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Thymine auxotrophy is associated with increased UV sensitivity in Escherichia coli and Bacillus subtilis
Mutation Research Letters
ELSEVIER
Mutation Research 347 (1995) 25-30
Thymine auxotrophy is associated with increased UV sensitivity in Escherichia coli and Bacillus subtilis Maria Mercedes Lojo
*
Cdtedra de Gen~tica Microbiana, CIGEBA (Centro de lnvestigaciones en Gen~tica Bdsica y Aplicada), Facultad de Ciencias Veterinarias, UniversidadNacional de La Plata, Calles 60 y 118, (1900) La Plata, Argentina
Received 20 December 1994;revised 20 March 1995;accepted 20 March 1995
Abstract
Thymine auxotrophy was shown to be associated with an increase in UV sensitivity both in Bacillus subtilis and in Escherichia coli. This UV sensitization became clearly evident in polA5 mutants of Bacillus subtilis: at UV doses of 16 J / m 2, a reduction of more than 10-fold in the survivor population is observed in thymine requiring spontaneous mutants (polA5 thyA thyB) compared to the parental strains (polA5). Reversion of either thyA or thyB mutation led to a partial recovery in the UV resistance. This result suggests that DNA repair polymerization might be improved by the biosynthesis of thymidylate or some effect associated with such activity, Keywords: Escherichia coil; Bacillus subtilis; UV sensitivity; polA; Thymidine auxotrophy
I. Introduction
Thymidylate synthesis in E. coli and B. subtilis
Thymidylate synthesis plays an important role in D N A metabolism in both prokaryotes and eukaryotes (Allen et al., 1980, 1983; Boorstein and Pardee, 1983). This synthesis is catalyzed by the enzyme thymidylate synthetase (EC 2.1.1.45) (TS). The enzyme, in turn, is part of the multienzymatic complex of deoxyribonucleotide (dNTP) synthesis which is coupled to the replication complex (Allen et al., 1983; Boorstein and Pardee, 1983; Laffan et al., 1990). TS uses tetrahydrofo-
* Corresponding author. Fax 54-21-253276.
late (THF) as cofactor, which is oxidized to dihydrofolate (DHF) during the course of the reaction. Thus, thymidylate synthesis requires the stoichiometric reduction of the formed D H F to THF, which is catalyzed by D H F reductase. D N A synthesis is closely coordinated with dNTP synthesis, so that bacterial TS activity prevents the incorporation of exogenous thymine into DNA, and thymine auxotrophs incorporate thymine much more efficiently than the wild type (WT) (Wilson et al., 1966). Thus, by contrast with W T strains, T h y - mutants are able to grow in the presence of D H F reductase inhibitors, such as aminopterin or trimethoprim, when thymine is present in the medium. This phenotypic feature makes possible the selection of spontaneous T h y mutants (Okada et al., 1960).
0165-7992/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-7992(95)00016-X
26
M.M. Lojo / Mutation Research 347 (1995) 25-30
Auxotrophy in E. coli is the result of mutation in the thyA gene, which encodes for the TS enzyme. Genetics in B. subtilis is more complex; there are two genes: thyA and thyB (Wilson et al., 1966), which map at 160 and 200 degrees, respectively (Piggot and Hoch, 1985). The thyA gene product (GPthyA) is a heat-resistant TS which is responsible for 95% of WT bacterial activity, whereas the thyB gene product (GPthyB) is a less active, heat-sensitive enzyme (Neuhard et al., 1978). Thymine auxotrophy is observed only in double mutants (thyA thyB) (Wilson et al., 1966). Although the function of GPthyA and GPthyB is still not completely understood, thyA mutants of B. subtilis (like in E. coli) are resistant to folate analogues in the presence of thymine. Instead, thyB mutants (like the WT) are sensitive (Wilson et al., 1966). It has been proposed that this behavior is related to the larger dTMP pool observed in thyA + thyB- mutants (Wilson et al., 1966).
Poll enzyme and the repair of UV-damaged DNA in E. coli and B. subtilis The structural and functional properties of Poll enzyme in B. subtilis (based on its size and substrate specificity) are remarkably similar to those of E. coli (Komberg and Baker, 1991). In both species mutation in the polA gene seems to affect the excision repair mechanism (Cooper and Hanawalt, 1972; Yasbin, 1977a) but this mechanism is not completely absent either in E. coli polA1 mutants (Cooper and Hanawalt, 1972; Youngs and Smith, 1973) or in B. subtilis polA5 mutants (Yasbin, 1977a; Friedman and Yasbin, 1983). B. subtilis polA5 mutation leads to a loss of Poll activity, measured by incorporation of dNTP into poly d(A-T) (Laipis and Ganesan, 1972). However, polA5 mutants of B. subtilis exhibit more polymerizing activity - 10% of the WT (Yehle and Ganesan, 1972) - than that observed in E. coli polA1 mutants - - 1% of the WT (Kornberg and Baker, 1991). It has been suggested that this additional polymerizing activity might account for the lesser sensitivity to UV light of B. subtilis polA5 compared to E. coli polA1 mutants (Laipis and Ganesan, 1972).
Table 1 Strains used Strain
Relevant genotype Source
Escherichia coli X697 X697T thyA W3110 W3110T thyA W3110TP thyA, polA1
Roy Curtis II1 This study a Laboratory collection Dr. A. Lobner Ollesen Dr. A. Lobner Ollesen
Bacillus subtilis HA101A 1A6 168TT IA215 IA215T1 ML311 ML312 SB1060 SB1060T7
Laboratory collection Bacillus Genetic Stock Center thyA, thyB, trpC Dr. N. Sueoka polA5 Bacillus Genetic Stock Center polA5, thyA, thyB This study b polA5,thyA This study c polA5,thyB This study d poIA5 Laboratory collection polA5, thyA, thyB This study b
Spontaneous thymine auxotroph isolated in a medium with trimethoprim plus thymine (see Materials and methods). b Spontaneous thymine auxotroph isolated at 40°C in a medium with trimethoprim plus thymine (see Materials and methods). c Spontaneous revertant to thymine prototrophy (resistant to trimethoprim in the presence of thymine). d Spontaneous revertant to thymine prototrophy (sensitive to trimethoprim in the presence of thymine).
