JOURNAL OF BACTERIOLOGY, JUlY 1977, p. 208-213 Copyright © 1977 American Society for Microbiology

Vol. 131, No. 1 Printed in U.S.A.

Effect of Transient Lambda Prophage Induction on Ultraviolet Light Resistance and Recombination in Escherichia colil ANDREW BRAUN2* AND DEBRA GLUCK Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154

Received for publication 27 January 1977

Transient induction of prophage increases the ultraviolet light resistance of most exponentially growing Escherichia coli lysogens. Resistance is increased in

wild-type, recB, recB recC, recB recC recF, and recB recC recL hosts. No enhancement in recA lysogens was found, nor was there enhancement in stationary cultures. Enhancement was dependent upon the Xred recombination system. Transient induction also increases the genetic recombination rate in recB lysogens as measured in Hfr x F- matings.

Dark repair of ultraviolet light (UV)-induced deoxyribonucleic acid (DNA) damage in bacterial cells appears to be mediated by at least two enzymatic mechanisms (6). One of these, the excision repair mechanism, removes the damaged region and then restores the integrity of the DNA by repair replication using the complementary, undamaged, DNA strand as a template. However, when normal DNA replication passes a damaged region, a second repair system, the post-replication pathway, becomes involved. This mechanism appears to be associated with the physical transfer of the lesion to daughter strands through a recombination-dependent mechanism (4, 16). Most Escherichia coli mutants that are defective in recombination are also found to be unusually UV sensitive (8, 16). Recently, Trgovcevic and Rupp have found that a recombination system coded for by bacteriophage X, the red system, can enhance the Xray survival of some Rec- E. coli mutants, presumably by increasing the recombination proficiency of these cells (21, 22). Transient induction of recB lysogens was found to substantially increase their radioresistance. This enhanced resistance was dependent on the presence of active red system gene products. In this communication, we extend the X-ray results of Trgovcevic and Rupp to the UV response of transiently induced Rec- lysogens. In addition, we investigated the effect of transient prophage induction on genetic recom-

bination in Hfr x F- (Rec-) matings. In both cases, a red-dependent enhancement was observed.

MATERIALS AND METHODS Bacterial and bacteriophage. Strains of bacteria and phage are listed in Tables 1 and 2, respectively. Media. Cells were grown with shaking in TYM medium: 1% tryptone (Difco), 1% NaCl, 0.04% maltose, and 0.01% yeast extract. Cells were spread on plates containing on 1% tryptone, 1% NaCl, and 1.2% agar. Recombinants were selected on minimal medium A plates (3) supplemented with 0.2% glucose and the appropriate amino acids. Dilutions were in DF salts: 0.01 M KH2PO4, 0.01 M K2HPO4, 0.001 M MgSO4, and 0.0001 M CaCl2. Transient induction. Cells were grown at 34°C in TYM to a concentration of about 2 x 108 cells per ml and then shifted to 42°C. At intervals, 0.1-ml samples were removed and diluted into 10 ml of ice-cold DF salts. Samples were kept on ice until used. UV irradiation. The diluted cells were poured into a petri dish and irradiated with a germicidal bulb (General Electric G15T8). To reduce UV doses to sensitive mutants, copper wire screens were placed between the bulb and the suspension. Doses were measured with a dosimeter (Blak-Ray, Ultraviolet Products, Inc., San Gabriel, Calif.). Irradiation and all subsequent steps were carried out in subdued light to minimize photoreactivation. The cells were spread on the tryptone salt plates, incubated for 1 or 2 days at 34°C, and counted. All platings were done in duplicate, and the results were averaged. Bacterial matings. Hfr donor and recipient cells were grown to a concentration of 2 x 108 per ml in I Publication number 1149 of the Department of Bio- TYM at 34°C. They were then mixed at a ratio of 1:20 and allowed to stand at 34°C in a large flask. After chemistry, Brandeis University. 2 Present address: Department of Radiation Therapy, 30 min, the mating was interrupted by Vortex mixHarvard Medical School, Boston, MA 02115. ing in a test tube (100 by 13 mm) for 1 min. 208

TRANSIENT x INDUCTION AND UV RESISTANCE

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209

TABLE 1. Bacterial strains used Bacterial strain

Relevant genotype

UV sensitiv- Recombina-

geoyeity

no.

