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An Inducible Pathway Is Required for Mutagenesis in ... for UV mutagenesis in S. typhimurium. .... and 6 x 109 per ml) on LB plates containing 100 ,ug of rifampin ...
JOURNAL

OF

Vol. 169, No. 6

BACTERIOLOGY, June 1987, p. 2885-2888

0021-9193/87/062885-04$02.00/0 Copyright C 1987, American Society for Microbiology

An Inducible Pathway Is Required for Mutagenesis in Salmonella typhimurium LT2 CRISTIAN ORREGOt AND ERIC EISENSTADT*

Department of Cancer Biology and Laboratory of Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115 Received 23 December 1986/Accepted 19 March 1986 UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA+ gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate (K. Overbye and P. Margolin, J. Bacteriol. 146:170-178, 1981; K. Overbye, S. M. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Mitomycin C induction of the Fb(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead us to deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability.

requirement for the plasmid-borne mucAB loci for optimal levels of bacterial mutagenesis (16, 18, 21), Weigle reactivation (21), and Weigle mutagenesis in S. typhimurium (33). These observations have led to the speculation that S. typhimurium has a defective umu-like locus (31, 34). This study established that overproduction of E. coli LexA protein in S. typhimurium LT2 reduced the frequency of UV-induced mutations. We also provide evidence that a second locus in addition to recA is required for mutability by UV. These data led us to deduce that S. typhimurium LT2 has an inducible pathway for UV mutagenesis which probably involves an analog of the E. coli umuDC loci. Bacteria and plasmids. S. typhimurium LT2 strains were PM155 and PM773 (24). Both strains carry the leuD21 allele. PM155 is topA+. Strain PM773 has the topA24 allele, a spontaneous deletion that extends from the cysB locus through topA and the entire trp operon. The allele designated topA24 corresponds to an allele previously designated supX24 (17, 24). The TopA phenotype was confirmed by examination of phage P22 plaque morphology. TopAstrains generate clear plaques, while TopA+ strains produce normal turbid plaques (24). Plasmids used were pBR322, pRB160 (a pBR322 derivative carrying a 950-base-pair insert containing the E. coli lexA+ gene; 3), and pSE205 (a lowcopy-number plasmid constructed by S. Elledge and carrying a sF(mucB'-lacZ') fusion; for a related construct, see reference 10). Plasmids were first moved from E. coli into S. typhimurium LB5010 (r- m+; 4) by transformation (14) and then into other S. typhimurium strains by P22-mediated transduction (5). UV irradiation. Transformants were grown overnight in LB broth (19) plus either 10 ,ug of tetracycline or 100 ,ug of ampicillin per ml (for plasmid maintenance) and suspended in 0.8% NaCl. Samples were exposed to UV, and titers were determined for viable cell number either on LB plates containing 50 ,ug of ampicillin per ml (Table 1) or on M9 minimal glucose plates (19) containing 40 ,ug of leucine per ml, 0.001% (vol/vol) Casamino Acids (Difco Laboratories, Detroit, Mich.), 10 jig of adenine per ml, and 6 ,ug of tetracycline per ml (Fig. 1). Colonies were counted after plates had been incubated at 30°C for 1 day (LB) or for 3 days (M9). For mutagenesis experiments, samples from

Induced mutations in Escherichia coli arise via at least two distinguishable processes (reviewed in reference 9). One class of mutation results from aberrant base pairing during replication of a DNA strand containing a modified base. This process does not apparently require any inducible functions. The second class of mutation arises as a consequence of nonreplicable lesions such as UV photoproducts. Mutagenesis in this latter case depends on the induction of a set of genes known as the SOS regulon. This family of coordinately regulated genes (din loci; 35) is induced by treatments that interfere with DNA replication and is negatively regulated by the lexA gene product (reviewed in reference 35). Repression is in turn modulated by the RecA protein, which mediates proteolysis of the LexA protein early during SOS induction. The umuDC operon is one of several controlled by the lexA repressor (1). umu mutations block SOS mutagenesis and modestly increase the sensitivity of bacteria when they are challenged with DNA-damaging agents (13, 20). Inducible responses to DNA damage in Salmonella typhimurium have not been as systematically characterized as in E. coli. Nonetheless, the following observations suggest that S. typhimurium has an SOS regulon which resembles that of E. coli: (i) UV light induces RecA synthesis in S. typhimurium (7, 30), (ii) recA mutants of S. typhimurium are nonmutable (15, 18), (iii) the RecA proteins of E. coli and S. typhimurium are structurally and functionally homologous (27, 31), (iv) introduction of either the lexA+ gene (30) or a truncated recA gene from E. coli (31) sensitizes S. typhimurium to the lethal effects of UV, and (v) din-lac fusions from E. coli are inducible by DNA damage when introduced into S. typhimurium (25). On the other hand, some specific features of mutagenesis and related responses to DNA damage distinguish the SOS response in S. typhimurium from that in E. coli. These differences include (i) weak Weigle reactivation and Weigle mutagenesis of P22 phage (33); (ii) two reports that S. typhimurium is nonmutable by UV (31, 32); and (iii) the Corresponding author. t Present address: School of Public Health, University of California, Berkeley, CA 94720. *