Results presented in this report show that both in E. coli and B. subtilis, thymine requirement is associated with an increased sensitivity to UV light. Studies performed on E. coli polA1 mutants regarding the effect of the absence of TS activity on the sensitivity towards UV light failed to show an additive effect. Nevertheless, a completely different effect was exhibited by mutant strains of B. subtilis, which showed a striking increase in the UV sensitivity of spontaneous Thy auxotrophic mutants (polA thyA thyB).
2. Material and methods
Bacterial strains The relevant genotypes of strains used in the present study are listed in Table 1. Media Bacteria were grown in CHT medium: 50 tzg/ml casein hydrolysate, 0.5% glucose, 1 ×
M.M. Lojo / Mutation Research 347 (1995) 25-30
27
Spizizen Minimum Salt Medium (SS 1 x ) (Anagnostopoulos and Spizizen, 1961). Thymine (20 / z g / m l ) a n d / o r tryptophan ( 2 0 / z g / m l ) was added as required.
dish with constant stirring. Surviving bacteria were determined by plating a suitable dilution in C H T medium containing the required components. All determinations were done in triplicate.
Mutant isolation Thymine auxotrophs of E. coli mutants were isolated as reported (Stacey and Simson, 1965), in a medium containing trimethoprim (10 / z g / m l ) and thymine ( 2 0 / z g / m l ) . T h y - spontaneous mutants of B. subtilis were isolated from poL45 strains according to Neuhard et al. (1978), with some modifications: 0.2 ml from late log cultures of polA5 strains (1A215 or SB1060) were plated in C H T medium containing both thymine (20 / z g / m l ) and trimethoprim (10 /zg/ml). Plates were incubated for 48 h at 40°C. In these conditions all the isolated clones - 1 x 10 -9 m u t a n t s / t o t a l bacteria (TB) - showed the T h y phenotype (they were unable to grow in medium without thymine). T h r e e clones from each one of the two strains (SB1060 and 1A215) were isolated in this way. Spontaneous revertants Thy ÷ from polA5 T h y - mutants were selected because of their ability to grow in a thymineless m e d i u m (Wilson et al., 1966). The genotypes of the isolated Thy ÷ clones were checked in medium with trimethoprim plus thymine (37°C). The resistant phenotype, which corresponds to the t h y A - t h y B + genotype (Wilson et al., 1966; Neuhard et al., 1978), occurred with a frequency of 1.5 x 10 -6 r e v e r t a n t s / T B , whereas the sensitive phenotype (thyA÷thyB - ) had a frequency of 3.5 x 10 -6 rev e r t a n t s / T B . Differences in colony morphology made possible the pre-selection of genotypes, since the colonies of t h y A - t h y B + mutants were small and had smooth borders, whereas those corresponding to thyA+thyB - were larger and showed rough borders. Six different clones were isolated in each case.
Qualitative assay Sensitivities of all isolated clones were qualitatively compared by irradiation in a solid medium. Cells from a log growing culture ( O D 5 0 0 n m = 1 . 0 ) were cross streaked on C H T plates plus thymine and irradiated with decreasing doses, as follows: 8 J / m 2, 4 J / m 2, the last one corresponding to the non-irradiated area. The growth was compared with that observed in the W T strain.
Irradiation Bacteria were irradiated according to Fields and Yasbin (1983), as follows: 2.5 ml of a culture in exponential growth (ODs00n m = 1.0) were centrifuged; bacteria were then suspended in 5.0 ml SS 1 × and were immediately irradiated in a petri
3. Results T h e six thyA mutants isolated from E. coil strain X697 exhibited a sensitivity towards U V light slightly higher than that of the parental strain (Table 2). This sensitivity could still be detected by qualitative assay. A similar behavior was observed with W3110 and W3110 thyA (Fig. 1). The Thy auxotroph mutants of B. subtilis (genotype thyA thyB) also showed a greater sensitivity to U V light (Fig. 2). In both species, differences with regard to survival percentages were statistically significant ( p < 0.001, by Student's test, 20 J / m z U V dose). These results make it possible to relate the T h y - phenotype to an increase in the sensitivity to U V light, thus suggesting that the activity of
Table 2 Effect of Thy- phenotype on the UV and B. subtilis Survivors (%) a 10 J/m 2 X697 (Thy+ ) 88.0 + 6.2 b X697 (Thy-) 46.4 + 8.1 b
sensitivity in E. coli
20 J/m 2 51.2 + 2.4 b 11.05:3.1 b
Exponentially growing cells ( O D s 0 0 n m = 0.5) were pelleted, suspended in saline to a final yield of ~ 1-3 x 107 CFU/ml and UV-irradiated with stirring. Appropriate dilutions were plated in CHT plus thymine medium. a 100% survivors= unirradiated cells. b Results are the mean value of data from at least three independent experiments (+ standard deviation, n > 9).
