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Induction of error-free DNA repair in Escherichia coli by thiamine deprivation
Mutation Research, 243 (1990) 165-171 Elsevier
165
MUTLET 0310
Induction of error-flee DNA repair in Escherichia coli by thiamine deprivation Nishi Sharma and Peter S. Fitt Department of Biochemistry, University of Ottawa, Ottawa, Ont. KIN 9B4 (Canada) (Accepted 10 September 1989)
Keywords: Stress response, inducible; DNA repair, error-free; Thiamine-deficient medium, E. coil
Summary Incubation of Escherichia coli AB 1157 in a thiamine-deficient medium causes a large, time-dependent increase in resistance to UV-radiation (254 nm) and a fall in its UV-induced mutation frequency to histidine p r o t o t r o p h y which are abolished in its uvrA mutant, but only delayed in Ion - and r e c A - cells. The response o f the lexA3 mutant resembles that of the parental cells. These effects are very similar to those we have shown to be induced by heat shock and are clearly due to an error-free, DNA-excision repair-dependent process. They may represent a general response to non-mutagenic stress in these cells.
Recent studies in this laboratory (Fitt and Sharma, 1989; Pardasani and Fitt, 1989a,b; Pardasani et al., 1989) have shown that heat shock leads to a large increase in the resistance of two strains of Escherichia coli K12 (AB1157 and JEI011) to short wavelength (254 nm) ultraviolet light (UV). This effect was dependent on protein synthesis, occurred in conditions where growth of the cells was faster than at the control temperature (Pardasani and Fitt, 1989a) and was accompanied by a large decrease in the UV-induced mutation frequency (Fitt and Sharma, 1989; Pardasani et al., 1989). It therefore appears to be caused by induction of an error-free DNA-repair process. In contrast, wildCorrespondence: Dr. P.S. Fitt, Department of Biochemistry, University of Ottawa, Ont. KIN 9B4 (Canada).
type E. coli B became more sensitive to UV after a similar treatment and it was suggested that this was due to its Ion- character (Pardasani and Fitt, 1989a). Subsequent studies with several mutants of E. coli AB1157 (Pardasani and Fitt, 1989b) confirmed that the Ion mutation abolished the effect and showed that the latter was also dependent on the uvrA, uvrB and recA genes. The UV resistance of uvrC and l e x A l ( I n d - ) mutants increased slightly after heat shock, but the effect was much less than observed with the parental strain. Two inducible pathways of D N A repair, the adaptive and SOS responses, have been studied in considerable detail (Friedberg, 1985; Walker, 1984, 1985). The former is induced by alkylation of D N A and is error-free, whereas the SOS response is elicited by various DNA-damaging treatments or
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166 interference with DNA replication (e.g., by thymine deprivation), involves the expression of more than 17 genes, and is thought to be the major cause of mutations in E. coll. In agreement with these findings, incubation of E. coli JE1011, a thymine auxotroph, in growth medium lacking thymine led to increases in both its sensitivity to UV-irradiation and in its UV-induced mutation frequency, in the latter case as would be expected if the SOS pathway were induced (Fitt and Sharma, 1989). However, when the cells were deprived of thiamine (vitamin B~), which they also require, the effect was similar to that of heat shock, so that a decrease in their sensitivity to UV was observed, accompanied by a fall in their UV-induced mutation frequency. In the present paper, we show that thiamine deprivation has a similar effect on E. coli AB1157 to what we have reported for E. coli JE1011. A study of several DNA-repair mutants of AB1157 has confirmed that the characteristics of the pathway induced by vitamin deprivation are very similar, possibly identical, to those observed for heat-induced DNA repair. The results show that the effects are caused by induction of an excisiondependent process that is clearly distinguishable from both the recombinational and SOS-repair systems. Further, error-free DNA repair was stimulated without any effect on the thermal resistance of E. coil AB1157 or any of the mutants studied. Materials and methods
Bacterial strains and culture. The bacterial strains used were obtained from the E. coli Genetic Stock Center, Yale University, New Haven, CN, through the courtesy of Dr. B.J. Bachmann and were the following derivatives of E. coli K12: (i) ABl157 (CGSC 1157), genotype: F - , argE3, hisG4, leuB6, proA2, thr-l, thi-1, ara-14, galK2, kdgK51, lacY1, mtl-1, xyl-5, mgl-51, rfbD1, supE44, rac-, rpsL31, tsx-33; (ii) AB1886 (CGSC 1886), the uvrA6 mutant of ABl157; (iii) AB1899 (CGSC 1899), the Ion mutant of ABl157; (iv) AB2463 (CGSC 2463), the recA13 mutant of
AB1157; (v) DM49 (CGSC 6367) the lexA3(Ind-) mutant of AB1157. Cultures were grown at 30°C in synthetic medium as previously described (Fitt and Sharma, 1989).
