Proc. Nat. Acad. Sci. USA Vol. 71, No. 2, pp. 503-506, February 1974

Interaction of Bacterial and Lambda Phage Recombination Systems in the X-Ray Sensitivity of Escherichia coli K-12 (DNA/repair/X bacteriophage red and gam genes/E. coli recB gene) 2ELJKO TRGOV6EVIC AND W. DEAN RUPP Departments of Therapeutic Radiology and Molecular Biochemistry, Yale University School of Medicine, New Haven, Connecticut, 06510 Communicated by Edward A. Adelberg, September 24, 1973

ABSTRACT E. coli cells lysogenic for the thermoin- fied by X red genes can effectively compensate for the lack of ducible prophage XcI857 can be transiently induced by a host-cell recB product in the repair of x-irradiated DNA. brief heat treatment. Although this treatment does not kill the cells, some X products normally formed during MATERIALS AND METHODS vegetative phage development are made that can alter the response of host cells to x-irradiation by causing an in- Bacterial Strains. The E. coli strains used in this study and crease in radioresistance. This increased resistance is par- their relevant genetic characteristics are as follows: ticularly striking in the recombination-deficient recB- strain, which is normally much more radiosensitive than AB1157 (7), a standard recombination-proficient strain, ob- its recB+ parent. After pulse-heating at 420, the survival tained from Dr. E. A. Adelberg, and carried in our collections curve of E. coli recB- lysogenized with XcI857 does not differ for many years. from that of the wild-type strain. Since X red mutants do AB2470 (4), recB2l derivative of AB1157. not increase the radioresistance of recB- strains, both X DM456 (8), Su- recombination-proficient strain, obtained red gene products, X exonuclease and dl-protein, are re- quired to compensate for the missing recB product. from Dr. K. B. Low. Furthermore, phage-induced radioresistance also occurs KL168 (9), Su- recB21, obtained from Dr. K. B. Low. in recB+ lysogens even when they carry X red-, but not when the X prophage is gam-. Thus, in wild-type cells, Bacteriophage Strains. The bacterial strains were made phage-induced radioresistance requires some interaction lysogenic by the following X mutants: between the bacterial recB gene product (exonuclease V) XcI857 (3): all the X strains used in this paper carry the and the phage -y-protein. marker cI857, which makes them thermoinducible. X- or gamma-ray-induced damage of bacterial cells is subject XcI857 redS (10): obtained from Dr. C. Radding. redS is to repair by several enzymatic systems that are specified by defective in the production of both X exonuclease and ,3 pro- bacterial genes, a number of which have been located on the tein. This phage was tested and found to be recombination- genetic map of Escherichia coli (1). defective by the spot test of Signer and Weil (12). One interesting example of repair is seen in the case of XcI857 redS29 (11): obtained from Dr. C. Radding. redS29 is phage-induced radioresistance of lysogenic bacteria (2). defective in the production of X exonuclease. It differs from other types of repair operating on bacterial XcI857 redll3 (10): obtained from Dr. C. Radding. redilS is DNA in that it appears to depend on products specified by defective in the production of B protein. the bacteriophage. This type of repair can easily be demon- XcI857 gam210 (13): obtained from Dr. C. Radding. gam210 strated in E. coli cells lysogenized with XcI857, a thermally in- is an amber mutation. Neither gam210 nor redS is able to grow ducible phage mutant that is not induced by either ultraviolet on E. coli strains carrying polA (13). We showed that gam210 (3) or gamma rays (2), and which was originally derived from and red3 can complement each other during growth on a polA X ind. Lysogens containing XcJ857 can survive a short heat strain, and used this test to confirm the presence of gam in this treatment in spite of the synthesis of some proteins normally phage strain. formed during vegetative phage development. Some of these XcI857 bio69 (14, 15): obtained from Dr. K. B. Low. The products, which are apparently harmless to bacteria, can del and the red exo genes are deleted from this strain. bring about a considerable increase in the survival of host cells XcI857 red270 gam2l0 del8 (Spi-; see ref. 14): obtained from exposed to ionizing radiation (2). Dr. J. Zissler. del8 is a pleiotropic mutation affecting the ex- Our present study was undertaken to identify the X gene(s) pression of amber red and gam mutations on an Su+ host. involved in phage-induced radioresistance of lysogens of wild Experimental Procedure. Lysogenic cells were grown over- type E. coli and also of the radiosensitive mutants carrying night for 18 hr at 300 with aeration in YET broth containing recB. Since the bacterial recombination system appears to 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, and 120 mg contribute to the repair of radiation damage (4-6), we have of NaOH per 1000 ml of water. All results in this paper are for tested the contribution of the X recombination system in phage- stationary phase cells grown in this way.* induced radioresistance of these strains.