AB1157 N99 JC5519 N3020 N100 JC3881 JC8877 KL321 KL322 Hfr3000

sup-37 sup+ recB21 recC22 sup-37 recB21 sup+ recA sup+ recB21 (recC22) recF143 recB21 (recC22) recL152 recB21 proB str recC22 proB str Hfr Hayes

Source

tion + + -

R R S S S S S S S R

Reference

B. Bachmann M. Gottesman A. J. Clark M. Gottesman M. Gottesman A. J. Clark A. J. Clark K. B. Low K. B. Low K. B. Low

-

-

-

+

1 *a

13 * * * * * * 1

a Asterisk indicates unpublished: N99 is W3102 (reference 1), and N100 is Meselson's strain 152.

TABLE 2. Bacteriophage A mutants used Defect

Genotype

Source Aexoa

cI857 cI857red3 cI857redA324 cI857redB603

a

+

+

-

+ +

+ +

c1857redB401 c1857redC314 c1857y210 cI85708 cI857029 cI857P80 cI857P80red3 Exonuclease.

+ + + +

+ + +

-

-

RESULTS Effects of transient induction on UV sensitivity. Since the thermolability of the XcI857 mutant repressor is partially reversible (20), lysogens containing this mutation can be held at 42°C for short intervals with little reduction in their colony-forming ability. As seen in the upper curve of Fig. 1, intervals of up to 6 min at 42°C result in no decline in viability. At longer intervals survival decreases. During the period at 42°C, many early X proteins, including the red gene products, are induced (10). The lower two curves of Fig. 1 show that the UV resistance of transiently induced recB recC lysogens increases with increasing time of induction. UV induction of the prophage does not occur since the cI857 repressor mutants used in this study are ind- (20). Control experiments indicate that transient heating of nonlysogenic recB recC cells does not alter their UV sensitivity. Other control experiments indicate that transiently induced recB recC lysogens retain their increased resistance to UV for at least 3 h after induction if stored on ice.

Reference

y

+ + + + + +

+ + + +

D. D. C. C. C. C. D. D. D. D. D.

Freifelder Freifelder Radding Radding Radding Radding Freifelder Freifelder Freifelder Freifelder Freifelder

20 19 14 14 14 23

2 2

Transient induction has no detectable effect on the UV sensitivity of lysogens in stationary phase. This is in marked contrast to the induced resistance to ionizing radiation found in stationary-phase lysogens by Trgovcevic and Rupp (21, 22). In Fig. 2, the UV dose-response curve of a recB recC lysogen, induced for 8 min, is seen to be biphasic. At low UV doses, survival of induced cells is similar to that of noninduced cells. However, at higher doses, the induced cells have a substantially increased UV resistance. A trivial explanation for this biphasic response is the presence of two populations of cells in the irradiated culture. For example, the culture might consist of 80% sensitive cells (such as nonlysogens) and 20%o resistant cells (such as lysogens). This possibility was checked by selecting 20 single-cell isolates from the culture. All were found to contain lambda immunity and to be nonviable at 42°C, and all became UV resistant when induced for 8 min. Thus, the biphasic dose-response curve of induced recB recC lysogens seems to be a charac-

210

J. BACTERIOL.

BRAUN AND GLUCK

The effect of transient X induction on the UV sensitivity of a wild-type host is shown in Fig. 3. Once again, survival was increased. However, the shape of the enhanced survival curve contrasts strikingly with that observed in a 107 recB recC host (compare with Fig. 2). Rather than an initial sensitive phase, the wild-type host response begins with an initial resistant stage. At high dose, survival appears to have the 0 same slope as in uninduced cells. Since greatly 100 -0- 06 enhanced survival is not observed when an amber y mutant of X is induced (y210, Fig. 3), we that the increased survival can be asbelieve -.1 cribed to the activity of this gene product. In10 -1 duction of the y210 mutant in wild-type lysoar gens containing an amber suppressor restores the increased UV resistance (Table 3, line 3). , Lysogens of recA mutant hosts were found to i a be nearly unaffected by transient induction (Table 3, lines 21, 22). Gillen and Clark have found that at least two pathways of recombination exist in E. coli (5). The pathway of the recB recC mutants accounts for the bulk of recombination observed in wildI 16 8 12 4 0 type cells. Residual recombination activity of recB recC mutants can be eliminated by addiMINUTES AT 42 tional recF or recL mutations. Triple recB recC FIG. 1. Enhanced UV resistance of JC551!9 recB recF and recB recC recL mutants are extremely recC (XcI857) lysogen as a function of the Xtime of UV sensitive (7). As indicated in Table 3, tran0

transient induction. Techniques were as descr ibed in Materials and Methods. Symbols: (0) concen tration of viable cells after induction, left scale; (0) concentration of viable cells after induction and UV i;rradiation with 20 J/m2, left scale; (A) fractional TV survival, right scale.