Determination of survival and mutation frequency. Survival and mutation frequency were measured by plating on the defined medium supplemented with agar, with the exception that the concentration of histidine was reduced to 107o(w/v) of its normal value (Fitt and Sharma, 1989; Pardasani et al., 1989; Witkin, 1974). Survival was determined by plating no more than 100-200 cells per dish, using appropriate dilutions in sterile, 0.9°7o (w/v) saline, whereas the mutation frequency was obtained by plating about 10s cells per dish (Demerec and Cahn, 1953; Fitt and Sharma, 1989; Witkin, 1974). The dishes were incubated at 37°C and colonies were counted after 1-2 days. Thiamine deprivation. Exponential phase cells were subjected to thiamine deprivation as described by Fitt and Sharma (1989). A fresh culture in complete medium was started using a 15070 (v/v) inoculum of an overnight culture, and grown with vigorous shaking to an A66Onm of 1.0 (about 3 h). The culture was divided into control and experimental samples and both were centrifuged at 2000 gmax for 8 min. The control pellet was resuspended in sterile, non-nutrient buffer, pH 7.0 (Fitt and Sharma, 1989) to an A66Onm of 1.0 (about 109 cells/ml). The experimental sample was resuspended to an A660nm of 1.0 in culture medium lacking thiamine and incubation was continued at 30°C with vigorous aeration for the appropriate time. The cells were then harvested and resuspended in non-nutrient dilution buffer as described for the control samples. The final suspensions of control or experimental cells in dilution buffer were irradiated immediately, and survival and mutation frequency were determined. Ultraviolet irradiation. Samples (10 ml) of the appropriate suspensions in non-nutrient dilution buffer containing about 109 cells/ml were it-
167 radiated with a germicidal lamp (254 nm) in uncovered, sterile, glass petri dishes (10 cm diam.), with gentle swirling on an orbital shaker. Doses were measured with a Blackray ultraviolet meter and survival and mutation frequency were determined as described above. All experiments were performed at least twice. Results
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Fig. 1. Effect of (a) thiamine deprivation and (b) heat shock on UV resistance ( ) and induced mutation frequency to histidine prototrophy (..... ) in Escherichia coli ABI157 (see Materials and Methods). (a) Control, O; 90 rain deprivation, ©; 3 h deprivation, Y]; 24 h deprivation, ~,. (b) 30°C control, O; 48°C/45 min, ©.
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discussion
The effect of thiamine deprivation on the resistance of Escherichia coli AB1157 to UV was first studied in the conditions previously shown to induce a large decrease in both the UV sensitivity and UV-induced mutation frequency o f E. coli JE1011 (Fitt and Sharma, 1989). Fig. la shows that the same effect was obtained with E. coli AB1157 and that maximum resistance to the radiation and reduction in the induced mutation frequency to histidine prototrophy were observed after 3 h of thiamine deprivation. Heat shock at 48°C for 45 min gave an effect similar to 90 min of thiamine deprivation (Fig. lb). More prolonged heat shock could not be tried, since the cells begin to die after 1 h at this temperature. In separate experiments (data not shown), it was found that addition of
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Fig. 2. Effect (a) thiamine deprivation and (b) heat shock on UV resistance and induced mutation frequencyin E. coil AB1899, a Ion- mutant of E. coli ABlI57. Methods and symbols as in Fig. 1. chloramphenicol (500 ~g/ml) to the medium halved the increase in UV resistance developed in E. coli AB1157 during 3 h of incubation in the absence of thiamine. The effect therefore depends on protein synthesis, as suggested by its time-dependence and in agreement with the results of Pardasani and Fitt (1989a), who showed that thermal induction of UV resistance in E. coil JE1011 required protein synthesis a n d / o r cell growth. Similar experiments were then undertaken with the Ion, u v r A , recA and l e x A ( I n d - ) mutants o f E. coli ABl157, since Pardasani and Fitt (1989b) and Pardasani et al. (1989) have shown that 48°C heat shock fails to increase the resistance of the first three strains to UV radiation, while the effect was considerably reduced in the case of the lexA1 mutant. The results of thiamine deprivation and heat shock experiments with the Ion - cells are shown in Fig. 2a, b. Incubation of the bacteria in minimal medium lacking thiamine caused an increase in UV resistance and a decrease in induced mutation frequency similar (Fig. 2a) to those observed with the parental strain (Fig. la), but the changes occurred more slowly. No effect was seen after 90 min in the thiamine-deficient medium and after 3 h the changes in radiation sensitivities and mutation frequency approached those given by ABI157 in 90 min, while the full effect was only seen in the samples held for 24 h in the absence of the vitamin.
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Fig. 3. Effect of thiamine deprivation on UV resistance and induced mutation frequency in E. coli AB1886, a u v r A - mutant of E. coli AB1157. Methods and symbols as in Fig. la. (Note: 3 h samples were not studied in this case.)