The data presented in this paper show that products of the * X In experiments with logarithmic phase cells not included here, genes of the recombination region of the phage can be utilized the results for recB- cells were qualitatively and quantitatively in the repair of bacterial DNA. In particular, proteins speci- similar to those in this paper; however, wild-type cells in the logarithmic phase of growth showed little or no phage-induced Abbreviation: YET, yeast extract-tryptone growth medium. radioresistance. 503 Downloaded by guest on October 1, 2021 504 Microbiology: Trgovbevi6 and Rupp Proc. Nat. Acad. Sci. USA 71 (1974)

109 100

on O~~~~~~~~krads C) .0 z z -j 106 lol S l 5 2

Symbols: nads. 10jI I

0 5 10 15 20 DOSE (krOds) FIG. 2. Surviving fraction of heated and unheated lysogens as a function of x-ray dose. Symbols: AB32470 recB21 lysogenic for XcI857, unheated (0), or pulse-heated at 420 for 6 min (-); culture was then diluted unheated AB1157 recB + lysogenic for XcI857 (A). N is number of An 0.1-ml aliquot of the overnight viable cells; No is numbers of cells before irradiation. 50-fold with prewarmed YET medium in order to achieve an immediate shift in temperature from 30° to 42°. The bacteria were thereafter incubated at 42° with aeration. At suitable bacterial host, we used lysogens carrying X mutants red and time intervals, 0.1-ml samples were withdrawn, chilled by gam. As illustrated in Fig. 3, the recB- lysogen carrying dilution with 0.9 ml of ice-cold YET broth, x-irradiated, and XcI857 redS does not show any detectable increase in radio- kept at 0° until plating. resistance upon heating. Similar results were also obtained X-irradiation was carried out with a Siemens Stabilipan 250 with recB strains carrying XcI857 red329 and XcI857 red1 13 operated at 250 kV and 15 mA without added filtration. The (data not shown); moreover, the latter (defective in the pro- dose rate was about 5000 rads/minute. duction of ,8-protein) was slightly more sensitive after heat Both nonirradiated and irradiated cell suspensions were fur- treatment. ther diluted with buffered saline and spread on YET agar Although not as striking as in recB- bacteria, phage-in- plates (YET medium plus 2%o agar). Colonies were counted duced radioresistance also occurs in wild-type cells lyso- after 1 day (AB1157) or 2 days (DM456, AB2470) or 3 days genized with XcI857. It might be expected that, for this in- (KL168) of incubation at 30°. creased resistance, the products of X red genes are needed too. The experiment presented in Fig. 4 shows, however, that this RESULTS is not the case: a similar increase in resistance after pulse- The Effect of Transient Induction of X< on Radiosensitivity of heating occurs in wild-type lysogens carrying either XcI857 the recB- Host. As shown in the upper curve of Fig. 1, the redS or XcI857 red+. phage AB2470 recB21 strain lysogenic for the thermoinducible The Role of In addition to the red gene products, any significant y-Protein. XcIS57 can be heated for 6 mmn at 42° without a protein coded by the X gene gam (13) is also of interest in the loss of colony forming ability. Since the c1557 repressor is in- study of phage-induced radioresistance because it may inter- activated within a few seconds (16), expression of normally recombination enzymes. Since gam- period. act with certain bacterial repressed X genes is expected to begin during this in our is an amber non- change 210, used study, mutation, appropriate The presence of viral products is clearly evident in the permissive hosts were lysogenized for XcI857 gam210. Their of the cells' response to x-rays. As a result of transient induc- radiosensitivities after pulse-heating were assayed. Fig. 5 shows tion, i.e., of pulse-heating at 42°, a striking increase in radio- that the recB- lysogen displays approximately the same in- resistance occurs (the lower curve of Fig. 1). Control experi- in after transient induction of either gam+ not crease survival ments show that pulse-heating of nonlysogenic cells does or gam- prophages. In marked contrast to this, the functional change the radiosensitivity (data not presented). The in- gam+ phage gene is required to produce an increase in radio- creased survival in the recB lysogen is more pronounced than resistance of the wild-type strain (Fig. 6). that described earlier for wild-type lysogens (2). As seen in for the gam gene is also shown in additional is The requirement Fig. 2, the survival of the recB- lysogen heated for 6 mmn experiments (data not shown). The wild-type cell lysogenized very similar to that of wild-type lysogens. Heating for longer in which the genes for del and red exo are the with XcI857 bio69, than 6 mmn can even increase the survival above that of deleted (14), shows the same increase in resistance after pulse- wild type, but such a treatment was not used in the experi- as either the red+ or red- lysogens (Fig. 4). How- number of colony induction ment shown in Fig. 2, because it reduces the ever, the triple mutant, del8 red270 gam210, does not display formers in the unirradiated control. the increased resistance upon pulse-heating. It is, therefore, The Role of X Red Products. In order to identify X genes, obvious that the absence of the gam gene product abolishes the whose products confer increased resistance on the recB phage-induced resistance in the wild-type cell. Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Recombination and Phage-Induced Radioresistance 505