teristic of these lysogens rather than a]n artifact due to a genetic inhomogeneity of the irradiated samples. Two loci, the exo and f8 genes, on the X genome are involved in phage-phage re combination. As seen in Fig. 2, the red-3 multation, which eliminates both these gene productts (18), also eliminates transient induction enhancement of UV resistance in recB recC host cl,ells. Several red mutants were examined to idetermine whether both these proteins weXre required to suppress the recB recC pheniotype. Table 3 includes a summary ofthese resu lts. No increased UV resistance was observed i]n lysogens lacking either the X exonuclease alone (redA, line 14, Table 3) or the proteinl alone (redB, lines 15 and 16, Table 3). These lresults indicate that both the exonuclease and the (8 protein are required for enhanced UV resistance. However, in contrast to the red-3 mutation, one mutant lacking both protein was found to have some increased UV resiistance (redC314, line 17, Table 3). We have no explanation for this aberrant result. is

100 red

red3i

Induced 8'

o

o

*

U Not Induced

10

..\

1

U V DOSE, JIM2

FIG. 2. Enhanced UV survival of transiently induced recB recC JC5519 (XcI857) as a function of UV dose.

TRANSIENT x INDUCTION AND UV RESISTANCE

VOL. 131, 1977

TABLE 3. Effect of transient induction on UV dose response' Host Wild type 1. AB1157 2. AB1157 3. AB1157 4. AB1157 5. N99 6. N99 recB recC or recBC 7. JC5519 8. JC5519 9. JC5519 10. N3020 11. N3020 12. N3020 13. N3020 14. N3020 15. N3020 16. N3020 17. N3020 18. KL321 19. KL321 20. KL32 recA 21. N100 22. N100 recBC recF 23. JC3881 24. JC3881 recBC recL 25. JC8877 26. JC8877

XcI857 lysogen°

Shape of enhanced dose-effect curvec

None +

y210 red3 +

.y210 None + red3 + + P80 + 029 red3 redA324 redB401 redB603 redC314 + P80 red3 P80 + P80

Shoulder Shoulder Shoulder Shoulder No shoulder Biphasic

Biphasic Biphasic Biphasic -

Slight, biphasic Biphasic Biphasic

+ red3 + red3

Biphasic

+ red3

Biphasic

211

lysogen as a recipient showed no enhanced recombination. DISCUSSION The central finding in this paper is that Xred gene products can suppress some of the phenotypic aspects of recB recC mutations in E. coli. We have presented two independent lines of evidence to support this proposition. First, the UV resistance of transiently induced lysogens is markedly increased. This increase is dependent on the presence of the X exonuclease and the ,( protein. In addition, we have shown that transient induction of X prophage can substantially increase genetic recombination in Hfr x F- crosses. This finding is consistent with that of Trgovcevic and Rupp, who found a similar relationship between transient X prophage induction and induced resistance to ionizing radiation in stationary cultures of E. coli (21, 22). Our finding of increased genetic recombination as a result of transient prophage induction is consistent with an observation by K. B. Low (personal communication) that increased recombination

a Dose-effect experiments similar to that shown in Fig. 2 are described. In all cases transient induction was for 8 min. b +, red+. c Biphasic curves are as shown in Fig. 2. -, No enhancement.

sient prophage induction reduced this sensitivity, an effect that was red dependent. Phage-induced UV resistance in recB lysogens was not dependent on X replication, as shown by the observation that resistance was present in induced 0 and P gene mutants (Table 3). Lambda replication is absent in 0 and P mutants (13). Genetic recombination after transient prophage induction. Increased red-dependent resistance of transiently induced Rec- lysogens suggests that these red functions increase the rate of recombination repair after irradiation. If this suggestion is correct, increased recombinant fornation in Hfr x F- matings should also be observed when lysogens are induced. This was tested, as seen in Fig. 4, by mating a HfrH donor with a recB (cI857P80) lysogen at 34°C and then transiently inducing the mixture. Recombinants were selected and found to increase in number with increased induction period. A similar experiment with a recB (cI857P80red3)

U V DOSE, J/M2

FIG. 3. Enhanced UV survival of transiently induced rec+ N99 (XcI857) as a function of UW dose. Open symbols: induced for 8 min; closed symbol: not induced. (0, 0), y+ lysogen; (0), 'y210 lysogen.