In the case of heat shock at 48°C for 45 min, no change in UV resistance occurred (Fig. 2b), in agreement with the results reported for the I o n cells grown in complex medium (Pardasani et al., 1989). However, a small decrease in the induced mutation frequency was always seen after heat shock (Fig. 2b). These results suggest that the failure of UV resistance to increase after heat shock was due to the impossibility of holding the cells at 48°C for longer than 45 min without loss of viability, a limitation that did not affect the thiaminedeprivation studies. Thus, although the lon gene product is clearly necessary for rapid induction of the error-free DNA-repair process responsible for these effects, it does not appear to play and essential role. The u v r A and r e c A mutants of E. coli AB1157 were studied next, since Pardasani and Fitt (1989b) also found that their resistance to UV was unchanged by heat shock. Thiamine deprivation failed to change the sensitivity of the u v r A mutant to UV, even after 24 h
of incubation (Fig. 3). A 20-30°7o decrease in mutation frequency was always observed after 90 min for some as yet unknown reason, but after 24 h in the absence of the vitamin the cells had the same induced mutation frequency as the control. The error-free system induced by thiamine deprivation is therefore dependent on excision repair, in agreement with our previous conclusion based on the effect of heat shock (Pardasani and Fitt, 1989). The results obtained with the r e c A mutant of E. coli AB1157 were unexpected. Pardasani and Fitt (1989b) showed that heat shock failed to increase the resistance of these cells to UV. This appeared to agree in part with the conclusions of Mitchel and Morrison (1983) based on their studies of the thermal induction of UV resistance in yeast, which they found to be dependent on recombinational repair and independent of DNA-excision repair. However, thiamine deprivation of the r e c bacteria (Fig. 4) caused an effect similar to that observed with the Ion - cells (Fig. 2): although little change in resistance to UV was observed after 90 min, there was a progressive increase to the maxi m u m resistance over 24 h, showing that RecA is
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Fig. 5. Effect of (a) thiamine deprivation and (b) heat shock on UV resistance and induced mutation frequency in E. coli DM49, a l e x A - ( I n d - ) m u t a n t of E. coli ABI157. Methods and symbols as in Fig. 1.
not essential for the process although necessary for its rapid induction. The mechanism responsible for these effects therefore differs fundamentally from the recombination-dependent increase in UV resistance seen in yeast. The r e c A - strain has a low natural or induced mutation frequency (Friedberg, 1985), owing to its inability to activate SOS repair, and thiamine deprivation had no detectable effect on it. Finally, the effect of the l e x A ( I n d - ) mutation on the induction of UV resistance by thiamine deficiency was studied. Pardasani and Fitt (1989b) used the l e x A l ( I n d - ) mutant, which has slightly different amino acid requirements than E. coli AB1157, in their work on the effect of heat shock, but we found that it grew very poorly in the appropriate defined medium. We therefore studied the l e x A 3 ( I n d - ) mutant (Mount et al., 1972), which grows as well as the parental strain, in addition to having identical nutritional requirements. The results in Fig. 5a show that the mutant developed resistance to UV during thiamine deprivation to give almost the same effect as the parental strain after 3 h and 24 h. However, the 90-min samples were consistently more sensitive to UV than the untreated controls, a result for which no explanation is available at present, but which may be related to the abnormally rapid degradation o f damaged D N A in these cells after they have
been exposed to UV (Mount et al., 1972). This nonspecific DNA degradation should be prevented once the repair system is induced sufficiently (after 3 h and 24 h o f thiamine deprivation) to permit rapid removal of the pyrimidine dimers. Despite this anomaly, it is clear that the lexA3(Ind - ) mutation does not prevent the development of UV resistance, although it may delay it somewhat. Heat shock (Fig. 5b) gave essentially the same results as previously reported for the lexA1 mutant (Pardasani and Fitt, 1989), with an incomplete induction of the repair process being observed, presumably as a result of the limit on the duration o f the treatment. The l e x A - cells have a low natural or induced mutation frequency, as in the case of the r e c A - strain, owing to their lack of SOS repair, and as expected neither thiamine deprivation nor heat shock had any measurable effect on it. We also studied the effect of thiamine deprivation on the ability of E. coli ABl157 and its Ion, uvrA, recA and lexA mutants to withstand challenge for 15 min by a lethal temperature (55°C). The experiments were performed as previously described (Pardasani and Fitt, 1989b), but thiamine deprivation did not affect the thermotolerance of any of these strains. We have previously shown (Pardasani and Fitt, 1989a) that induction of UV resistance in E. coli JE1011 by heat shock takes place without any increase in thermal resistance and the present results confirm that the systems involved are independent. However, it is known that induction of the heat shock regulon can occur without production of thermotolerance (VanBogelen et al., 1987), while a mutation in the dnaK gene, whose product is a major heat shock protein of E. coli, does not prevent the thermal induction of heat resistance (Ramsey, 1988), so the relationship between the heat shock regulon and the inducible, error-free DNA-repair system we have studied remains uncertain. It can be seen from these studies that thiamine deprivation induces an error-free DNA-repair process in E. coli ABI157, leading to a large increase in the resistance of the bacteria to UV irradiation and a corresponding decrease in their UV-induced
