow light from General Electric Gold fluorescent lamps. Selection method. Cells of AB1886 were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine as described by Adelberg et al. (1) to 33% survival and grown for 5 h. The mutagenized stock was subcultured in K medium overnight, diluted 1:10 in the same medium, and grown at 370C to a density of 10' cells per ml. The cells were washed with and suspended in four-fifths volume of MI medium and irradiated with UV (254 nm) at a total fluence of 2 J/m2, and penicillin was added to a final concentration of 400 U/ml. The cell suspension was divided into two aliquots, and one part was photoreactivated at 370C for 30 min, whereas the other was kept in the dark at 37°C for the same time as a reference control. Prewarmed M2 medium (onefifth volume) was added to both suspensions, and the cultures were grown at 43°C for 90 min with vigorous aeration. Cells of both cultures were then subjected to osmotic shock by diluting 1/80 in water at 500C and keeping them at this temperature for 15 min (see 9). Samples from both treated cultures were plated to determine the cell survivals. One-fourth volume of 5x KMS medium was added to the photoreactivated cell suspensions, and the culture was grown at 37°C to a titer of 10' cells per ml (approximately 4 h), when the next cycle of UV irradiation, photoreactivation, penicillin treatment, and osmotic shock were repeated. The culture from the final penicillin cycle was diluted and plated on five LB agar plates which were incubated at 370C overnight. Single colonies were
779
780
SANCAR AND RUPERT
picked (about 15 colonies from each plate) for growth in 2 ml of LB agar and tested for photoreactivation by the method of Harm and Hillebrandt (6) using T4vl phage. Survival curves. Bacterial colony-forming UV survival and phage plaque-forming UV survival were determined by usual methods. For plasmid survival, 0.1 ml of irradiated R6K DNA (10 ,g/ml) was mixed with 0.2 ml of competent cells in 0.05 M CaCl2 and transformation was carried out by the method of Cohen et al. (2). The transformation mixture was kept on ice for 40 min, heat shocked at 420C for 90 s, and put on ice again. The cell suspensions were then diluted 1/10 in 0.15 M NaCl and either photoreactivated or kept dark at 370C for 50 min. After this treatment, the cells were centrifuged, suspended in 3 ml of LB agar, and grown for 3 h at 370C before plating on LB plates containing 200 jg of ampicillin per ml.
RESULTS Penicillin killing of UV-rradiated and UV-irradiated-photoreactivated cells. For isolation of phr mutants, a uvr parental strain (AB1886) was desirable to increase the differential UV response between phr+ and phr cells. However, the multiauxotrophic character of this strain, chosen to facilitate later genetic analysis, necessitated use of K medium during penicillin treatment, and in this medium the cells do not show appreciable growth delay (indicated by optical density change) after UV fluences producing 10 to 30% survival. They do, however, show division delay (17), viable counts remaining constant for about 90 min after irradiation, before resuming at the normal division rate. The survival of UV-irradiated cells after photoreactivation was about 95%, with a division delay of only about 15 min. Consequently, it was possible to use the unirradiated cells as an approximate phr+ control in some reconstruction experiments described below. The division septum of growing cells is more sensitive to penicillin than the rest of the cell envelope (15). If cells are grown in low penicillin concentrations, dividing cells are preferentially affected and become sensitive to osmotic shock (18). In the reconstruction experiment shown in Fig. 1, we subjected the cells to osmotic shock after treatment with 320 to 330 U of penicillin per ml. The UV fluence (2.0 J/m2) and the penicillin concentration were selected by trial and error to optimize for enrichment of Phrcells. The results shown in the figure are typical, enrichment factors for Phr- cells ranging from 10 to 30 in different experiments. Separate tests showed that, if the penicillin was removed 100 min after irradiation-the max2imum time before resumption of penicillin sensitivity-the enrichment produced by the treatment was maintained quantitatively through at least eight generations of subsequent