212

BRAUN AND GLUCK

K.

MINUTES FIG. 4. Enhanced recombination in recB KL321 (XcI857) as a function of time of transient induction. Cells were mated with Hfr3000 (Hayes) for 30 min as described in Materials and Methods. After interruption the mixture was held at 42°C for the indicated time, and portions were plated on selective medium. Proline+ recombinants per surviving recipient cells are plotted. Two independent experiments are shown: (0, *) red -P80 lysogens; (El, *) red3P80 lysogens.

is found in matings which involve the transfer of X prophage into a recB recipient. The recB survivors of the resultant zygotic induction were found to have greatly increased rates of genetic recombinant formation, presumably due to the transiently induced prophage. When the prophage was red defective, the increased recombination rate was not observed. Our results are also consistent with the observation by Mizuuchi and Fusasawa that chromosome mobilization is enhanced in Rec- merodiploids infected by x and that this enhancement is partially red dependent (12). Gillen and Clark have shown that at least two pathways of recombination exist in E. coli. (5) One, mediated by the RecB and RecC gene products (exonuclease V), accounts for about 90% of genetic recombination. A suppressor of recB recC mutations, sbcA, restores the rate of recombinant formation and UV resistance of recB recC mutants to wild-type levels. The sbcA mutation also enhances the UV resistance and recombination proficiency of triple recB recC recF and recB recC recL mutants (L. Margossian, K. McLeod, and A. J. Clark, personal communication).

J. BACTERIOL.

Thus, it appears that transient prophage induction and the sbcA mutation are analogous in their effects on UV resistance and recombination proficiency in Rec- hosts. This similarity extends to the biochemical manifestation of the sbcA mutation: the appearance of a new exonuclease, exonuclease VIII. In terms of its biochemical activity, exonuclease VIII is indistinguishable from the X exonuclease. Only in their antigenic properties do these two nucleases appear to differ (11). Indeed, Clark has suggested that the two enzymes be renamed EcoExoVIII and LamExoVIm (personal communication). Thus, it is not unexpected that the presence of the X-coded nuclease is capable of increasing the UV resistance and recombination proficiency of recB recC mutants reminicent ofsbcA. However, the requirement for the 18 protein for this enhancement is unexpected. It is possible that the sbcAmediated recombination pathway requires the presence of a ,8-like protein. Such a protein has not yet been reported. Another unexpected finding in this report is the X-dependent enhancement of UV resistance in wild-type lysogens by transient X induction. It is clear from this result and similar results with ionizing radiation by Trgovcevic and Rupp (21, 22) that, in terms of survival, wild-type E. coli does not have the optimal mechanism for DNA repair after UV or ionizing irradiation. The presence of the y gene product enhances survival by several orders of magnitude above that in uninduced wild-type lysogens. The only known activity of the fy product is the inhibition of the recB recC nuclease (exonuclease V) (17). It is difficult to suggest a mechanism for this survival enhancement based on exonuclease V inhibition since total absence of exonuclease V in recB recC mutants renders them more sensitive to UV and ionizing radiation. Trgovcevic and Rupp have suggested that this inhibition at early times of the repair sequence is crucial, whereas, at later times, the inhibition is overcome and other exonuclease V-mediated processes occur (21). Presumably, during the early postirradiation stages, uninhibited exonuclease V causes lethal lesions at damaged sites. The possibility that the y product has some other protective property unrelated to the recB recC nuclease is unlikely since there is no y-dependent enhancement of UV resistance in recB recC lysogens. Although the effect of transient prophage induction on UV sensitivity is similar to the Xray response, enhanced UV resistance has only been found in exponentially growing lysogens. Trgovcevic and Rupp found that recB lysogen

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TRANSIENT x INDUCTION AND UV RESISTANCE

resistance to X rays could be induced in both exponential- and stationary-phase cultures, whereas y gene-dependent resistance in wildtype hosts was observed only in stationary cultures (21). We can only speculate that these discrepancies with the current results are related to the different relationship of DNA replication or metabolic activity of the host to X-ray and UV damage repair. It is possible that the induced repair systems described here may be a model for the SOS repair system suggested by Radman (15). This repair system has been hypothesized to become induced when cells are irradiated with UV or treated with certain other agents that affect DNA replication or repair. Although it yields increased survival, the SOS system is presumed to be error-prone and leads to mutational alterations in the DNA. ACKNOWLEDGMENTS We wish to thank David Freifelder for providing facilities, phage strains, and general encouragement. We also wish to thank A. J. Clark and Brooks Low for communicating unpublished results and W. D. Rupp and B. Low for stimulating discussions. Supported by Public Health Service grant GM-14358 from the National Institute of General Medical Sciences, grant BMS75-03154 from the National Science Foundation, and contract no. E(11-1)-3233 from the Energy Research and Development Administration to David Freifelder.

