Proc. Natl. Acad. Sci. USA Vol. 74, No. 9, pp. 3947-3950, September 1977 Genetics

Mutator action by Escherichia coli strains carrying dnaE mutations (spontaneous mutation/DNA replication)

C. G. SEVASTOPOULOS* AND D. A. GLASERt Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720

Contributed by Donald A. Glaser, June 17, 1977

ABSTRACT Several newly isolated temperature-sensitive dnaE mutants of Escherichia coli exhibit powerful mutagenic action at permissive temperatures. Mutation rates for the two most active mutants were assayed at four different temperatures and compared to wild-type behavior. Temperature-resistant revertants of the original temperature-sensitive dnaE mutants exhibited lower, nearly normal, mutation rates, but no antimutator strains were found.

The appearance of new mutants in a population is an essential part of the evolution of a species. It is now believed that most, if not all, spontaneous mutations arise as errors in DNA replication, recombination, or repair. Mutation rates are genetically controlled and genes that lead to an appreciable alteration in the rate of mutation of other genes have been recognized and studied in several organisms (1-6). These mutation-controlling genes, which can carry mutations themselves, are known as mutator or antimutator genes, depending on whether their effect results in an increase or decrease of spontaneous mutation rates. In bacteriophage T4, for example, some mutant alleles of gene 43 (the structural gene for the phage DNA polymerase) act as mutators (3, 7); other mutations in the same gene 43 act as antimutators (7). These studies suggest that the wild-type rate in T4 is not the theoretically possible minimum, again suggesting that mutation rates can be adjusted by selection. How the polymerase is involved in control of mutation rates is not fully understood, but recent results with T4 suggest that spontaneous mutation rates reflect relative rates of polymerization and proofreading during DNA synthesis (8); mutations altering the normal ratio of 5'-to-3' polymerase activity to 3'to-5' exonuclease "editing" activity might act as mutators when the ratio is increased, and as antimutators when the ratio is decreased. In Escherichia coli, at least two classes of mutants with altered DNA polymerase, polA (9) and dnaE (10), have been shown to increase the spontaneous mutation rate to a small extent, i.e., they exhibit mild mutator activity. There are at least six other known mutator genes in E. coli, all causing dramatic increases in mutation rates; one of these, mutT, may be involved in DNA replication because DNA synthesis is required for mutT action (6, 11). Little is known about the enzymatic functions of the E. coli mutator genes (6). Because no antimutator activity has ever been observed in E. coil, we decided to screen our collection of newly isolated temperature-sensitive strains (12), defective in DNA replication (dnats mutants), for mutator/antimutator effects at permissive temperatures. The mutagenic character of these dnat, strains was studied by measuring mutation frequencies at 340 for three independent mutational events: reversion to leucine independence (Leu - Leu+) and acquisition of resistance to L-valine The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemnent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

and 6-azauracil (Vals -, VaIR, Azas - AzaR). These events were selected for experimental convenience and also to facilitate comparisons with previously reported mutator effects in dna mutants (10).

MATERIALS AND METHODS Bacterial Strains. All dna mutants were derived from strain AT2255 (also called DG17) as described elsewhere (12). DG17 has the following genotype: argG6, metBi, his-i, leu-6, thyA3, thi-1, mtl-2, xyl-7, malAl, gal-6, lacYl, strA104, tonA2. Media. Liquid minimal medium (solution K) was previously described by Wehr et al. (13). Unless stated otherwise, nutrients were added to minimal media at the following final concentrations: L-amino acids, 25 ttg/ml; glucose, 0.2%; thymine, 10 Alg/ml. L-Valine was added at 40 tg/ml when valine resistance was assayed. Similarly, 6-azauracil was added at 40 Ag/ml. Luria (L) broth (14) with CaCI2 omitted was used as a complex medium. For assaying resistance to ampicillin or rifampicin, L medium was supplemented with 0.2% glucose and thymine at 10 ,ug/ml (LTG medium). Drugs were added at 10 ,qg/ml for DL-ampicillin or 100,ug/ml for rifampicin (15). Membrane filters (47-mm diameter, 0.2-ytm pore size, 3 X 108 pores per cm2 pore density) were obtained from Nucleopore Corp., Pleasanton, CA 94566. Mutation Frequencies. The procedure of Hoess and Herman (2) was adapted as follows. Modified fluctuation tests were carried out by starting a single colony in L broth (supplemented with thymine at 10 ,g/ml) and allowing the cells to reach stationary phase. The cells were then diluted so that a series of 10 tubes, each containing 2 ml of L broth, was inoculated with about 200 cells per tube and subsequently incubated with shaking for 21 hr at 34°. To assay for spontaneous Leu+, ValR, or AzaR mutants, 0. 1-ml aliquots of appropriate dilutions were plated on selective plates; two samples per tube were taken to assay for each type of mutational event. Viable cells were counted by combining 0.2 ml from each of the 10 tubes in the series and plating cells on complete plates. Frequencies were then calculated by averaging the number of revertant or resistant colonies and dividing by the viable cell count. To avoid plates representing "jackpots," revertant colony counts of more than twice the mean were not included. Mutation Rates. It has been known since the early 1950s that bacterial populations growing on the surface of membrane filters can be transferred easily from one solid medium to another without disturbing the spatial relationship among members of the population. This technique, which is conAbbreviations: Leu- and Leu+, leucine-requiring and leucine-independent; Ts+, temperature-resistant; Val, Aza, Rif, and Amp refer to valine, 6-azauracil, rifampicin, and ampicillin; S, sensitive; R, resis-