J. BACTERIOL.
zZO
a \
0 IO
D' lo-2\
-
-3 l____ l____ l____ l____ l_ 0
20
40
60
80
100
TIME AFTER ADDITION OF PENICILLIN(min)
FIG. 1. Penicillin survival of UV-irradiated AB1886. A 20-ml culture grown in K medium was washed and suspended in 16 ml of Ml medium containing400 U ofpenicillin per ml, and UVirradiated at 2 Jlm2. Half was photoreactivated for 30 min at 37°C, whereas the remainder was kept dark. A 2-ml volume ofprewarmed M2 medium was added to each aliquot at time zero, and the cultures were further incubated with shaking. At the indicated times, 0.1ml samples were taken into tubes containing 10 ml of water at 50°C and blended in a Vortex mixer vigorously for 30 s, and then dilutions were made and plated on LB plates. To determine the survival of unirradiated cells, 330 U of penicillin per ml was added to an exponential culture in K medium, and samples were diluted as above and plated. Symbols: *, UV-irradiated cells; 0, UV-irradiated and photoreactivated cells; A, non-irradiated cells.
growth, and therefore presumably for an indefinite time. In other trials we found that the parental uvr' strain (AB1157) showed no detectable division delay from irradiation with 2.0 J/m2. Thus, these uvr+ cells should behave like the unirradiated cells in Fig. 1, indicating that enrichment for uvr mutants (by about 100-fold) should also be possible by means of this treatment. Isolation of phr mutants. An AB1886 culture mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine was divided into three aliquots. One, the "selection culture," was subjected to UV irradiation, photoreactivation, penicillin treatment, and osmotic shock, as described in Materials and Methods. A second aliquot, the "control culture," was subjected to the same treatments, except that the photoreactivation
PENICILLIN SELECTION OF DNA REPAIR MUTANTS
VOL. 138, 1979
was omitted. The fraction of cells surviving in each treated culture was then determined by plating in comparison with the untreated aliquot. Survival was higher in the non-photoreactivated control because continuing growth delay in the absence of photorepair protected against penicillin action. The ratio of control culture survival to selection culture survival gives the degree of enrichment experienced by any phr mutants which are present in the latter (since all the cells become phenotypically Phr- in the absence of photoreactivating light). After such a cycle of treatments, the selection culture was regrown and divided into aliquots, and the cycle was repeated. Table 1 shows the result of a five-cycle experiment. The calculated TABLE 1. Penicillin enrichment ofphr mutants EnrichSurviving fraction Cycle ment facno. Selection culture Control culture tora 1 6x 10-2 6X 10' 10 1 X 10-1 2 6.24 x 10' 6.24 3 6.6 X 10-2 3.7 x 10-' 5.6 4 5
2X 10'
4X
10-1
6.5X 10-' 1.0
3.2 2.5
a Enrichment factor for phr mutants is estimated by the ratio of the control culture survival to the selection culture survival as described in the text. It is inherently assumed that the phr cells of interest will show the same growth properties as the bulk of the culture, a fact confirmed by the final isolates.