M.M. Lojo / Mutation Research 347 (1995) 25-30
28
100.0~
P
ii
10.0
0 ::3 O3
1.0 W3110PT 0,1
0
i
i
i
I
4
8
12
16
20
UV-Doae (d/m 2) Fig. 1. Effect of thyA mutation on the UV sensitivity of E. coli polA mutant. Exponentially growing cells of E. coli mutants ( O D s 0 0 n m = 0.5) were pelleted, suspended in saline to a final yield of ~ 1-3 × 10 7 CFU/ml and UV-irradiated at the indicated doses. The survivors were determined by plating an appropriate dilution on CHT medium with thymine. Each datum is the mean value of data from at least three independent experiments (standard error < 15%, n = 9). Strains: E. coli W3110 ©; W3110 thyA e; W3110 polA1 zx; W3110 polA1 thyA A.
lO0.Ol~!~=~:~:~!l~
the TS e n z y m e could c o n t r i b u t e to a higher efficiency in the repair synthesis of D N A . Loss of TS activity in the W3110 polA1 m u t a n t of E. coli a l t e r e d the s h o u l d e r of the inactivation curve, w h e r e a s its slope r e m a i n e d u n c h a n g e d (Fig. 1), suggesting that thyA m u t a t i o n does n o t have a n additive effect o n the U V sensitivity of the polA1 strain. A l t h o u g h in p o l A 5 m u t a n t s of B. subtilis the p e r c e n t a g e of surviving b a c t e r i a was slightly lowered by the U V doses studied, the differences were n o t statistically significant in either of the two strains (SB1060 a n d 1A215), in t h r e e indep e n d e n t e x p e r i m e n t s at U V doses up to 16 J / m 2 ( T a b l e 3 a n d Fig. 2). Nevertheless, T h y - m u t a n t s ( p o l A 5 thyA thyB genotype) isolated from these strains showed a m a r k e d increase in sensitivity to U V light. R e s u l t s o b t a i n e d with m u t a n t SB1060T7 can b e seen in Fig. 2B. Similar results were o b t a i n e d with the rest of the t h y m i n e r e q u i r i n g clones analyzed in a total of t h r e e i n d e p e n d e n t e x p e r i m e n t s p e r f o r m e d for each clone (data n o t shown) s u p p o r t i n g the idea that the increase in U V sensitivity is d e t e r m i n e d by the loss of TS activity. M u t a n t 1A215T1 exhibited a similar be-
A
1
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.
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~
B
A215
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~
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E
0 °>.
P
-i (/3
1~1~1
~
1.0
0.1
0
' 4-
' 8
' 12
UV-Doae (J/m 2)
16
20
$1ollo17
1.0
0.1
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4
. . . . . 8 12 16
20
IN-Dose (J/m 2)
Fig. 2. Effect of thyA and thyB mutations on the UV sensitivity of B. subtilis polA5 mutant. Exponentially growing cells of B. subtilis mutants (ODs00nm = 1.0) were pelleted, suspended in saline to a final yield of ~ 1-3 × 10 7 CFU/ml and UV-irradiated at the indicated doses. The survivorswere determined by plating an appropriate dilution on CHT medium plus thymine. Each point is the mean value of data from at least three independent experiments (standard error < 20%, n = 9). (A) B. subtilis strains: HA101A (WT) o, 1A6 (thyA thyB) e, 1A215 (po/A5) /,, 1A215T1 (polA5 thyA thyB) A. (B) B. subtilis strains: HA101A (WT) ©, 168"VI" ( thyA thyB ) e, SB1060 ( polA5 ) A, SB1060T7 ( polA5 thyA thyB ) A .
M.M. Lojo / Mutation Research 347 (1995) 25-30 Table 3 Effect of reversion of thyA and thyB mutation on the U V sensitivity of B. subtilis strain 1A215T1 (poM5 thyA thyB) Survivors (%) a 4 J/m 2
8 J/m 2
16 J / m 2
1A215(po/A5) 78.0+6.0 64.2+ 2.1 47.1+9.1 1A215T1 (polA thyA thyB) 41.5+5.4 13.3+ 2.6 3.2+ 1.7 ML311 (polA thyA) 65.3+3.2 29.6+ 4.2 18.7+5.3 ML312(polAthyB) 70.6+4.6 54.3+11.7 21.7+6.2 Exponentially growing cells (OD50on m = 1.0) were pelleted, suspended in saline to a final yield of ~ 1-5 × 107 C F U / m l and UV-irradiated with stirring. Results are the m e a n value of data from at least three independent experiments expressed as % survivors ( + standard deviation, n = 9). a 100% survivors = unirradiated cells.
havior (Table 3 and Fig. 2A), as was also the case with the rest of the Thy- mutants isolated from strain 1A215. Dose-response studies showed that reversion of both mutations (thyA or thyB) leads to partial recovery of the resistance to UV light observed in the parental strain. Results presented in Table 3 correspond to two of the mutants isolated in the present study (ML311 and ML312); these results have been confirmed with the rest of the clones isolated in the other experiments. In all cases the simple revertants showed a sensitivity which was intermediate between those of strains polA5 and polA5 thyA thyB.