tant. * Present address: Becton Dickinson Electronics Laboratory, 506 Clyde Avenue, Mountain View, CA 94043. t To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Frequencies of mutation to nonrequirement of leucine (Leu- Leu+), L-valine resistance (ValS - VaIR), and 6-azauracil resistance (AzaS - AzaR), at 340, for SG strains carrying dnat. mutations near or at dnaE AzaS - AzaR ValS - VaiR Leu- - Leu+ SG strain Factor* AzaR X 105 VaiR X 108 Factor* Factor* Leu+ X 109 (dna allele) -

175 (667) 230 (668) 345 (669) 406 (670) 790 (671) 902 (672) 921 (673) 980 (674) 1319 (675) 1424 (676) 1484 (677) 1556 (678) 1758 (679) 1788 (680) 1794 (681) 1887 (682) 2095 (683)

DG17 (wild type)

3.9 6.0 91 20 38 142 8.4 4.4 3.5 110 2.5 22 8.6 6.2 4.7 3.9 7.2

1.1 1.7 26 5.7 11 41 2.4 1.2 1.0 31 0.7 6.3 2.4 1.7 1.3 1.1 2.1

3.2 15 512 205 349 2220 136 7.2 151 1350 1.9 644 144 20 96 7.9 202

1.4 6.8 233 93 159 1009 62 3.3 69 614 0.9 293 65 9.1 44 3.6 92

0.1 0.2 12 2.9 3.3 28 1.2 0.3 3.1 6.7 0.1 6.0 0.4 0.1 0.2 0.2 3.2

1 2 120 29 33 280 12 3 31 67 1 60 4 1 2 2 32

3.5

1.0

2.2

1.0

0.1

1.0

* Stimulation factor equals mutation frequency of SG strain divided by wild-type frequency for the same mutational event; it is a measure of mutator activity.

ceptually similar to the original spreading experiment by Newcombe (16), has been used to determine spontaneous mutation rates in E. cols (17). In order to measure rates of mutation to ampicillin and rifampicin resistance in strains SG902 and DG17, the procedure of Matney (17) was adapted as follows. Overnight cultures were diluted to an approximate concentration of 105 cells per ml. A 5-ml aliquot of this suspension was drawn through a sterile membrane filter held in a filtration unit. In each of 12 separate experiments, 30-50 replicate populations each of SG902 and DG17 were deposited on filters in this manner, and the membranes were transferred with sterile forceps to the surface of soft (0.7% agar) LTG petri dishes. They were then incubated at the appropriate temperature (250, 30°, 340, or 37°). After various incubation intervals,'four to six membranes for each strain were transferred to LTG plates (0.7% agar) containing either DL-ampicillin at 10 Ag/ml or rifampicin at 100 /Ag/ml.o Simultaneously, the population size was determined by placing additional replicate membranes into 125-ml Erlenmayer flasks containing 20 ml of diluting fluid (solution K); the cells were removed by vigorous shaking, and viable plate counts (on LTG medium) were made of appropriate dilutions of the resulting suspension. (Full recovery, as measured by viable counts, was obtained when a population of known size was removed from the membrane as described here.) The transferred membranes were incubated on ampicillin or rifampicin plates. The progeny of resistant mutants produced colonies that grew out of the sensitive population film and were counted with a standard colony counter (Nucleopore membranes are translucent). With this procedure mutation rates (as opposed to frequencies) can be determined by relating the increase in the number of resistant clones, and hence of mutations, that has arisen over a given time interval, to the increase in the total number of bacteria and, therefore, to the number of bacterial generations that occurred over the same time period. The method is unaffected by differences in growth rate between $ Ampicillin- and rifampicin-resistant clones can be obtained from cells growing on rich media supplemented with the drug (15).