781
enrichment factors for phr mutants after each cycle are shown in the right-hand column, along with the overall enrichment (about 3,000-fold) calculated from their product. When single colonies were isolated from this multiply cycled culture, 3 out of 77 tested were phr, whereas none of 151 colonies tested from a mutagenized nonenriched culture of AB1886 was phr. Characteristics of the Phr- mutants. The phr mutants isolated by this procedure were designated CSR06 (phr-1), CSR58 (phr-2), and CSR70 (phr-3). When tested for photoreactivating activity by cell survival, phage survival, and transforming DNA (plasmid) survival methods, CSR06 and the CSR70 behaved identically in all three assays, whereas CSR58 showed some residual photoreactivating activity. Figure 2 gives the results of photoreactivating assays for CSR06, and for the parental type, showing the absence of detectable photoreactivating activity by all three tests. (Still unpublished studies by W. Harm [personal communication] on a triple mutant CSR603 [recAl uvrA6 phr-1] indicate, nevertheless, a curious kind of leakiness: although 99% of the cells contain no photoreactivating enzyme whatever, about 1% contain essentially a single active molecule apiece.) DISCUSSION The selection method described here for isolation of UV-sensitive mutants of E. coli has
A. CELL
z 0 0 4 LL
z >
o0-
C,)
(J/m2 ) FIG. 2. Photoreactivation of cells, phage, and transforming DNA. AB1886 (uvrA6) and CSR06 (uvrA6phr1) cells were UV irradiated, or infected with UV-irradiated phage, or transformed with UV-irradiated R6K plasmid DNA, and then exposed to photoreactivating light for 50 min. (A) Cell survival. (B) T4vl phage plaque-forming survival. (C) R6K transforming DNA survival (ampicillin resistance marker). Solid symbols indicate no photoreactivation; open symbols indicate approximately maxiimal photoreactivation. Circles indicate CSR06 cells, orphages orplasmid DNA in CSR06 as host; triangles indicate AB1886 cells, orphages or plasmid DNA in AB1886 as host. U V FLUENCE
782
SANCAR AND RUPERT
achieved a 3,000-fold enrichment forphr cells in a culture mutagenized with N-methyl-N'-nitroN-nitrosoguanidine. The method, based on a physiological response (growth and/or division delay) to UV, of repair-deficient cells, should also be applicable to isolation of UV-sensitive mutants lacking dark repair functions. Although Howard-Flanders and Theriot's ingenious technique for the isolation of uvr mutants is relatively fast and has been widely used, it requires more than average technical skill, and by its very nature (reactivation of an infecting phage) it has limitations as to the type of mutants it can detect. Our method is simpler and faster, and being based on the response of the cells to pyrimidine dimers in their own DNA, rather than dimers in an infecting phage DNA, it may reveal mutants not detectable by the "suicidal-phagereactivation method." Moreover, its principle (selecting for growth/division delay) might be adaptable to selection of UV-sensitive mutants from eucaryotes (e.g., by killing growing, repairproficient cells with white light treatment after
bromodeoxyuridine incorporation). Our use of UV-irradiated transforming (plasmid) DNA as an assay for in vivo photoreactivation in E. coli is novel, and should have applications in studying the dark repair mechanisms of this bacterium. It is noteworthy that this assay enabled us to photoreactivate UVirradiated transforming DNA in vivo, a phenomenon which has not been observed since the "classic transformable species," e.g., Haemophilus influenzae, Bacillus subtilus, and Diplococcus pneumoniae, are not photoreactivable (13). ACKNOVWLEDGMENT1 We thank R. C. Clowes for many helpful suggestions. This work was supported by Public Health Service research grant GM-1647 from the National Institute for General Medical Sciences. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'nitro-N-nitrosoguanidine in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 18:788-795. 2. Cohen, S. N., A. C. Y. Chang, and C. L. Hlu. 1972. Non-chromosomal antibiotic resistance in bacterial genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114.