4. Discussion
TS enzyme is a component of the multienzymatic complex that may serve to channel dNTPs to sites of DNA replication (Reedy and Pardee, 1980; Mathews et al., 1988). Research involving both prokaryotes and eukaryotes suggests that thymidylate synthesis by TS contributes to the optimal efficiency of the replication process (Allen et al., 1983; Boorstein and Pardee, 1983; Reedy and Pardee, 1980). Data presented in this report indicate that loss of TS activity is associated with an increased UV sensitivity both in E. coli and in B. subtilis and this behavior could be taken as evidence of the possible role of this
29
enzyme in the modulation of DNA repair polymerization. Many agents apparently inhibiting repair also affect DNA precursor pools, suggesting a possible role of dNTP pools in the inhibition of DNA repair (Meuth, 1984). Since lowered levels of dTTP pools have been reported in Thy- mutants of B. subtilis (Neuhard et al., 1978) and in E. coli (Pritchard and Zaritsky, 1970), these differences from WT strains might account for the lessened repair efficiency observed in all the Thy- mutants studied in this report. Loss of TS activity in the W3110 polA1 mutant of E. coli alters the shoulder of the inactivation curve, whereas its slope remains unchanged (Fig. 1), suggesting that thyA mutation does not have an additive effect on the UV sensitivity of this E. coli polA1 mutant. At variance with E. coli polA1, polA5 mutants of B. subtilis exhibit lower sensitivity to UV light (Laipis and Ganesan, 1972), as well as to other DNA-damaging agents, such as methyl methanesulfonate and ethyl methanesulfonate (Friedman and Yasbin, 1983). Thus, when polA5 strains of B. subtilis (SB1060 and 1A215) were irradiated, only a slight increase in UV sensitivity was observed, compared to the repair-proficient strain, at UV doses up to 16 J / m 2 (Fig. 2 and Table 3). Since polA5 mutants contain 10% of the WT DNA polymerization activity (Yehle and Ganesan, 1972) it could be thought that this residual activity might operate efficiently in our mutants at the UV doses studied so far. Another possible explanation would be that the activity of B. subtilis Poll enzyme could be efficiently replaced by another DNA polymerase, such as PollII, which has already been shown to be essential for the mechanism of W-reactivation in B. subtilis (Fields and Yasbin, 1980; Friedman and Yasbin, 1983; Yasbin et al., 1991). In any case, the remaining activity was clearly affected by the loss of TS activity (Fig. 2 and Table 3). Moreover, at the assayed UV doses, Thy- mutants were more UV-sensitive than the polA5 counterpart (Fig. 2) and when TS activity was absent, polA5 mutants of B. subtilis were nearly as sensitive to UV light as the E. coli counterpart (Figs. 1 and 2). In B. subtilis, as regards the UV sensitivity of
30
M.M. Lojo / Mutation Research 347 (1995) 25-30
polA5 mutants, both TSs seemed to contribute to the improvement of DNA repair (Table 3), an interesting feature in a species which has evolved conserving two genes for presumptively the same function.
Acknowledgements The author warmly thanks Dr. O. Grau for his advice, support and encouragement and Mrs. S.A. Moya-Galli for typing the manuscript.
References Allen, J.R., G.P.V. Reedy, G.W. Lasser and Ch.K. Mathews (1980) T4 ribonucleotide reductase: Physical and kinetic linkage to other enzymes of deoxyribonucleotide biosynthesis, J. Biol. Chem., 255, 7583-7588. Allen, J.R., G.W. Lasser, D.A. Goldman and Ch.K. Mathews (1983) T4 phage deoxyribonucleotide synthesizing enzyme complex, J. Biol. Chem., 258, 5746-5753. Anagnostopoulos, C. and J. Spizizen (1961) Requirement for transformation in Bacillus subtilis, J. Bacteriol., 81, 741746. Boorstein, R.J. and A.B. Pardee (1983) Coordinate inhibition of DNA synthesis and thymidylate synthase activity following DNA damage and repair, Biochem. Biophys. Res. Commun., 117, 30-36. Cooper, P.K. and P.C. Hanawalt (1972) Role of DNA polymerase I and the rec system in excision repair in Escherichia coli, Proc. Natl. Acad. Sci. USA, 69, 1156-1160. Fields, P.I. and R.E. Yasbin (1980) Involvement of deoxyribonucleic acid polymerase III in w-reactivation in Bacillus subtilis, J. Bacteriol., 144, 473-475. Fields, P.I. and R.E. Yasbin (1983) DNA repair in Bacillus subtilis: an inducible dimer specific w-reactivation system, Mol. Gen. Genet., 190, 481-486. Friedman, B. and R.E. Yasbin (1983) The genetics and specificity of the constitutive excision repair system of Bacillus subtilis, Mol. Gen. Genet., 190, 475-480. Iwakura, M., M. Kawata, K. Tsuda and T. Tanaka (1988) Nucleotide sequence of the tbymidylate synthase B and dihydrofolate reductase genes contained in one Bacillus subtilis operon, Gene, 64, 9-20. Kornberg, A. and T.A. Baker (1991) in: DNA Replication, W.H. Freeman and Co., New York, pp. 113-164. Laffan, J.J., I.L. Skolnik, D.A. Hadley, M. Bouyea and W. Firshein (1990) Characterization of a multienzyme c o m -
plex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors, J. Bacteriol., 172, 5724-5731. Laipis, P.J. and A.T. Ganesan (1972) A deoxyribonucleic acid polymerase I deficient mutants of Bacillus subtilis, J. Biol. Chem., 247, 5867-5871. Mathews, Ch.K., L.K. Moen, Y. Wang and R.G. Sargent (1988) Intracellular organization of DNA precursor biosynthetic enzymes, Trends Biochem. Sci., 13, 394-397. Meuth, M. (1984). The relevance of DNA precursor pools to repair, in: A. Collins, C.S. Dowries and R.T. Johnson (Eds.), DNA Repair and its Inhibition, pp. 217-228. Neuhard, J., A.R. Price, L. Schack and E. Thomassen (1978) Two thymidylate synthetase in Bacillus subtilis, Proc. Natl. Acad. Sci. USA, 75, 1194-1198. Okada, T., K. Yanagisawa and F.J. Ryan (1960) Elective production of thymine-less mutants, Nature, 188, 340-341. Piggot, P.J. and J.A. Hoch (1985) Revised genetic linkage map of Bacillus subtilis, Microbiol. Rev., 49, 158-179. Pritchard, R.H. and A. Zaritsky (1970) Effect of thymine concentration on the replication velocity of DNA in a thymineless mutant of Escherichia coli, Nature, 226, 126131. Reedy, G.P.V. and A.B. Pardee (1980) Multienzyme complex for metabolic channeling in mammalian DNA replication, Proc. Natl. Acad. Sci. USA, 77, 3312-3316. Stacey, K.A. and E. Simson (1965) Improved method for the isolation of thymine-requiring mutants of Escherichia coli, J. Bacteriol., 90, 554-555. Wilson, M.C., J.L. Farmer and F.J. Rothman (1966) Thymidylate synthesis and aminopterine resistance in Bacillus subtilis, J. Bacteriol., 92, 186-196. Yasbin, R.E. (1977a) DNA repair in Bacillus subtilis. I. The presence of an inducible system, Mol. Gen. Genet., 153, 211-218. Yasbin, R.E. (1977b) DNA repair in Bacillus subtilis. II. Activation of the inducible system in competent bacteria, Mol. Gen. Genet., 153, 218-225. Yasbin, R.E., D. Cheo and K.W. Bayles (1991) The SOB system of Bacillus subtilis: a global regulation involved in DNA repair and differentiation, Res. Microbiol., 142, 885-892. Yehle, C.O. and A.T. Ganesan (1972) Deoxyribonucleic acid synthesis in bacteriophage SPOl-infected Bacillus subtilis. I. Bacteriophage deoxyribonucleic acid synthesis and fate of host deoxyribonucleic acid in normal and polymerasedeficient strains, J. Virol. 9, 263-272. Youngs, D.A. and K.C. Smith (1973) Involvement of DNA polymerase in excision repair after ultraviolet irradiation, Nature New Biol., 244, 240-241.