LITERATURE CITED 1. Bachmann, B. 1972. Pedigrees of some mutant strains ofEscherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Campbell, A. 1961. Sensitive mutants of bacteriophage A. Virology 14:22-32. 3. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia coli requiring methionine or vitamin B,2. J. Bacteriol. 60:17-28. 4. Ganesan, A. K. 1974. Persistence of pyrimidine dimers during post-replication repair in ultraviolet light irradiated Escherichia coli K12. J. Mol. Biol. 87:103119. 5. Gillen, J. R., and A. J. Clark. 1974. The Rec E pathway of bacterial recombination, p. 123-136. In R. F. Grell (ed.), Mechanisms in recombination. Plenum Press, New York.

6. Grossman, L., A. G. Braun, R. Feldberg, and I. Mahler. 1975. Enzymatic repair of DNA. Annu. Rev. Biochem. 44:19 43. 7. Horii, Z., and A. J. Clark. 1973. Genetic analysis of the Rec F pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J. Mol. Biol. 80:327-344. 8. Howard-Flanders, P., and R. P. Boyce. 1966. DNA repair and genetic recombination: studies on mutants

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of Escherichia coli defective in these processes. Radiat. Res. 6(Suppl.):156-184. Howard-Flanders, P., and L. Theriot. 1968. Mutants of Escherichia coli K12 defective in DNA repair in genetic recombination. Genetics 53:1137-1150. Kourisky, P., M. F. Bourguignon, and F. Gross. 1971. Kinetics of viral transcription after induction of prophage, p. 647-666. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Kushner, S. R., H. Nagaishi, and A. J. Clark. 1974. Isolation of exonuclease VIII: the enzyme associated with the sbc A indirect suppressor. Proc. Natl. Acad. Sci. U.S.A. 71:3593-3597. Mizuuchi, K., and T. Fusasawa. 1969. Chromosome mobilization in rec-merodiploids of Escherichia coli K12 following infection with bacteriophage A. Virology 39:467-481. Ogawa, T., and J. Tomizawa. 1968. Replication of bacteriophage DNA. I. Replication of DNA of lambda phage defective in early functions. J. Mol. Biol. 38:217-225. Radding, C. M. 1970. The role of exonuclease and , protein of bacteriophage X in genetic recombination. I. Effects of red mutants on protein structure. J. Mol. Biol. 52:491-500. Radman, M. 1975. SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis, p. 355-367. In P. C. Hanawalt and R. B. Setlow (ed.), Molecular mechanisms for repair of DNA, part A. Plenum Press, New York. Rupp, W. D., C. E. Wilde III, D. L. Reno, and P. Howard-Flanders. 1971. Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J. Mol. Biol. 61:25-44. Sakaki, Y., A. E. Karu, S. Linn, and H. Echols. 1973. Purification and properties of the X protein specified by bacteriophage X in inhibitor of the host rec BC recombination enzyme. Proc. Natl. Acad. Sci. U.S.A.

70:2215-2219. 18. Shulman, M. J., L. M. Hallick, H. Echols, and E. R. Signer. 1970. Properties of recombination-deficient mutants of bacteriophage lambda. J. Mol. Biol. 52:501-520. 19. Signer, E. R., and J. Weil. 1968. Recombination in bacteriophage. I. Mutants deficient in general recombination. J. Mol. Biol. 34:261-271. 20. Sussman, R., and F. Jacob. 1962. Sur un systeme de repression thermosensible chez le bacteriphage d'Escherichia coli. C. R. Acad. Sci. Paris 254:15171519. 21. Trgovcevic, Z., and W. D. RuW. 1974. Interaction of bacterial and lambda phage recombination systems in the X-ray sensitivity of Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 71:503-506. 22. Trgovcevic, Z., and W. D. Rupp. 1975. Lambda bacteriophage gene products and X-ray sensitivity ofEscherichia coli: comparison of red-dependent and gamdependent radioresistance. J. Bacteriol. 123:212-221. 23. Zisler, J., E. Signer, and F. Schaefer. 1971. The role of recombination in growth of bacteriophage lambda. I. The gamma gene, p. 455-468. In A. D. Hershey (ed.), The bacteriophage lambda. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.