J. BACTERIOL. 3. Davis, B. D. 1949. Isolation of biochemically deficient mutants ofbacteria by penicillin. J. Am. Chem Soc. 70: 4267. 4. H lo, B. A, and P. A. Swenson. 1969. Effects of ultraviolet radiation on respiration and growth in radiation-resistant and radiation-sensitive strains of Ewcherichia coli B. J. Bacteriol. 9:815-823. 5. Harm, W. 1963. Mutants of phage T4 with increased sensitivity to ultraviolet. Virology 19:66-71. 6. Harm, W., and B. Hillebrandt. 1962. A non-photoreactivable mutant of Escherichia coli B. Photochem. Photobiol. 1:271-272. 7. Howard-Flanders, P., R. P. Boyce, and L. Theriot. 1966. Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53:1119-1136. 8. Howard-Flanders, P., and L Theriot. 1962. A method for selecting radiation-sensitive mutants of Escherichia co& Genetics 47:1219-1224. 9. Hurwtz, C., J. M. Reiner, and J. V. Lanau. 1958. Studies on the physiology and biochemistry of penicillin induced spheroplasts of Escherichia coli. J. Bacteriol. 76:612-717. 10. Jagger, J. 1961. A small and inexpensive ultraviolet doserate meter useful in biological experiments. Radiat. Res. 14:394-403. 11. Jagger, J., W. C. Wise, and R. S. Stafford. 1964. Delay in growth and division induced by near ultraviolet radiation in Escherichia coli and its role in photoprotection and liquid holding recovery. Photochem. Photobiol. 3:11-24. 12. Kelner, A. 1953. Growth, respiration and nucleic acid synthesis in ultraviolet irradiated and in photoreactivated Escherichia coli. J. Bacteriol. 65:252-262. 13. Kelner, A. 1964. Correlation between genetic transformability and nonphotoreactivability in Bacillus subtilis. J. Bacteriol. 87:1295-1303. 14. Kontomichalou, P., M Mitani, and R. C. Clowes. 1970. Circular R-factor molecules controlling penicillinase synthesia replicating in Escherichia coli under either relaxed or stringent control. J. Bacteriol. 104:34-44. 15. Lederberg, J. 1957. Mechanism of action of penicillin. J. Bacteriol. 73:144. 16. Lederberg, J., and N. Zinder. 1948. Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc. 70:4267-4268. 17. Rupp, W. D., C. E. Wilde m, D. L Reno, and P. Howard-Flanders. 1971. Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J. Mol. Biol. 61:25-44. 18. Schwarz, U., A. Asmus, and H. Frank. 1969. Autolytic enzymes and cell division of Escherichia coli. J. Mol. Biol. 41:419-429. 19. Swenson, P. A., and R. L Schenley. Respiration, growth and viability of repair-deficient mutants of Escherichia coli after ultraviolet irradiation. Int. J. Radiat. Biol. 25:51-60. 20. Takebe, H., and J. Jagger. 1969. Action spectrum for growth delay induced in Escherichia coli B/r by farultraviolet radiation. J. Bacteriol. 98:677-682.
JOURNAL OF BACTERIOLOGY, June 1979, p. 779-782
0021-9193/79/06-0779/04$02.00/0
Penicillin Selection of Escherichia coli Deoxyribonucleic Acid Repair Mutants AZIZ SANCARt* AND CLAUD S. RUPERT
University of Texas at Dallas, Programs in Biology, Richardson, Texas 75080 Received for publication 22 March 1979
A rapid method has been developed for isolation ofultraviolet-sensitive mutants of Escherichia coli, by inducing delay in the growth and/or division of repairdeficient cells with low fluences of far-ultraviolet radiation, and killing with penicillin the repair-proficient cells, which continue to grow and divide. With this technique, we have achieved about a 3,000-fold enrichment for photoreactivationless (phr) cells and have isolated and characterized three phr mutants.
In seeking photoreactivationless (phr) mutants of Escherichia coli K-12 for genetic analysis, random screening was too slow to provide the desired numbers, whereas positive selection for repair deficiency by the "suicidal-phage reactivation" method (8) was cumbersome, because of the need to photoreactivate phage-bacterium complexes. Other cell properties correlated with repair deficiencies give no help in selecting phr mutants. We consequently adapted the penicillin selection technique (3, 16), commonly used to isolate auxotrophs, for selection of repair mutants. UV (254 nm) irradiation will cause growth, and/or division delay at nonlethal doses (12; see also 11 and 20), and the UV fluences required for this effect are somewhat lower in the absence of DNA repair (4, 19). Since penicillin acts only on growing and/or dividing cells, it was possible that repair-proficient cells could be preferentially killed after suitable, low UV fluences. Development of this technique has allowed isolation of several phr mutants from E. coli K-12. The method is also applicable to isolation of other UV-sensitive mutants as well. MATERIALS AND METHODS Bacterial strains, plasmid, and phage. The E. coli K-12 strains AB1157 (uvrA +) and AB1886 (uvrA6) are described elsewhere (7). DNA from plasmnid R6K (amp+ str+; see 14) was supplied by R. C. Clowes, and the mutant phage T4vl (5) was supplied by W. Harm. Media. LB medium consists of the following (per liter): tryptone (Difco), 10 g; yeast extract (Difco), 5 g; NaCl, 10 g. K medium (17) is M9 medium plus decolorized vitamin-free amino acids (Difco), and 1 jg of thiamine per ml. Ml medium contains (per liter): Na2HPO4, 7.5 g; KH2PO4, 3.75 g; NaCl, 0.6 g; and sucrose, 120 g. M2 medium contains (per liter): decolorized vitamin-free Casamino Acids, 50 g; glucose, 30 t Present address: Yale University School of Medicine, Radiobiology Laboratories, New Haven, CT 06510.