10'.1 101 0

0 0 z l1r,-2

a>- -J -4

U) A

I IV IA 0 3 6 9 12 15 0 3 6 9 12 15 PREINCUBATION AT 42-1 (min) PREINCUBATION AT 42 (min) FIG. 3. Effect of red gene on the survival of pulse-heated FIG. 5. Effect of X gam gene on the survrival of pulse-heated recB21 E. coli irradiated with 10 krads. Symbols: AB2470 recB21 recB21 E. coli irradiated with 10 krads. Symbols: KLI68 recB21 lysogenic for XcI857 red+ (0); AB2470 recB21 lyosgenic for lysogenic for )XcI857 red+ gam+ (0); KL168 recB21 lysogenic for XcI857 red3(A). XcI857 red + gam210 (A).

DISCUSSION can contribute to bacterial recombinant formation in recB- or recC- cells, are in accord with our findings. The results can be summarized as follows. First, transiently The experiments with the wild-type host that demonstrate induced X can recB- provide the bacterial host with some an effect of the phage ey-protein are more difficult to interpret. products that increase the cell survival to at least wild-type Since the results of 3 and 5 show that Sy-protein has no level. X red X Figs. Functional genes for both exonuclease and , pro- effect on the survival of x-irradiated recB strains, the changes tein are needed to produce this effect. in sensitivity observed in the wild-type strain require the Second, an increase in radioresistance of wild-type lysogens simultaneous presence of and the bacterial recB+ is observed upon transient heat induction, and this requires ey-protein gene product, and may be a consequence of an interaction be- the gam+ gene product of phage X but not the products of the tween them. It has been previously suggested that ef-protein red genes. inhibits exonuclease the that is absent from recB An interpretation of the results obtained with the recB- V, protein and recC cells (19-21), and that -y-protein converts a recB+ host can be easily suggested. It is known that the phage X can cell to a RecB - phenotype as a consequence of this inhibition recombine by means of the host-cell recombination enzymes (22y, 23). From this model, it is predicted that the transient (12, 17). The host-cell recombination enzymes also can par- induction of the red-gam+ phage in the wild-type bacterium ticipate in the repair of ultraviolet lesions of the X DNA (6, should inhibit exonuclease V and increase the radiosensitivity 18). Our results show that the X red products can effectively to that of the recB strain. in marked contrast to the substitute for the However, bacterial recB product in the repair of x- response expected for the RecB- phenotype, the survival of irradiated host DNA. The results of K. B. Low (personal com- the x-irradiated wild strain increased after transient in- munication), which demonstrate that the red gene proteins type X duction of the X red-gam+ phage (Fig. 4). Thus, when survival