170
mutation frequency. The process is dependent on excision repair involving the UvrABC endonuclease and presumably involves an increase in the activity of this enzyme. Neither the Ion nor the recA gene products are essential, but both are required for rapid induction of the system. Mutations in the lexA gene that render its product resistant to the RecA protease do not prevent induction of this error-free DNA-repair pathway, proving that it is not subject to r e c A - l e x A control. In most respects, this system appears very similar to the one we have previously shown to be induced by heat shock. The differences between them arise mainly when long term thiamine deprivation is studied and almost certainly reflect the need with E. coli ABl157 for a heat shock temperature of 48°C (Pardasani et al., 1989), which cannot be prolonged beyond 45 rain without killing the cells so that only effects that develop relatively quickly can be observed. The simplest hypothesis is that both types of stress lead to the induction of a single regulon or gene network (Gottesman, 1984) involving the uvrA, B and C genes. The fact that the uvrA and B genes are part of the SOS regulon appears to be a complicating factor, but it is known that the uvrB gene, at least, is transcribed from two different promoters, only one of which is regulated by LexA (Sancar et al., 1982). The stress-induced increase in UvrABC endonuclease activity in the parental strain would permit a more efficient removal of pyrimidine dimers from its DNA leading to an increased cell survival, together with a decreased mutation frequency owing to the elimination o f the signal for SOS regulon induction. In the u v r A - strain, excision repair is absent and non-inducible, so stress would not increase survival or change the high natural and induced mutation frequencies of these cells. A non-essential, enhancing role for the Ion and recA gene products is harder to envisage. One possibility is that the optimum rate of increase in UvrABC endonuclease activity requires stabilization of the enzyme by one gene product and stabilization of one or more of the UvrABC mRNAs by the seco~id. Absence of the appropriate protein would permit slower accumulation of the
enzyme at its normal rate of synthesis in the presence of the stabilizer in one case or at the higher induced rate in the absence of stabilization in the other. Our earlier suggestion (Pardasani et al., 1989) that the Lon protease might degrade a repressor controlling the error-free repair system seems less likely on the basis of our present evidence that it is not essential to the process. Induction of the error-free system by thiamine deprivation occurs without a simultaneous increase in thermotolerance and we have previously shown (Pardasani and Fitt, 1989a) that this is also true of its induction by heat shock in E. coli JEl011. Since the heat shock regulon can be induced without affecting thermotolerance (VanBogelen et al., 1987), it would be of interest to study the effect of other known inducers of the heat-shock response on the system we have described. More generally, it seems possible that the induction of error-free DNA repair may be a relatively non-specific response to a wide variety of non-mutagenic chemical, nutritional or physical stresses.
Acknowledgement We thank the Natural Science and Engineering Research Council of Canada for financial support.
References Demerec, C., and E. Cahn (1953) Studies of mutability in nutritionally deficient strains of Escherichia coli, J. Bacteriol., 65, 27-36. Fitt, P.S., and N. Sharma (1989) Induction of error-free DNA repair in Escherichia coli by heat shock or nutritional stress, Curt. Microbiol., in press. Friedberg, E.C. (1985) DNA Repair, Freeman, New York. Gottesman, S. (1984) Bacterial regulation: global regulatory networks, Annu. Rev. Genet., 18,415-4~1. Mitchel, R.E.J., and D.P. Morrison (1983) Heat-shock induction of ultraviolet resistance in Saccharomyces cerevisiae, Radiat. Res., 96, 95-99. Mount, D.W., K.B. Low and S.T. Edmiston (1972) Dominant mutations (lex) in Escherichia coli K-12 which affect radiation sensitivity and frequency of ultraviolet light-induced mutations, J. Bacteriol., 112, 886-893.
171 Pardasani, D., and P.S. Fitt (1989a) Strain-dependent induction by heat shock of resistance to ultraviolet light in Escherichia coli, Curr. Microbiol., 18, 99-103. Pardasani, D., and P.S. Fitt (1989b) Study of the effect of mutations in DNA repair genes on the thermal induction of error-free repair in Escherichia coli, Curr. Microbiol., in press. Pardasani, D., N. Sharma and P.S. Fitt (1989) Dependence on the Ion gene of the thermal induction of resistance to ultraviolet light in Escherichia coli, Curr. Microbiol., in press. Ramsey, N. (1988) A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance, Mol. Gen. Genet., 211, 332-334. Sancar, G.B., A. Sancar, J.W. Little and W.D. Rupp (1982)
The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters. Cell., 28, 523-530. VanBogelen, R.A., M.A. Acton and F.C. Neidhardt (1987) Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli, Genes Develop., 1,525-531. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Walker, G.C. (1985) Inducible DNA repair systems, Annu. Rev. Biochem., 54, 425-457. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907.