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FIG. 1. Survival of S. typhimurium LT2 after treatment with Strain PM155 with plasmid pBR322; 0, various doses of UV. strain PM155 with plasmid pRB160 carrying the lexA gene from E. coli. 0,

exposed and unexposed cultures were diluted into fresh LB plus tetracycline or ampicillin and grown overnight. The number of rifampin-resistant mutants in the overnight cultures was determined by spreading 0.1-ml samples of saturated overnight cultures (cell densities were between 4 x 109 and 6 x 109 per ml) on LB plates containing 100 ,ug of rifampin per ml and by counting colonies after plates had been incubated for 1 to 2 days at 30°C. Inhibition of UV mutability by excess LexA. The presence of excess LexA increased by a factor of at least 8 the rate of inactivation of cells by UV irradiation (Fig. 1). The increased UV sensitivity imparted by excess LexA might be due to blocked induction of excision repair (the inducibility of excision repair in S. typhimurium has not yet been demonstrated; 35). In similar irradiation experiments, we measured the frequency of forward mutations to rifampin resistance (100 jig of rifampin per ml) after UV exposure (Table 1). UV irradiation of strain PM155 carrying pBR322 induced Rif' mutants to a frequency of almost 10-6. In the presence of pRB160, however, the induction of Rifr mutants by UV was greatly diminished and reached a frequency no higher than 2.5 x 10-8 (Table 1). As comparable numbers of surviving bacteria were monitored in these experiments, the lower yield of UV-induced mutants in the presence of pRB160 cannot be accounted for solely by the increased UV sensitivity of these bacteria. Reversion of the leuD21 phenotype to leucine independence and induction of streptomycinresistant mutants were also suppressed by pRB160 (results not shown). These results led to the conclusion that UV mutability in S. typhimurium is repressible by LexA. Sedgwick and Goodwin (30) had previously described

sensitization to UV by a plasmid-encoded E. coli lexA+ gene introduced into S. typhimurium LT22. Even in the absence of the plasmid, however, S. typhimurium LT22 showed virtually no induction of rifampin-resistant mutants by UV (30). Nonetheless, UV mutability in S. typhimurium LT2 is well established (6, 12, 22, 24, 29), and the results in Table 1 confirm this fact. The apparent nonmutability of the LT22 strain reported by Sedgwick and Goodwin (31) might have been due to UV induction of P22 prophage present in this strain (37). TopA and SOS induction in S. typhimurium. At least three inducible gene products are required for UV mutagenesis in E. coli. These products are activated RecA, UmuD, and UmuC (36). In S. typhimurium, only RecA and its structural gene have been identified (27). Yet Overbye et al. (23) and Overbye and Margolin (24) observed that S. typhimurium topA mutants (including a suppressible amber allele of topA) were nonmutable by either UV or methyl methanesulfonate and that mutability by UV or methyl methanesulfonate could be restored by suppressing the amber allele of topA with either of two amber suppressors, by complementing the topA defect with E. coli topA on an F' factor, or by introducing plasmid pKM101. These observations imply that at least one inducible locus other than recA is required for mutagenesis. The reasoning behind this interpretation is straightforward. (i) The nonmutability of topA bacteria is likely to be a result of decreased expression of genes whose products are required for DNA repair and mutability (8, 24); (ii) the mucAB locus on plasmid pKM101 is inducible by DNA damage via activation of RecA and subsequent cleavage of LexA (10); and (iii) as UV mutability in E. coli requires activated RecA in addition to its ability to cleave LexA (2, 11, 28), restoration of UV mutability by pKM101 to topA bacteria implies that RecA activation and expression of mucAB are both occurring in a topA background. Indeed, Overbye et al. (23) state that "high-level synthesis of [RecA TABLE 1. Suppression of UV-induced mutagenesis in strain PM155 by a high-copy-number plasmid carrying the E. coli lexA+ gene Plasmid and expt no.a