g; sucrose, 120 g; thiamine, 5 mg; and MgSO4, 12.5 g. KMS medium is a mixture of Ml and M2 in a ratio of 4 to 1 (giving essentially K medium plus 0.01 M MgSO4 and 12% sucrose). UV irradiation and photoreactivation. The UV radiation was from a low-pressure mercury lamp (Osram HNS 12) emitting mostly at 254 nm. UV fluence rates were measured with a Jagger meter (10), calibrated against a standard lamp (U.S. Bureau of Standards) and thermopile. Photoreactivation was carried out in a glass-bottomed water bath with three 15-W Westinghouse Daylight fluorescent lamps at 2-cm distance. Unless otherwise stated, all experiments involving UV irradiation were done under yellow light from General Electric Gold fluorescent lamps. Selection method. Cells of AB1886 were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine as described by Adelberg et al. (1) to 33% survival and grown for 5 h. The mutagenized stock was subcultured in K medium overnight, diluted 1:10 in the same medium, and grown at 370C to a density of 10' cells per ml. The cells were washed with and suspended in four-fifths volume of MI medium and irradiated with UV (254 nm) at a total fluence of 2 J/m2, and penicillin was added to a final concentration of 400 U/ml. The cell suspension was divided into two aliquots, and one part was photoreactivated at 370C for 30 min, whereas the other was kept in the dark at 37°C for the same time as a reference control. Prewarmed M2 medium (onefifth volume) was added to both suspensions, and the cultures were grown at 43°C for 90 min with vigorous aeration. Cells of both cultures were then subjected to osmotic shock by diluting 1/80 in water at 500C and keeping them at this temperature for 15 min (see 9). Samples from both treated cultures were plated to determine the cell survivals. One-fourth volume of 5x KMS medium was added to the photoreactivated cell suspensions, and the culture was grown at 37°C to a titer of 10' cells per ml (approximately 4 h), when the next cycle of UV irradiation, photoreactivation, penicillin treatment, and osmotic shock were repeated. The culture from the final penicillin cycle was diluted and plated on five LB agar plates which were incubated at 370C overnight. Single colonies were
779
780
SANCAR AND RUPERT
picked (about 15 colonies from each plate) for growth in 2 ml of LB agar and tested for photoreactivation by the method of Harm and Hillebrandt (6) using T4vl phage. Survival curves. Bacterial colony-forming UV survival and phage plaque-forming UV survival were determined by usual methods. For plasmid survival, 0.1 ml of irradiated R6K DNA (10 ,g/ml) was mixed with 0.2 ml of competent cells in 0.05 M CaCl2 and transformation was carried out by the method of Cohen et al. (2). The transformation mixture was kept on ice for 40 min, heat shocked at 420C for 90 s, and put on ice again. The cell suspensions were then diluted 1/10 in 0.15 M NaCl and either photoreactivated or kept dark at 370C for 50 min. After this treatment, the cells were centrifuged, suspended in 3 ml of LB agar, and grown for 3 h at 370C before plating on LB plates containing 200 jg of ampicillin per ml.