Communicated by R.H. Haynes
165
MUTLET 0310
Induction of error-flee DNA repair in Escherichia coli by thiamine deprivation Nishi Sharma and Peter S. Fitt Department of Biochemistry, University of Ottawa, Ottawa, Ont. KIN 9B4 (Canada) (Accepted 10 September 1989)
Keywords: Stress response, inducible; DNA repair, error-free; Thiamine-deficient medium, E. coil
Summary Incubation of Escherichia coli AB 1157 in a thiamine-deficient medium causes a large, time-dependent increase in resistance to UV-radiation (254 nm) and a fall in its UV-induced mutation frequency to histidine p r o t o t r o p h y which are abolished in its uvrA mutant, but only delayed in Ion - and r e c A - cells. The response o f the lexA3 mutant resembles that of the parental cells. These effects are very similar to those we have shown to be induced by heat shock and are clearly due to an error-free, DNA-excision repair-dependent process. They may represent a general response to non-mutagenic stress in these cells.
Recent studies in this laboratory (Fitt and Sharma, 1989; Pardasani and Fitt, 1989a,b; Pardasani et al., 1989) have shown that heat shock leads to a large increase in the resistance of two strains of Escherichia coli K12 (AB1157 and JEI011) to short wavelength (254 nm) ultraviolet light (UV). This effect was dependent on protein synthesis, occurred in conditions where growth of the cells was faster than at the control temperature (Pardasani and Fitt, 1989a) and was accompanied by a large decrease in the UV-induced mutation frequency (Fitt and Sharma, 1989; Pardasani et al., 1989). It therefore appears to be caused by induction of an error-free DNA-repair process. In contrast, wildCorrespondence: Dr. P.S. Fitt, Department of Biochemistry, University of Ottawa, Ont. KIN 9B4 (Canada).
type E. coli B became more sensitive to UV after a similar treatment and it was suggested that this was due to its Ion- character (Pardasani and Fitt, 1989a). Subsequent studies with several mutants of E. coli AB1157 (Pardasani and Fitt, 1989b) confirmed that the Ion mutation abolished the effect and showed that the latter was also dependent on the uvrA, uvrB and recA genes. The UV resistance of uvrC and l e x A l ( I n d - ) mutants increased slightly after heat shock, but the effect was much less than observed with the parental strain. Two inducible pathways of D N A repair, the adaptive and SOS responses, have been studied in considerable detail (Friedberg, 1985; Walker, 1984, 1985). The former is induced by alkylation of D N A and is error-free, whereas the SOS response is elicited by various DNA-damaging treatments or
0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166 interference with DNA replication (e.g., by thymine deprivation), involves the expression of more than 17 genes, and is thought to be the major cause of mutations in E. coll. In agreement with these findings, incubation of E. coli JE1011, a thymine auxotroph, in growth medium lacking thymine led to increases in both its sensitivity to UV-irradiation and in its UV-induced mutation frequency, in the latter case as would be expected if the SOS pathway were induced (Fitt and Sharma, 1989). However, when the cells were deprived of thiamine (vitamin B~), which they also require, the effect was similar to that of heat shock, so that a decrease in their sensitivity to UV was observed, accompanied by a fall in their UV-induced mutation frequency. In the present paper, we show that thiamine deprivation has a similar effect on E. coli AB1157 to what we have reported for E. coli JE1011. A study of several DNA-repair mutants of AB1157 has confirmed that the characteristics of the pathway induced by vitamin deprivation are very similar, possibly identical, to those observed for heat-induced DNA repair. The results show that the effects are caused by induction of an excisiondependent process that is clearly distinguishable from both the recombinational and SOS-repair systems. Further, error-free DNA repair was stimulated without any effect on the thermal resistance of E. coil AB1157 or any of the mutants studied. Materials and methods
Bacterial strains and culture. The bacterial strains used were obtained from the E. coli Genetic Stock Center, Yale University, New Haven, CN, through the courtesy of Dr. B.J. Bachmann and were the following derivatives of E. coli K12: (i) ABl157 (CGSC 1157), genotype: F - , argE3, hisG4, leuB6, proA2, thr-l, thi-1, ara-14, galK2, kdgK51, lacY1, mtl-1, xyl-5, mgl-51, rfbD1, supE44, rac-, rpsL31, tsx-33; (ii) AB1886 (CGSC 1886), the uvrA6 mutant of ABl157; (iii) AB1899 (CGSC 1899), the Ion mutant of ABl157; (iv) AB2463 (CGSC 2463), the recA13 mutant of
AB1157; (v) DM49 (CGSC 6367) the lexA3(Ind-) mutant of AB1157. Cultures were grown at 30°C in synthetic medium as previously described (Fitt and Sharma, 1989).