10 1 I A A~~~~~~~~~~~~~~ 8~~~ 0- z0 z z I II I 10-2 -I 0

U) a) A ~~~~A 10 Int a a

.%_ 0 3 6 9 12 15 0 3 6 9 12 15 PREINCUBATION AT 42- (min) PREINCUBATION AT 420 (min ) X gene on FIG. 4. Effect of red the survival of pulse-heated FIG. 6. Effect of X gam gene on the survival of pulse-heated recB+ E. coli irradiated with 20 krads. Symbols: AB1157 recB+ reeB+ E. coli irradiated with 20 krads. Symbols: DM456 recB+ lysogenic for XcI857 red+ (0); AB1157 recB+ lysogenic for lysogenic for XcI857 red+ gam+ (0); DM456 recB+ lysogenic for XcI857 red3 (A). XcI857 red + yam21O (A). Downloaded by guest on October 1, 2021 506 Microbiology: Trgovbevi6 and Rupp Proc. Nat. Acad. Sci. USA 71 (1974)

of x-irradiated cells is used as the criterion, yv-protein does not 8. Mount, W. D. (1971) "Isolation and genetic analysis of a seem to convert recB+cells to a RecB- phenotype. strain of E. coli K-12 with an amber recA mutation," J. Since recB+ cells are more resistant than recB cells, the Bacteriol. 107, 388-389. 9. Bachmann, B. J. (1972) Pedigrees of some mutant strains of presence of exonuclease V benefits the x-irradiated bacteria. Escherichia coli K-12," Bacteriol. Rev. 36, 525-557. However, the simultaneous presence of y-protein and exo- 10. Shulman, M. J., Hallick, L. M., Echols, H. & Signer, E. R. nuclease V is even better, since there is higher survival than in (1970) "Properties of recombination-deficient mutants of the absence of 'y-protein. Although there are various inter- bacteriophage lambda," J. Mol. Biol. 52, 501-520. 11. Radding, C. M. (1970) "The role of exonuclease-and , pro- pretations of this effect, we shall consider three of them that tein of bacteriophage X in genetic recombination," J. Mol. involve interaction of the '-yprotein and exonuclease V. The Biol. 52, 491499. first possibility is a differential inhibition by y-protein of the 12. Signer, E. R. & Weil, J. (1968) "Recombination in bac- exonuclease V activities. The purified exonuclease V is a com- teriophage X. I. Mutants deficient in general recombina- plex enzyme with at least four different known activities tion," J. Mol. Biol. 34, 261-271. 13. Zissler, J., Signer, E. & Schaefer, F. (1971) "The role of re- (24-26). Perhaps one (or more) of these activities increases combination in growth of bacteriophage lambda. I. The the survival of x-irradiated cells while others decrease survival. gamma gene," in The Bacteriopage Lambda, ed. Hershey, A. In the absence of 'v-protein, the balance of these activities D. (Cold Spring Harbor Laboratory, Cold Spring Harbor, would lie slightly toward the survival-promoting activity. N. Y.), pp. 455468. 14. Zissler, J., Signer, E. & Schaefer, F. (1971) "The role of If '-protein prefehtially inhibits the detrimental activity, the recombination in growth of bacteriophage lambda. II. beneficial activity would then become more pronounced and Inhibition of growth by prophage P2," The Bacteriophage result in the higher survival of the transiently induced x-irra- Lambda, ed. Hershey, A. 1). (Cold Spring Harbor Labora- diated cells. tory, Cold Spring Harbor, N. Y.), pp. 469475. 15. Manly, K. F., Signer, E. R. & Radding, C. M. (1969) "Non- Another possibility is that the presence of 'y-protein results essential functions of bacteriophage X," Virology 37, 177- in only a partial inhibition of exonuclease V, and that an 188. intermediate level of the exonuclease V activity is better for 16. Kourilsky, P., Bourguignon, M. F. & Gros, F. (1971) "Ki- the x-irradiated cell than either a high level or its complete netics of viral transcription after induction of prophage," The absence. For example, a limited nuclease attack might be Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring Har- bor Laboratory, Cold Spring Harbor, N. Y. ), pp. 647-666. required for successful DNA repair while extensive DNA 17. Franklin, C. N. (1967) "Deletions and functions of the cen- degradation could prevent the successful completion of repair. ter of the o80-X phage genome. Evidence for a phage func- This possibility is consistent with recent results of Sakaki et al. tion promoting genetic recombination," Genetics 57, 301- (27), who have purified the '-protein and shown that it in- 318. hibits all known activities of the RecBC nuclease. 