RESULTS Penicillin killing of UV-rradiated and UV-irradiated-photoreactivated cells. For isolation of phr mutants, a uvr parental strain (AB1886) was desirable to increase the differential UV response between phr+ and phr cells. However, the multiauxotrophic character of this strain, chosen to facilitate later genetic analysis, necessitated use of K medium during penicillin treatment, and in this medium the cells do not show appreciable growth delay (indicated by optical density change) after UV fluences producing 10 to 30% survival. They do, however, show division delay (17), viable counts remaining constant for about 90 min after irradiation, before resuming at the normal division rate. The survival of UV-irradiated cells after photoreactivation was about 95%, with a division delay of only about 15 min. Consequently, it was possible to use the unirradiated cells as an approximate phr+ control in some reconstruction experiments described below. The division septum of growing cells is more sensitive to penicillin than the rest of the cell envelope (15). If cells are grown in low penicillin concentrations, dividing cells are preferentially affected and become sensitive to osmotic shock (18). In the reconstruction experiment shown in Fig. 1, we subjected the cells to osmotic shock after treatment with 320 to 330 U of penicillin per ml. The UV fluence (2.0 J/m2) and the penicillin concentration were selected by trial and error to optimize for enrichment of Phrcells. The results shown in the figure are typical, enrichment factors for Phr- cells ranging from 10 to 30 in different experiments. Separate tests showed that, if the penicillin was removed 100 min after irradiation-the max2imum time before resumption of penicillin sensitivity-the enrichment produced by the treatment was maintained quantitatively through at least eight generations of subsequent
J. BACTERIOL.
zZO
a \
0 IO
D' lo-2\
-
-3 l____ l____ l____ l____ l_ 0
20
40
60
80
100
TIME AFTER ADDITION OF PENICILLIN(min)
FIG. 1. Penicillin survival of UV-irradiated AB1886. A 20-ml culture grown in K medium was washed and suspended in 16 ml of Ml medium containing400 U ofpenicillin per ml, and UVirradiated at 2 Jlm2. Half was photoreactivated for 30 min at 37°C, whereas the remainder was kept dark. A 2-ml volume ofprewarmed M2 medium was added to each aliquot at time zero, and the cultures were further incubated with shaking. At the indicated times, 0.1ml samples were taken into tubes containing 10 ml of water at 50°C and blended in a Vortex mixer vigorously for 30 s, and then dilutions were made and plated on LB plates. To determine the survival of unirradiated cells, 330 U of penicillin per ml was added to an exponential culture in K medium, and samples were diluted as above and plated. Symbols: *, UV-irradiated cells; 0, UV-irradiated and photoreactivated cells; A, non-irradiated cells.
growth, and therefore presumably for an indefinite time. In other trials we found that the parental uvr' strain (AB1157) showed no detectable division delay from irradiation with 2.0 J/m2. Thus, these uvr+ cells should behave like the unirradiated cells in Fig. 1, indicating that enrichment for uvr mutants (by about 100-fold) should also be possible by means of this treatment. Isolation of phr mutants. An AB1886 culture mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine was divided into three aliquots. One, the "selection culture," was subjected to UV irradiation, photoreactivation, penicillin treatment, and osmotic shock, as described in Materials and Methods. A second aliquot, the "control culture," was subjected to the same treatments, except that the photoreactivation
PENICILLIN SELECTION OF DNA REPAIR MUTANTS
VOL. 138, 1979
was omitted. The fraction of cells surviving in each treated culture was then determined by plating in comparison with the untreated aliquot. Survival was higher in the non-photoreactivated control because continuing growth delay in the absence of photorepair protected against penicillin action. The ratio of control culture survival to selection culture survival gives the degree of enrichment experienced by any phr mutants which are present in the latter (since all the cells become phenotypically Phr- in the absence of photoreactivating light). After such a cycle of treatments, the selection culture was regrown and divided into aliquots, and the cycle was repeated. Table 1 shows the result of a five-cycle experiment. The calculated TABLE 1. Penicillin enrichment ofphr mutants EnrichSurviving fraction Cycle ment facno. Selection culture Control culture tora 1 6x 10-2 6X 10' 10 1 X 10-1 2 6.24 x 10' 6.24 3 6.6 X 10-2 3.7 x 10-' 5.6 4 5
2X 10'
4X
10-1
6.5X 10-' 1.0
3.2 2.5
a Enrichment factor for phr mutants is estimated by the ratio of the control culture survival to the selection culture survival as described in the text. It is inherently assumed that the phr cells of interest will show the same growth properties as the bulk of the culture, a fact confirmed by the final isolates.