Determination of survival and mutation frequency. Survival and mutation frequency were measured by plating on the defined medium supplemented with agar, with the exception that the concentration of histidine was reduced to 107o(w/v) of its normal value (Fitt and Sharma, 1989; Pardasani et al., 1989; Witkin, 1974). Survival was determined by plating no more than 100-200 cells per dish, using appropriate dilutions in sterile, 0.9°7o (w/v) saline, whereas the mutation frequency was obtained by plating about 10s cells per dish (Demerec and Cahn, 1953; Fitt and Sharma, 1989; Witkin, 1974). The dishes were incubated at 37°C and colonies were counted after 1-2 days. Thiamine deprivation. Exponential phase cells were subjected to thiamine deprivation as described by Fitt and Sharma (1989). A fresh culture in complete medium was started using a 15070 (v/v) inoculum of an overnight culture, and grown with vigorous shaking to an A66Onm of 1.0 (about 3 h). The culture was divided into control and experimental samples and both were centrifuged at 2000 gmax for 8 min. The control pellet was resuspended in sterile, non-nutrient buffer, pH 7.0 (Fitt and Sharma, 1989) to an A66Onm of 1.0 (about 109 cells/ml). The experimental sample was resuspended to an A660nm of 1.0 in culture medium lacking thiamine and incubation was continued at 30°C with vigorous aeration for the appropriate time. The cells were then harvested and resuspended in non-nutrient dilution buffer as described for the control samples. The final suspensions of control or experimental cells in dilution buffer were irradiated immediately, and survival and mutation frequency were determined. Ultraviolet irradiation. Samples (10 ml) of the appropriate suspensions in non-nutrient dilution buffer containing about 109 cells/ml were it-
167 radiated with a germicidal lamp (254 nm) in uncovered, sterile, glass petri dishes (10 cm diam.), with gentle swirling on an orbital shaker. Doses were measured with a Blackray ultraviolet meter and survival and mutation frequency were determined as described above. All experiments were performed at least twice. Results
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Fig. 1. Effect of (a) thiamine deprivation and (b) heat shock on UV resistance ( ) and induced mutation frequency to histidine prototrophy (..... ) in Escherichia coli ABI157 (see Materials and Methods). (a) Control, O; 90 rain deprivation, ©; 3 h deprivation, Y]; 24 h deprivation, ~,. (b) 30°C control, O; 48°C/45 min, ©.
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discussion
The effect of thiamine deprivation on the resistance of Escherichia coli AB1157 to UV was first studied in the conditions previously shown to induce a large decrease in both the UV sensitivity and UV-induced mutation frequency o f E. coli JE1011 (Fitt and Sharma, 1989). Fig. la shows that the same effect was obtained with E. coli AB1157 and that maximum resistance to the radiation and reduction in the induced mutation frequency to histidine prototrophy were observed after 3 h of thiamine deprivation. Heat shock at 48°C for 45 min gave an effect similar to 90 min of thiamine deprivation (Fig. lb). More prolonged heat shock could not be tried, since the cells begin to die after 1 h at this temperature. In separate experiments (data not shown), it was found that addition of
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Fig. 2. Effect (a) thiamine deprivation and (b) heat shock on UV resistance and induced mutation frequencyin E. coil AB1899, a Ion- mutant of E. coli ABlI57. Methods and symbols as in Fig. 1. chloramphenicol (500 ~g/ml) to the medium halved the increase in UV resistance developed in E. coli AB1157 during 3 h of incubation in the absence of thiamine. The effect therefore depends on protein synthesis, as suggested by its time-dependence and in agreement with the results of Pardasani and Fitt (1989a), who showed that thermal induction of UV resistance in E. coil JE1011 required protein synthesis a n d / o r cell growth. Similar experiments were then undertaken with the Ion, u v r A , recA and l e x A ( I n d - ) mutants o f E. coli ABl157, since Pardasani and Fitt (1989b) and Pardasani et al. (1989) have shown that 48°C heat shock fails to increase the resistance of the first three strains to UV radiation, while the effect was considerably reduced in the case of the lexA1 mutant. The results of thiamine deprivation and heat shock experiments with the Ion - cells are shown in Fig. 2a, b. Incubation of the bacteria in minimal medium lacking thiamine caused an increase in UV resistance and a decrease in induced mutation frequency similar (Fig. 2a) to those observed with the parental strain (Fig. la), but the changes occurred more slowly. No effect was seen after 90 min in the thiamine-deficient medium and after 3 h the changes in radiation sensitivities and mutation frequency approached those given by ABI157 in 90 min, while the full effect was only seen in the samples held for 24 h in the absence of the vitamin.
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Fig. 3. Effect of thiamine deprivation on UV resistance and induced mutation frequency in E. coli AB1886, a u v r A - mutant of E. coli AB1157. Methods and symbols as in Fig. la. (Note: 3 h samples were not studied in this case.)