Communicated by T.R. Skopek
ELSEVIER
Mutation Research 347 (1995) 25-30
Thymine auxotrophy is associated with increased UV sensitivity in Escherichia coli and Bacillus subtilis Maria Mercedes Lojo
*
Cdtedra de Gen~tica Microbiana, CIGEBA (Centro de lnvestigaciones en Gen~tica Bdsica y Aplicada), Facultad de Ciencias Veterinarias, UniversidadNacional de La Plata, Calles 60 y 118, (1900) La Plata, Argentina
Received 20 December 1994;revised 20 March 1995;accepted 20 March 1995
Abstract
Thymine auxotrophy was shown to be associated with an increase in UV sensitivity both in Bacillus subtilis and in Escherichia coli. This UV sensitization became clearly evident in polA5 mutants of Bacillus subtilis: at UV doses of 16 J / m 2, a reduction of more than 10-fold in the survivor population is observed in thymine requiring spontaneous mutants (polA5 thyA thyB) compared to the parental strains (polA5). Reversion of either thyA or thyB mutation led to a partial recovery in the UV resistance. This result suggests that DNA repair polymerization might be improved by the biosynthesis of thymidylate or some effect associated with such activity, Keywords: Escherichia coil; Bacillus subtilis; UV sensitivity; polA; Thymidine auxotrophy
I. Introduction
Thymidylate synthesis in E. coli and B. subtilis
Thymidylate synthesis plays an important role in D N A metabolism in both prokaryotes and eukaryotes (Allen et al., 1980, 1983; Boorstein and Pardee, 1983). This synthesis is catalyzed by the enzyme thymidylate synthetase (EC 2.1.1.45) (TS). The enzyme, in turn, is part of the multienzymatic complex of deoxyribonucleotide (dNTP) synthesis which is coupled to the replication complex (Allen et al., 1983; Boorstein and Pardee, 1983; Laffan et al., 1990). TS uses tetrahydrofo-
* Corresponding author. Fax 54-21-253276.
late (THF) as cofactor, which is oxidized to dihydrofolate (DHF) during the course of the reaction. Thus, thymidylate synthesis requires the stoichiometric reduction of the formed D H F to THF, which is catalyzed by D H F reductase. D N A synthesis is closely coordinated with dNTP synthesis, so that bacterial TS activity prevents the incorporation of exogenous thymine into DNA, and thymine auxotrophs incorporate thymine much more efficiently than the wild type (WT) (Wilson et al., 1966). Thus, by contrast with W T strains, T h y - mutants are able to grow in the presence of D H F reductase inhibitors, such as aminopterin or trimethoprim, when thymine is present in the medium. This phenotypic feature makes possible the selection of spontaneous T h y mutants (Okada et al., 1960).
0165-7992/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165-7992(95)00016-X
26
M.M. Lojo / Mutation Research 347 (1995) 25-30
Auxotrophy in E. coli is the result of mutation in the thyA gene, which encodes for the TS enzyme. Genetics in B. subtilis is more complex; there are two genes: thyA and thyB (Wilson et al., 1966), which map at 160 and 200 degrees, respectively (Piggot and Hoch, 1985). The thyA gene product (GPthyA) is a heat-resistant TS which is responsible for 95% of WT bacterial activity, whereas the thyB gene product (GPthyB) is a less active, heat-sensitive enzyme (Neuhard et al., 1978). Thymine auxotrophy is observed only in double mutants (thyA thyB) (Wilson et al., 1966). Although the function of GPthyA and GPthyB is still not completely understood, thyA mutants of B. subtilis (like in E. coli) are resistant to folate analogues in the presence of thymine. Instead, thyB mutants (like the WT) are sensitive (Wilson et al., 1966). It has been proposed that this behavior is related to the larger dTMP pool observed in thyA + thyB- mutants (Wilson et al., 1966).
Poll enzyme and the repair of UV-damaged DNA in E. coli and B. subtilis The structural and functional properties of Poll enzyme in B. subtilis (based on its size and substrate specificity) are remarkably similar to those of E. coli (Komberg and Baker, 1991). In both species mutation in the polA gene seems to affect the excision repair mechanism (Cooper and Hanawalt, 1972; Yasbin, 1977a) but this mechanism is not completely absent either in E. coli polA1 mutants (Cooper and Hanawalt, 1972; Youngs and Smith, 1973) or in B. subtilis polA5 mutants (Yasbin, 1977a; Friedman and Yasbin, 1983). B. subtilis polA5 mutation leads to a loss of Poll activity, measured by incorporation of dNTP into poly d(A-T) (Laipis and Ganesan, 1972). However, polA5 mutants of B. subtilis exhibit more polymerizing activity - 10% of the WT (Yehle and Ganesan, 1972) - than that observed in E. coli polA1 mutants - - 1% of the WT (Kornberg and Baker, 1991). It has been suggested that this additional polymerizing activity might account for the lesser sensitivity to UV light of B. subtilis polA5 compared to E. coli polA1 mutants (Laipis and Ganesan, 1972).