18. Echols, H. & Gingery, R. (1968) "Mutants of bacteriophage X defective in vegetative genetic recombination," J. Mol. Finally, a third possibility is that '-yprotein combines with Biol. 34, 239-249. exonuclease V to produce a modified enzyme with a new ac- 19. Buttin, G. & Wright, M. (1968) "Enzymatic DNA degrada- tivity different from the original activities of exonuclease V. tion in E. coli: Its relationship to synthetic processes at the According to this notion, the altered enzyme is more efficient chromosome level," Cold Spring Harbor Symp. Quant. Biol. 33, 259-269. than the original exonuclease V in increasing the survival of 20. Oishi, M. (1969) "An ATP-dependent deoxyribonuclease x-irradiated cells. from Escherichia coli with a possible role in genetic recom- bination," Proc. Nat. Acad. Sci. USA 64, 1292-1299. This work was supported by U.S. Public Health Service Grants 21. Barbour, S. D. & Clark, A. J. (1970) "Biochemical and CA 06519 and GM 18587. genetic studies of recombination proficiency in Escherichia 1. Howard-Flanders, P. & Boyce, R. P. (1966) "DNA repair coli. I. Enzymatic activity associated with recB and and genetic recombination: Studies on mutants of Esche- recC ' genes," Proc. Nat. Acad. Sci. USA 65, 955-961. richia coli defective in these processes," Radiat. Res. Suppl. 22. Unger, R. C., Echols, H. & Clark, A. J. (1972) "Interaction 6, 156-184. of the recombination pathways of bacteriophage X and host 2. .Trgoveevi6, 2., Petranovie, D. & Zgaga, V. (1971) "Phage Escherichia coli: Effects on X recombination," J. Mol. Biol. induced radioresistance of lysogenic bacteria," Biochem. 70, 531-537. 23. R. C. & Clark, A. J. (1972) "Interaction of the re- Biophys. Res. Commun. 43, 688-693. Unger, host 3. Sussman, R. & Jacob, F. (1962) "Sur un systeme de repres- combination pathways of bacteriophage X and its sion thermosensible chez le bacteriophage X d'Escherichia Escherichia coli K12: Effects on exonuclease V activity," coli," C. R. H. Acad. Sci. 254, 1517-1519. J. Mol. Biol. 70, 539-548. 4. Howard-Flanders, P. & Theriot, L. (1966) "Mutants of 24. Goldmark, P. J. & Linn, S. (1970) "An endonuclease activity Escherichia coli K-12 defective in DNA repair and in genetic from Escherichia coli absent from certain rec strains," Proc. recombination," Genetics 53, 1137-1150. Nat. Acad. Sci. USA 67, 434-441. "Discontinui- .25. P. J. & Linn, S. (1972) "Purification and proper- 5. Rupp, W. D. & Howard-Flanders, P. (1968) Goldmark, Biol. ties in the DNA synthesized in an excision-defective strain ties of the recBC DNase of Escherichia coli K-12," J. of Escherichia coli following ultraviolet irradiation," J. Mol. Chem. 247, 1849-1860. Biol. 31, 291-304. 26. Karu, A. E., MacKay, V., Goldmark, P. J. & Linn, S. (1973) "The recBC of Escherichia coli K-12. 6. Radman, M., Cordone, L., Krsmanovic-Simic, D. & Errera, deoxyribonuclease Biol. M. (1970) "Complementary action of recombination and Substrate specificity and reaction intermediates," J. excision in the repair of ultraviolet-irradiation damage to Chem. 248, 48744884. (1973). DNA," J. Mol. Biol. 49, 203-212. 27. Sakaki, Y., Karu, A. E., Linn, S. & Echols, H. (1973) 7. Howard-Flanders, P. & Theriot, L. (1962) "A method for "Purification and properties of the a-protein specified by selecting radiation-sensitive mutants of Escherichia coli," bacteriophage X: an inhibitor of the host RecBC recombina- Genetics 47, 1219-1224. tion enzyme," Proc. Nat. Acad. Sci. USA 70, 2215-2219. Downloaded by guest on October 1, 2021