In the case of heat shock at 48°C for 45 min, no change in UV resistance occurred (Fig. 2b), in agreement with the results reported for the I o n cells grown in complex medium (Pardasani et al., 1989). However, a small decrease in the induced mutation frequency was always seen after heat shock (Fig. 2b). These results suggest that the failure of UV resistance to increase after heat shock was due to the impossibility of holding the cells at 48°C for longer than 45 min without loss of viability, a limitation that did not affect the thiaminedeprivation studies. Thus, although the lon gene product is clearly necessary for rapid induction of the error-free DNA-repair process responsible for these effects, it does not appear to play and essential role. The u v r A and r e c A mutants of E. coli AB1157 were studied next, since Pardasani and Fitt (1989b) also found that their resistance to UV was unchanged by heat shock. Thiamine deprivation failed to change the sensitivity of the u v r A mutant to UV, even after 24 h
of incubation (Fig. 3). A 20-30°7o decrease in mutation frequency was always observed after 90 min for some as yet unknown reason, but after 24 h in the absence of the vitamin the cells had the same induced mutation frequency as the control. The error-free system induced by thiamine deprivation is therefore dependent on excision repair, in agreement with our previous conclusion based on the effect of heat shock (Pardasani and Fitt, 1989). The results obtained with the r e c A mutant of E. coli AB1157 were unexpected. Pardasani and Fitt (1989b) showed that heat shock failed to increase the resistance of these cells to UV. This appeared to agree in part with the conclusions of Mitchel and Morrison (1983) based on their studies of the thermal induction of UV resistance in yeast, which they found to be dependent on recombinational repair and independent of DNA-excision repair. However, thiamine deprivation of the r e c bacteria (Fig. 4) caused an effect similar to that observed with the Ion - cells (Fig. 2): although little change in resistance to UV was observed after 90 min, there was a progressive increase to the maxi m u m resistance over 24 h, showing that RecA is
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Fig. 4. Effect of thiamine deprivation on UV resistance and induced mutation frequency in E. coli AB2463, a r e c A - mutant of E. coli AB1157. Methods and symbols as in Fig. la.
169 20
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Fig. 5. Effect of (a) thiamine deprivation and (b) heat shock on UV resistance and induced mutation frequency in E. coli DM49, a l e x A - ( I n d - ) m u t a n t of E. coli ABI157. Methods and symbols as in Fig. 1.
not essential for the process although necessary for its rapid induction. The mechanism responsible for these effects therefore differs fundamentally from the recombination-dependent increase in UV resistance seen in yeast. The r e c A - strain has a low natural or induced mutation frequency (Friedberg, 1985), owing to its inability to activate SOS repair, and thiamine deprivation had no detectable effect on it. Finally, the effect of the l e x A ( I n d - ) mutation on the induction of UV resistance by thiamine deficiency was studied. Pardasani and Fitt (1989b) used the l e x A l ( I n d - ) mutant, which has slightly different amino acid requirements than E. coli AB1157, in their work on the effect of heat shock, but we found that it grew very poorly in the appropriate defined medium. We therefore studied the l e x A 3 ( I n d - ) mutant (Mount et al., 1972), which grows as well as the parental strain, in addition to having identical nutritional requirements. The results in Fig. 5a show that the mutant developed resistance to UV during thiamine deprivation to give almost the same effect as the parental strain after 3 h and 24 h. However, the 90-min samples were consistently more sensitive to UV than the untreated controls, a result for which no explanation is available at present, but which may be related to the abnormally rapid degradation o f damaged D N A in these cells after they have
been exposed to UV (Mount et al., 1972). This nonspecific DNA degradation should be prevented once the repair system is induced sufficiently (after 3 h and 24 h o f thiamine deprivation) to permit rapid removal of the pyrimidine dimers. Despite this anomaly, it is clear that the lexA3(Ind - ) mutation does not prevent the development of UV resistance, although it may delay it somewhat. Heat shock (Fig. 5b) gave essentially the same results as previously reported for the lexA1 mutant (Pardasani and Fitt, 1989), with an incomplete induction of the repair process being observed, presumably as a result of the limit on the duration o f the treatment. The l e x A - cells have a low natural or induced mutation frequency, as in the case of the r e c A - strain, owing to their lack of SOS repair, and as expected neither thiamine deprivation nor heat shock had any measurable effect on it. We also studied the effect of thiamine deprivation on the ability of E. coli ABl157 and its Ion, uvrA, recA and lexA mutants to withstand challenge for 15 min by a lethal temperature (55°C). The experiments were performed as previously described (Pardasani and Fitt, 1989b), but thiamine deprivation did not affect the thermotolerance of any of these strains. We have previously shown (Pardasani and Fitt, 1989a) that induction of UV resistance in E. coli JE1011 by heat shock takes place without any increase in thermal resistance and the present results confirm that the systems involved are independent. However, it is known that induction of the heat shock regulon can occur without production of thermotolerance (VanBogelen et al., 1987), while a mutation in the dnaK gene, whose product is a major heat shock protein of E. coli, does not prevent the thermal induction of heat resistance (Ramsey, 1988), so the relationship between the heat shock regulon and the inducible, error-free DNA-repair system we have studied remains uncertain. It can be seen from these studies that thiamine deprivation induces an error-free DNA-repair process in E. coli ABI157, leading to a large increase in the resistance of the bacteria to UV irradiation and a corresponding decrease in their UV-induced