781
enrichment factors for phr mutants after each cycle are shown in the right-hand column, along with the overall enrichment (about 3,000-fold) calculated from their product. When single colonies were isolated from this multiply cycled culture, 3 out of 77 tested were phr, whereas none of 151 colonies tested from a mutagenized nonenriched culture of AB1886 was phr. Characteristics of the Phr- mutants. The phr mutants isolated by this procedure were designated CSR06 (phr-1), CSR58 (phr-2), and CSR70 (phr-3). When tested for photoreactivating activity by cell survival, phage survival, and transforming DNA (plasmid) survival methods, CSR06 and the CSR70 behaved identically in all three assays, whereas CSR58 showed some residual photoreactivating activity. Figure 2 gives the results of photoreactivating assays for CSR06, and for the parental type, showing the absence of detectable photoreactivating activity by all three tests. (Still unpublished studies by W. Harm [personal communication] on a triple mutant CSR603 [recAl uvrA6 phr-1] indicate, nevertheless, a curious kind of leakiness: although 99% of the cells contain no photoreactivating enzyme whatever, about 1% contain essentially a single active molecule apiece.) DISCUSSION The selection method described here for isolation of UV-sensitive mutants of E. coli has
A. CELL
z 0 0 4 LL
z >
o0-
C,)
(J/m2 ) FIG. 2. Photoreactivation of cells, phage, and transforming DNA. AB1886 (uvrA6) and CSR06 (uvrA6phr1) cells were UV irradiated, or infected with UV-irradiated phage, or transformed with UV-irradiated R6K plasmid DNA, and then exposed to photoreactivating light for 50 min. (A) Cell survival. (B) T4vl phage plaque-forming survival. (C) R6K transforming DNA survival (ampicillin resistance marker). Solid symbols indicate no photoreactivation; open symbols indicate approximately maxiimal photoreactivation. Circles indicate CSR06 cells, orphages orplasmid DNA in CSR06 as host; triangles indicate AB1886 cells, orphages or plasmid DNA in AB1886 as host. U V FLUENCE
782
SANCAR AND RUPERT
achieved a 3,000-fold enrichment forphr cells in a culture mutagenized with N-methyl-N'-nitroN-nitrosoguanidine. The method, based on a physiological response (growth and/or division delay) to UV, of repair-deficient cells, should also be applicable to isolation of UV-sensitive mutants lacking dark repair functions. Although Howard-Flanders and Theriot's ingenious technique for the isolation of uvr mutants is relatively fast and has been widely used, it requires more than average technical skill, and by its very nature (reactivation of an infecting phage) it has limitations as to the type of mutants it can detect. Our method is simpler and faster, and being based on the response of the cells to pyrimidine dimers in their own DNA, rather than dimers in an infecting phage DNA, it may reveal mutants not detectable by the "suicidal-phagereactivation method." Moreover, its principle (selecting for growth/division delay) might be adaptable to selection of UV-sensitive mutants from eucaryotes (e.g., by killing growing, repairproficient cells with white light treatment after
bromodeoxyuridine incorporation). Our use of UV-irradiated transforming (plasmid) DNA as an assay for in vivo photoreactivation in E. coli is novel, and should have applications in studying the dark repair mechanisms of this bacterium. It is noteworthy that this assay enabled us to photoreactivate UVirradiated transforming DNA in vivo, a phenomenon which has not been observed since the "classic transformable species," e.g., Haemophilus influenzae, Bacillus subtilus, and Diplococcus pneumoniae, are not photoreactivable (13). ACKNOVWLEDGMENT1 We thank R. C. Clowes for many helpful suggestions. This work was supported by Public Health Service research grant GM-1647 from the National Institute for General Medical Sciences. LITERATURE CITED 1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. Optimal conditions for mutagenesis by N-methyl-N'nitro-N-nitrosoguanidine in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 18:788-795. 2. Cohen, S. N., A. C. Y. Chang, and C. L. Hlu. 1972. Non-chromosomal antibiotic resistance in bacterial genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114.