Table 1 Strains used Strain
Relevant genotype Source
Escherichia coli X697 X697T thyA W3110 W3110T thyA W3110TP thyA, polA1
Roy Curtis II1 This study a Laboratory collection Dr. A. Lobner Ollesen Dr. A. Lobner Ollesen
Bacillus subtilis HA101A 1A6 168TT IA215 IA215T1 ML311 ML312 SB1060 SB1060T7
Laboratory collection Bacillus Genetic Stock Center thyA, thyB, trpC Dr. N. Sueoka polA5 Bacillus Genetic Stock Center polA5, thyA, thyB This study b polA5,thyA This study c polA5,thyB This study d poIA5 Laboratory collection polA5, thyA, thyB This study b
Spontaneous thymine auxotroph isolated in a medium with trimethoprim plus thymine (see Materials and methods). b Spontaneous thymine auxotroph isolated at 40°C in a medium with trimethoprim plus thymine (see Materials and methods). c Spontaneous revertant to thymine prototrophy (resistant to trimethoprim in the presence of thymine). d Spontaneous revertant to thymine prototrophy (sensitive to trimethoprim in the presence of thymine).
Results presented in this report show that both in E. coli and B. subtilis, thymine requirement is associated with an increased sensitivity to UV light. Studies performed on E. coli polA1 mutants regarding the effect of the absence of TS activity on the sensitivity towards UV light failed to show an additive effect. Nevertheless, a completely different effect was exhibited by mutant strains of B. subtilis, which showed a striking increase in the UV sensitivity of spontaneous Thy auxotrophic mutants (polA thyA thyB).
2. Material and methods
Bacterial strains The relevant genotypes of strains used in the present study are listed in Table 1. Media Bacteria were grown in CHT medium: 50 tzg/ml casein hydrolysate, 0.5% glucose, 1 ×
M.M. Lojo / Mutation Research 347 (1995) 25-30
27
Spizizen Minimum Salt Medium (SS 1 x ) (Anagnostopoulos and Spizizen, 1961). Thymine (20 / z g / m l ) a n d / o r tryptophan ( 2 0 / z g / m l ) was added as required.
dish with constant stirring. Surviving bacteria were determined by plating a suitable dilution in C H T medium containing the required components. All determinations were done in triplicate.
Mutant isolation Thymine auxotrophs of E. coli mutants were isolated as reported (Stacey and Simson, 1965), in a medium containing trimethoprim (10 / z g / m l ) and thymine ( 2 0 / z g / m l ) . T h y - spontaneous mutants of B. subtilis were isolated from poL45 strains according to Neuhard et al. (1978), with some modifications: 0.2 ml from late log cultures of polA5 strains (1A215 or SB1060) were plated in C H T medium containing both thymine (20 / z g / m l ) and trimethoprim (10 /zg/ml). Plates were incubated for 48 h at 40°C. In these conditions all the isolated clones - 1 x 10 -9 m u t a n t s / t o t a l bacteria (TB) - showed the T h y phenotype (they were unable to grow in medium without thymine). T h r e e clones from each one of the two strains (SB1060 and 1A215) were isolated in this way. Spontaneous revertants Thy ÷ from polA5 T h y - mutants were selected because of their ability to grow in a thymineless m e d i u m (Wilson et al., 1966). The genotypes of the isolated Thy ÷ clones were checked in medium with trimethoprim plus thymine (37°C). The resistant phenotype, which corresponds to the t h y A - t h y B + genotype (Wilson et al., 1966; Neuhard et al., 1978), occurred with a frequency of 1.5 x 10 -6 r e v e r t a n t s / T B , whereas the sensitive phenotype (thyA÷thyB - ) had a frequency of 3.5 x 10 -6 rev e r t a n t s / T B . Differences in colony morphology made possible the pre-selection of genotypes, since the colonies of t h y A - t h y B + mutants were small and had smooth borders, whereas those corresponding to thyA+thyB - were larger and showed rough borders. Six different clones were isolated in each case.
Qualitative assay Sensitivities of all isolated clones were qualitatively compared by irradiation in a solid medium. Cells from a log growing culture ( O D 5 0 0 n m = 1 . 0 ) were cross streaked on C H T plates plus thymine and irradiated with decreasing doses, as follows: 8 J / m 2, 4 J / m 2, the last one corresponding to the non-irradiated area. The growth was compared with that observed in the W T strain.
Irradiation Bacteria were irradiated according to Fields and Yasbin (1983), as follows: 2.5 ml of a culture in exponential growth (ODs00n m = 1.0) were centrifuged; bacteria were then suspended in 5.0 ml SS 1 × and were immediately irradiated in a petri
3. Results T h e six thyA mutants isolated from E. coil strain X697 exhibited a sensitivity towards U V light slightly higher than that of the parental strain (Table 2). This sensitivity could still be detected by qualitative assay. A similar behavior was observed with W3110 and W3110 thyA (Fig. 1). The Thy auxotroph mutants of B. subtilis (genotype thyA thyB) also showed a greater sensitivity to U V light (Fig. 2). In both species, differences with regard to survival percentages were statistically significant ( p < 0.001, by Student's test, 20 J / m z U V dose). These results make it possible to relate the T h y - phenotype to an increase in the sensitivity to U V light, thus suggesting that the activity of
Table 2 Effect of Thy- phenotype on the UV and B. subtilis Survivors (%) a 10 J/m 2 X697 (Thy+ ) 88.0 + 6.2 b X697 (Thy-) 46.4 + 8.1 b
sensitivity in E. coli
20 J/m 2 51.2 + 2.4 b 11.05:3.1 b
Exponentially growing cells ( O D s 0 0 n m = 0.5) were pelleted, suspended in saline to a final yield of ~ 1-3 x 107 CFU/ml and UV-irradiated with stirring. Appropriate dilutions were plated in CHT plus thymine medium. a 100% survivors= unirradiated cells. b Results are the mean value of data from at least three independent experiments (+ standard deviation, n > 9).