170
mutation frequency. The process is dependent on excision repair involving the UvrABC endonuclease and presumably involves an increase in the activity of this enzyme. Neither the Ion nor the recA gene products are essential, but both are required for rapid induction of the system. Mutations in the lexA gene that render its product resistant to the RecA protease do not prevent induction of this error-free DNA-repair pathway, proving that it is not subject to r e c A - l e x A control. In most respects, this system appears very similar to the one we have previously shown to be induced by heat shock. The differences between them arise mainly when long term thiamine deprivation is studied and almost certainly reflect the need with E. coli ABl157 for a heat shock temperature of 48°C (Pardasani et al., 1989), which cannot be prolonged beyond 45 rain without killing the cells so that only effects that develop relatively quickly can be observed. The simplest hypothesis is that both types of stress lead to the induction of a single regulon or gene network (Gottesman, 1984) involving the uvrA, B and C genes. The fact that the uvrA and B genes are part of the SOS regulon appears to be a complicating factor, but it is known that the uvrB gene, at least, is transcribed from two different promoters, only one of which is regulated by LexA (Sancar et al., 1982). The stress-induced increase in UvrABC endonuclease activity in the parental strain would permit a more efficient removal of pyrimidine dimers from its DNA leading to an increased cell survival, together with a decreased mutation frequency owing to the elimination o f the signal for SOS regulon induction. In the u v r A - strain, excision repair is absent and non-inducible, so stress would not increase survival or change the high natural and induced mutation frequencies of these cells. A non-essential, enhancing role for the Ion and recA gene products is harder to envisage. One possibility is that the optimum rate of increase in UvrABC endonuclease activity requires stabilization of the enzyme by one gene product and stabilization of one or more of the UvrABC mRNAs by the seco~id. Absence of the appropriate protein would permit slower accumulation of the
enzyme at its normal rate of synthesis in the presence of the stabilizer in one case or at the higher induced rate in the absence of stabilization in the other. Our earlier suggestion (Pardasani et al., 1989) that the Lon protease might degrade a repressor controlling the error-free repair system seems less likely on the basis of our present evidence that it is not essential to the process. Induction of the error-free system by thiamine deprivation occurs without a simultaneous increase in thermotolerance and we have previously shown (Pardasani and Fitt, 1989a) that this is also true of its induction by heat shock in E. coli JEl011. Since the heat shock regulon can be induced without affecting thermotolerance (VanBogelen et al., 1987), it would be of interest to study the effect of other known inducers of the heat-shock response on the system we have described. More generally, it seems possible that the induction of error-free DNA repair may be a relatively non-specific response to a wide variety of non-mutagenic chemical, nutritional or physical stresses.
Acknowledgement We thank the Natural Science and Engineering Research Council of Canada for financial support.
References Demerec, C., and E. Cahn (1953) Studies of mutability in nutritionally deficient strains of Escherichia coli, J. Bacteriol., 65, 27-36. Fitt, P.S., and N. Sharma (1989) Induction of error-free DNA repair in Escherichia coli by heat shock or nutritional stress, Curt. Microbiol., in press. Friedberg, E.C. (1985) DNA Repair, Freeman, New York. Gottesman, S. (1984) Bacterial regulation: global regulatory networks, Annu. Rev. Genet., 18,415-4~1. Mitchel, R.E.J., and D.P. Morrison (1983) Heat-shock induction of ultraviolet resistance in Saccharomyces cerevisiae, Radiat. Res., 96, 95-99. Mount, D.W., K.B. Low and S.T. Edmiston (1972) Dominant mutations (lex) in Escherichia coli K-12 which affect radiation sensitivity and frequency of ultraviolet light-induced mutations, J. Bacteriol., 112, 886-893.
171 Pardasani, D., and P.S. Fitt (1989a) Strain-dependent induction by heat shock of resistance to ultraviolet light in Escherichia coli, Curr. Microbiol., 18, 99-103. Pardasani, D., and P.S. Fitt (1989b) Study of the effect of mutations in DNA repair genes on the thermal induction of error-free repair in Escherichia coli, Curr. Microbiol., in press. Pardasani, D., N. Sharma and P.S. Fitt (1989) Dependence on the Ion gene of the thermal induction of resistance to ultraviolet light in Escherichia coli, Curr. Microbiol., in press. Ramsey, N. (1988) A mutant in a major heat shock protein of Escherichia coli continues to show inducible thermotolerance, Mol. Gen. Genet., 211, 332-334. Sancar, G.B., A. Sancar, J.W. Little and W.D. Rupp (1982)
The uvrB gene of Escherichia coli has both lexA-repressed and lexA-independent promoters. Cell., 28, 523-530. VanBogelen, R.A., M.A. Acton and F.C. Neidhardt (1987) Induction of the heat shock regulon does not produce thermotolerance in Escherichia coli, Genes Develop., 1,525-531. Walker, G.C. (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli, Microbiol. Rev., 48, 60-93. Walker, G.C. (1985) Inducible DNA repair systems, Annu. Rev. Biochem., 54, 425-457. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli, Bacteriol. Rev., 40, 869-907.
Communicated by R.H. Haynes