pBR322 Expt 1

Expt 2

UV dose

Surviving

(J/m2)

fraction

0 2 4 8 0 12 24 36 48

1.0 0.96 0.74 0.60 1.0 0.81 0.63 0.52 0.36

Mutation

frequencyb

x 107) 107) 107) 107) 107) 107) 107) 107) 107)

0.2 (7.8 4.8 (7.5 33.0 (5.8 63.0 (4.7 1.8 (9.6 37.1 (7.8 63.1 (6.0 66.4 (5.8 86.2 (3.5

x x x x x x x x

(9.5 (1.1 (1.5 (4.5 (8.0 (3.1 (1.1 (9.7

x x x x x x x x

pRB160 (lexA+) Expt 1

Expt 2

a

0 2 4 8 0 0.8 2 2.8

1.0 0.16 0.02 0.006 1.0 0.39 0.14 0.12

2.7 4.1 4.1 3.6 0.6 0.3 2.5 1.2

108) 108) 107) 106) 107) 107) 107) 106)

In experiment 1, bacteria were diluted and grown overnight in LB plus 10

jig of tetracycline per ml. In experiment 2, bacteria were diluted and grown overnight in LB plus 100 p.g of ampicillin per ml. bRif' mutants 108 cells. Numbers in parentheses are numbers of viable bacteria that were added to 5 ml of LB plus antibiotic and grown overnight to allow for expression of rifampin resistance (Rifr).

NOTES

VOL. 169, 1987 |

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FIG. 2. Induction of 4F(mucB'-lacZ') fusion on plasmid pSE205 in topA+ (0 and 0) or topA (A and A) strains of S. typhimurium LT2. Strains were grown in M9 medium plus 1% Casamino Acids to mid-log phase. Each culture was split in half, and 0.2 ,ug of mitomycin C (MMC) per ml was added to one portion at the time indicated by the arrow. 0, A, No mitomycin C; 0, A, 0.2 ,ug of mitomycin C

per

ml.

was] approximately the

[TopA+] cells (data

same

in UV-irradiated

[TopA-] and

not shown)."

To directly examine the expression of mucAB in topA bacteria, we introduced plasmid pSE205 carrying a F(mucB'-lacZ') fusion into topA and topA+ bacteria and measured 0-galactosidase activity (19) after the addition of 0.2 ,ug of mitomycin C per ml to cells growing exponentially in M9 medium plus 1% Casamino Acids. The results (Fig. 2) clearly show that the fusion protein was induced by mitomycin C in both genetic backgrounds. The faster onset of induction in topA mutants compared with that in topA+ strains is reproducible. Thus, it would appear that topA bacteria do not express at least one chromosomal locus whose product is essential for the generation of UV-induced mutations. The suppression of mutability by excess LexA (Table 1) suggests that expression of this locus (or these loci) will be inducible by DNA damage. It is reasonable to imagine that one of these loci will turn out to be a homolog of the E. coli umuDC loci. In view of the low degree of base sequence homology (52%) between the functionally equivalent umuDC and mucAB loci (26), a reported failure to detect hybridization of S. typhimurium DNA to a probe containing the E. coli umuDC operon (31) cannot be considered definitive evidence for the absence of a umuDC homolog in S. typhimurium. We are grateful to P. Margolin, S. Elledge, and P. L. Foster for providing bacterial strains and plasmids. Comments by Foster about an earlier draft of this paper were helpful. This work was supported by Public Health Service grant GM34258 from the National Institutes of Health.

LITERATURE CITED 1. Bagg, A., C. J. Kenyon, and G. C. Walker. 1981. Inducibility of a gene product required for UV and chemical mutagenesis in Escherichia coli. Proc. Natl. Acad. Sci. USA 78:5749-5753.