M.M. Lojo / Mutation Research 347 (1995) 25-30
28
100.0~
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ii
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i
i
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16
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UV-Doae (d/m 2) Fig. 1. Effect of thyA mutation on the UV sensitivity of E. coli polA mutant. Exponentially growing cells of E. coli mutants ( O D s 0 0 n m = 0.5) were pelleted, suspended in saline to a final yield of ~ 1-3 × 10 7 CFU/ml and UV-irradiated at the indicated doses. The survivors were determined by plating an appropriate dilution on CHT medium with thymine. Each datum is the mean value of data from at least three independent experiments (standard error < 15%, n = 9). Strains: E. coli W3110 ©; W3110 thyA e; W3110 polA1 zx; W3110 polA1 thyA A.
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the TS e n z y m e could c o n t r i b u t e to a higher efficiency in the repair synthesis of D N A . Loss of TS activity in the W3110 polA1 m u t a n t of E. coli a l t e r e d the s h o u l d e r of the inactivation curve, w h e r e a s its slope r e m a i n e d u n c h a n g e d (Fig. 1), suggesting that thyA m u t a t i o n does n o t have a n additive effect o n the U V sensitivity of the polA1 strain. A l t h o u g h in p o l A 5 m u t a n t s of B. subtilis the p e r c e n t a g e of surviving b a c t e r i a was slightly lowered by the U V doses studied, the differences were n o t statistically significant in either of the two strains (SB1060 a n d 1A215), in t h r e e indep e n d e n t e x p e r i m e n t s at U V doses up to 16 J / m 2 ( T a b l e 3 a n d Fig. 2). Nevertheless, T h y - m u t a n t s ( p o l A 5 thyA thyB genotype) isolated from these strains showed a m a r k e d increase in sensitivity to U V light. R e s u l t s o b t a i n e d with m u t a n t SB1060T7 can b e seen in Fig. 2B. Similar results were o b t a i n e d with the rest of the t h y m i n e r e q u i r i n g clones analyzed in a total of t h r e e i n d e p e n d e n t e x p e r i m e n t s p e r f o r m e d for each clone (data n o t shown) s u p p o r t i n g the idea that the increase in U V sensitivity is d e t e r m i n e d by the loss of TS activity. M u t a n t 1A215T1 exhibited a similar be-
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Fig. 2. Effect of thyA and thyB mutations on the UV sensitivity of B. subtilis polA5 mutant. Exponentially growing cells of B. subtilis mutants (ODs00nm = 1.0) were pelleted, suspended in saline to a final yield of ~ 1-3 × 10 7 CFU/ml and UV-irradiated at the indicated doses. The survivorswere determined by plating an appropriate dilution on CHT medium plus thymine. Each point is the mean value of data from at least three independent experiments (standard error < 20%, n = 9). (A) B. subtilis strains: HA101A (WT) o, 1A6 (thyA thyB) e, 1A215 (po/A5) /,, 1A215T1 (polA5 thyA thyB) A. (B) B. subtilis strains: HA101A (WT) ©, 168"VI" ( thyA thyB ) e, SB1060 ( polA5 ) A, SB1060T7 ( polA5 thyA thyB ) A .
M.M. Lojo / Mutation Research 347 (1995) 25-30 Table 3 Effect of reversion of thyA and thyB mutation on the U V sensitivity of B. subtilis strain 1A215T1 (poM5 thyA thyB) Survivors (%) a 4 J/m 2
8 J/m 2
16 J / m 2
1A215(po/A5) 78.0+6.0 64.2+ 2.1 47.1+9.1 1A215T1 (polA thyA thyB) 41.5+5.4 13.3+ 2.6 3.2+ 1.7 ML311 (polA thyA) 65.3+3.2 29.6+ 4.2 18.7+5.3 ML312(polAthyB) 70.6+4.6 54.3+11.7 21.7+6.2 Exponentially growing cells (OD50on m = 1.0) were pelleted, suspended in saline to a final yield of ~ 1-5 × 107 C F U / m l and UV-irradiated with stirring. Results are the m e a n value of data from at least three independent experiments expressed as % survivors ( + standard deviation, n = 9). a 100% survivors = unirradiated cells.
havior (Table 3 and Fig. 2A), as was also the case with the rest of the Thy- mutants isolated from strain 1A215. Dose-response studies showed that reversion of both mutations (thyA or thyB) leads to partial recovery of the resistance to UV light observed in the parental strain. Results presented in Table 3 correspond to two of the mutants isolated in the present study (ML311 and ML312); these results have been confirmed with the rest of the clones isolated in the other experiments. In all cases the simple revertants showed a sensitivity which was intermediate between those of strains polA5 and polA5 thyA thyB.