J. BACTERIOL. 3. Davis, B. D. 1949. Isolation of biochemically deficient mutants ofbacteria by penicillin. J. Am. Chem Soc. 70: 4267. 4. H lo, B. A, and P. A. Swenson. 1969. Effects of ultraviolet radiation on respiration and growth in radiation-resistant and radiation-sensitive strains of Ewcherichia coli B. J. Bacteriol. 9:815-823. 5. Harm, W. 1963. Mutants of phage T4 with increased sensitivity to ultraviolet. Virology 19:66-71. 6. Harm, W., and B. Hillebrandt. 1962. A non-photoreactivable mutant of Escherichia coli B. Photochem. Photobiol. 1:271-272. 7. Howard-Flanders, P., R. P. Boyce, and L. Theriot. 1966. Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53:1119-1136. 8. Howard-Flanders, P., and L Theriot. 1962. A method for selecting radiation-sensitive mutants of Escherichia co& Genetics 47:1219-1224. 9. Hurwtz, C., J. M. Reiner, and J. V. Lanau. 1958. Studies on the physiology and biochemistry of penicillin induced spheroplasts of Escherichia coli. J. Bacteriol. 76:612-717. 10. Jagger, J. 1961. A small and inexpensive ultraviolet doserate meter useful in biological experiments. Radiat. Res. 14:394-403. 11. Jagger, J., W. C. Wise, and R. S. Stafford. 1964. Delay in growth and division induced by near ultraviolet radiation in Escherichia coli and its role in photoprotection and liquid holding recovery. Photochem. Photobiol. 3:11-24. 12. Kelner, A. 1953. Growth, respiration and nucleic acid synthesis in ultraviolet irradiated and in photoreactivated Escherichia coli. J. Bacteriol. 65:252-262. 13. Kelner, A. 1964. Correlation between genetic transformability and nonphotoreactivability in Bacillus subtilis. J. Bacteriol. 87:1295-1303. 14. Kontomichalou, P., M Mitani, and R. C. Clowes. 1970. Circular R-factor molecules controlling penicillinase synthesia replicating in Escherichia coli under either relaxed or stringent control. J. Bacteriol. 104:34-44. 15. Lederberg, J. 1957. Mechanism of action of penicillin. J. Bacteriol. 73:144. 16. Lederberg, J., and N. Zinder. 1948. Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc. 70:4267-4268. 17. Rupp, W. D., C. E. Wilde m, D. L Reno, and P. Howard-Flanders. 1971. Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J. Mol. Biol. 61:25-44. 18. Schwarz, U., A. Asmus, and H. Frank. 1969. Autolytic enzymes and cell division of Escherichia coli. J. Mol. Biol. 41:419-429. 19. Swenson, P. A., and R. L Schenley. Respiration, growth and viability of repair-deficient mutants of Escherichia coli after ultraviolet irradiation. Int. J. Radiat. Biol. 25:51-60. 20. Takebe, H., and J. Jagger. 1969. Action spectrum for growth delay induced in Escherichia coli B/r by farultraviolet radiation. J. Bacteriol. 98:677-682.