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2. Blanco, M., G. Herrera, P. Collado, J. Rebollo, and L. M. Botella. 1982. Influence of recA protein on induced mutagenesis. Biochimie 64:633-636. 3. Brent, R., and M. Ptashne. 1980. The lexA gene product represses its own promoter. Proc. Natl. Acad. Sci. USA 77:1932-1936. 4. Bullas, L. R., and J.-I. Ryu. 1983. Salmonella typhimurium LT2 strains which are r- m+ for all three chromosomally located systems of DNA restriction and modification. J. Bacteriol. 156:471-474. 5. Davis, R. W., D. Botstein, and J. R. Roth (ed.). 1980. Advanced bacterial genetics, p. 78-79. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 6. Demerec, M., Z. Hartman, P. E. Hartman, T. Yura, J. S. Gots, M. Ozeki, and S. W. Glover. 1956. Genetic studies with bacteria, p. 107-120. Carnegie Institute of Washington publication no. 621. Carnegie Institute of Washington, Washington, D.C. 7. Dobson, P. P., and G. C. Walker. 1980. Plasmid (pKM101)mediated Weigle reactivation in Escherichia coli K12 and Salmonella typhimurium LT2. Genetic dependence, kinetics of induction, and effect of chloramphenicol. Mutat. Res. 71:2541. 8. Drlica, K. 1984. Biology of bacterial deoxyribonucleic acid topoisomerases. Microbiol. Rev: 48:273-289. 9. Eisenstadt, E. 1987. Analysis of mutagenesis, p. 1016-1033. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.). Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 10. Elledge, S. J., and G. C. Walker. 1983. The muc genes of pKM101 are induced by DNA damage. J. Bacteriol. 155: 1306-1315. 11. Ennis, D. G., B. Fisher, S. Edniston, and D. W. Mount. 1985. Dual role for Escherichia coli RecA protein in SOS mutagenesis. Proc. Natl. Acad. Sci. USA 82:3325-3329. 12. Kanazir, D. T., P. E. Hartnan, and D. Savic. 1970. Ultravioletinduced reversions of Salmonella his frameshift mutations. J. Bacteriol. 101:649-651. 13. Kato, T., and Y. Shinoura. 1977. Isolation and characterization of mutants of Escherichia coli deficient in induction of mutations by ultraviolet light. Mol. Gen. Genet. 156:121-131. 14. Lee, G. S., and G. F.-L. Ames. 1984. Analysis of promoter mutations in the histidine transport operon of Salmonella typhimurium: use of hybrid M13 bacteriophages for cloning, transformation, and sequencing. J. Bacteriol. 159:1000-1005. 15. MacPhee, D. G. 1973. Effect of rec mutations on the ultraviolet protecting and mutation-enhancing properties of the plasmid R-Utrecht in Salmonella typhimurium. Mutat. Res. 19:357-359. 16. MacPhee, D. G. 1973. Effect of an R factor and caffeine on ultraviolet mutability in Salmonella typhimurium. Mutat. Res. 18:367-370. 17. Margolin, P., L. Zumstein, R. Sternglanz, and J. C. Wang. 1985. The Escherichia coli supX locus is topA, the structural gene for DNA topoisomerase I. Proc. Natl. Acad. Sci. USA 82: 5437-5441. 18. McCann, J., N. E. Spingarn, J. Kobori, and B. N. Ames. 1975. Detection of carcinogens as mutagens: bacterial tester strains with R-factor plasmids. Proc. Natl. Acad. Sci. USA 72:979-983. 19. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. Miller, S. S., and E. Eisenstadt. 1985. Enhanced sensitivity of Escherichia coli umuC to photodynamic inactivation by angelicin (isopsoralen). J. Bacteriol. 162:1307-1310. 21. Mortelmans, K. E., and B. A. D. Stocker. 1976. Ultraviolet light protection, enhancement of ultraviolet light mutagenesis, and mutator effect of plasmid R46 in Salmonella typhimurium. J. Bacteriol. 128:271-282. 22. Nakano, E., H. Ichikawa-Ryo, and S. Kondo. 1982. Comparative mutability of the Ames tester strains of Salmonella typhimurium by ultraviolet radiation and by 4-nitroquinoline 1-oxide. Mutat. Res. 93:35-44. 23. Overbye, K. M., S. K. Basu, and P. Margolin. 1983. Loss of DNA topoisomerase I activity alters many cellular functions in

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