4. Discussion
TS enzyme is a component of the multienzymatic complex that may serve to channel dNTPs to sites of DNA replication (Reedy and Pardee, 1980; Mathews et al., 1988). Research involving both prokaryotes and eukaryotes suggests that thymidylate synthesis by TS contributes to the optimal efficiency of the replication process (Allen et al., 1983; Boorstein and Pardee, 1983; Reedy and Pardee, 1980). Data presented in this report indicate that loss of TS activity is associated with an increased UV sensitivity both in E. coli and in B. subtilis and this behavior could be taken as evidence of the possible role of this
29
enzyme in the modulation of DNA repair polymerization. Many agents apparently inhibiting repair also affect DNA precursor pools, suggesting a possible role of dNTP pools in the inhibition of DNA repair (Meuth, 1984). Since lowered levels of dTTP pools have been reported in Thy- mutants of B. subtilis (Neuhard et al., 1978) and in E. coli (Pritchard and Zaritsky, 1970), these differences from WT strains might account for the lessened repair efficiency observed in all the Thy- mutants studied in this report. Loss of TS activity in the W3110 polA1 mutant of E. coli alters the shoulder of the inactivation curve, whereas its slope remains unchanged (Fig. 1), suggesting that thyA mutation does not have an additive effect on the UV sensitivity of this E. coli polA1 mutant. At variance with E. coli polA1, polA5 mutants of B. subtilis exhibit lower sensitivity to UV light (Laipis and Ganesan, 1972), as well as to other DNA-damaging agents, such as methyl methanesulfonate and ethyl methanesulfonate (Friedman and Yasbin, 1983). Thus, when polA5 strains of B. subtilis (SB1060 and 1A215) were irradiated, only a slight increase in UV sensitivity was observed, compared to the repair-proficient strain, at UV doses up to 16 J / m 2 (Fig. 2 and Table 3). Since polA5 mutants contain 10% of the WT DNA polymerization activity (Yehle and Ganesan, 1972) it could be thought that this residual activity might operate efficiently in our mutants at the UV doses studied so far. Another possible explanation would be that the activity of B. subtilis Poll enzyme could be efficiently replaced by another DNA polymerase, such as PollII, which has already been shown to be essential for the mechanism of W-reactivation in B. subtilis (Fields and Yasbin, 1980; Friedman and Yasbin, 1983; Yasbin et al., 1991). In any case, the remaining activity was clearly affected by the loss of TS activity (Fig. 2 and Table 3). Moreover, at the assayed UV doses, Thy- mutants were more UV-sensitive than the polA5 counterpart (Fig. 2) and when TS activity was absent, polA5 mutants of B. subtilis were nearly as sensitive to UV light as the E. coli counterpart (Figs. 1 and 2). In B. subtilis, as regards the UV sensitivity of
30
M.M. Lojo / Mutation Research 347 (1995) 25-30
polA5 mutants, both TSs seemed to contribute to the improvement of DNA repair (Table 3), an interesting feature in a species which has evolved conserving two genes for presumptively the same function.
Acknowledgements The author warmly thanks Dr. O. Grau for his advice, support and encouragement and Mrs. S.A. Moya-Galli for typing the manuscript.
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plex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors, J. Bacteriol., 172, 5724-5731. Laipis, P.J. and A.T. Ganesan (1972) A deoxyribonucleic acid polymerase I deficient mutants of Bacillus subtilis, J. Biol. Chem., 247, 5867-5871. Mathews, Ch.K., L.K. Moen, Y. Wang and R.G. Sargent (1988) Intracellular organization of DNA precursor biosynthetic enzymes, Trends Biochem. Sci., 13, 394-397. Meuth, M. (1984). The relevance of DNA precursor pools to repair, in: A. Collins, C.S. Dowries and R.T. Johnson (Eds.), DNA Repair and its Inhibition, pp. 217-228. Neuhard, J., A.R. Price, L. Schack and E. Thomassen (1978) Two thymidylate synthetase in Bacillus subtilis, Proc. Natl. Acad. Sci. USA, 75, 1194-1198. Okada, T., K. Yanagisawa and F.J. Ryan (1960) Elective production of thymine-less mutants, Nature, 188, 340-341. Piggot, P.J. and J.A. Hoch (1985) Revised genetic linkage map of Bacillus subtilis, Microbiol. Rev., 49, 158-179. Pritchard, R.H. and A. Zaritsky (1970) Effect of thymine concentration on the replication velocity of DNA in a thymineless mutant of Escherichia coli, Nature, 226, 126131. Reedy, G.P.V. and A.B. Pardee (1980) Multienzyme complex for metabolic channeling in mammalian DNA replication, Proc. Natl. Acad. Sci. USA, 77, 3312-3316. Stacey, K.A. and E. Simson (1965) Improved method for the isolation of thymine-requiring mutants of Escherichia coli, J. Bacteriol., 90, 554-555. Wilson, M.C., J.L. Farmer and F.J. Rothman (1966) Thymidylate synthesis and aminopterine resistance in Bacillus subtilis, J. Bacteriol., 92, 186-196. Yasbin, R.E. (1977a) DNA repair in Bacillus subtilis. I. The presence of an inducible system, Mol. Gen. Genet., 153, 211-218. Yasbin, R.E. (1977b) DNA repair in Bacillus subtilis. II. Activation of the inducible system in competent bacteria, Mol. Gen. Genet., 153, 218-225. Yasbin, R.E., D. Cheo and K.W. Bayles (1991) The SOB system of Bacillus subtilis: a global regulation involved in DNA repair and differentiation, Res. Microbiol., 142, 885-892. Yehle, C.O. and A.T. Ganesan (1972) Deoxyribonucleic acid synthesis in bacteriophage SPOl-infected Bacillus subtilis. I. Bacteriophage deoxyribonucleic acid synthesis and fate of host deoxyribonucleic acid in normal and polymerasedeficient strains, J. Virol. 9, 263-272. Youngs, D.A. and K.C. Smith (1973) Involvement of DNA polymerase in excision repair after ultraviolet irradiation, Nature New Biol., 244, 240-241.
Communicated by T.R. Skopek