mutant (drug-resistant) and wild-type (drug sensitive) bacteria; it also allows selection to be imposed on solid media without disturbing the growing clones.

RESULTS AND DISCUSSION Mutation Frequencies of dnaE Mutants. Of all dna mutants tested, only those that were mapped near or at dnaE showed a considerable variation in mutation frequencies when compared with their wild-type parent (strain DG17, which presumably is isogenic to the SG mutants except for its dna + character). Measured frequencies of dnaE SG strains are listed in Table 1, together with DG17 values for the same mutational events (Leu- Leu+, ValS ValR, AzaS - AzaR). A stimulation factor is calculated by dividing each SG frequency by the corresponding value obtained for DG17. Hence stimulation factors will be greater than unity for mutator strains and less than unity for antimutators. The results in Table 1 confirm previous observations that dnaE strains act as mutators in permissive temperatures (10); moreover, several of these mutants (especially SG345, SG902, SG1424, and SG1556) exhibited powerful mutator action for all three independent events tested at levels (stimulation factors) observed previously with mut strains only, or T4 phage defective in gene 43 (2, 3, 18). It is believed that the mutator action exhibited by dnaE mutants at permissive temperatures is caused by erratic functioning of DNA polymerase III (dnaE gene product) during DNA replication, as is the case with gene 43 polymerase in phage T4. However, unlike T4, no antimutator effect for dnaE mutants has been observed, and this is also evident from results in Table 1. (Stimulation factors for strain SG1484 are less than unity, but the deviations from wild-type frequencies are well within experimental error, hence SG1484 is probably not an antimutator.) Mutation Frequencies of Temperature-Resistant Revertants. In order'to show that the mutator effect of SG345, SG902, and SG1424 was caused by the temperature-sensitive mutation in their dnaE gene, spontaneous temperature-resistant rever,

Proc. Natl. Acad. Sci. USA 74 (1977)

Genetics: Sevastopoulos and Glaser

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Table 2. Mutation frequencies (at 340) in temperature-resistant derivatives of SG dnaE strains Azas _ AzaR Vals- VaIR Leu - Leu+ Factort Leu+ X 109 Factort VaIR X 108 Factort AzaR X 105 Ts+ strain* 345-1 345-2 345-3 902-1 902-2 902-3 1424-1 1424-2 1424-3 1424-4

5.8 9.1 6.7 12.1 19.6 9.9 3.6 4.2 7.1 6.3

1.6 2.6 1.9 3.4 5.6 2.8 1.0 1.2 2.0 1.8

4.5 10.2 2.8 20.4 18.5 13.0 3.3 4.6 5.0 5.1

DG17 (wild type)

3.5

1.0

2.2

2.0 4.6 1.3 9.3 8.4 5.9 1.5 2.1 2.3 2.3 1.0

0.1 0.1 0.1 0.5 1.1 0.9 0.1 0.1 0.2 0.2

1.0 1.0 1.0 5.0 11.0 9.0 1.0 1.0 2.0 2.0

0.1

1.0

* Temperature-resistant (Ts+) derivatives of SG345, SG902, and SG1424 were obtained by plating cells on an L plate and incubating at 410. Cultures to measure frequencies were started from independent Ts+ colonies. t See definition for Table 1.

Table 3. Temperature dependence of rates of spontaneous mutation to ampicillin resistance (AmpS - AmpR) and rifampicin resistance (Rifs- RifR) for strains SG902 dnaE672 and DG17 dna+* Rifs - RifR AmpS - AmpR Factort SG902, rate X 109 DG17, rate X 109 Factort SG902, rate X 109 DG17, rate X 109 57 19 0.10 1,100 1,600 160 48 110 0.40 5,300 1,300 520 153 150 1.1 23,000 1,636 1,800 305 200 2.2 61,000 1,773 3,900 * Values in this table are averages from three independent experiments for both strains at all four temperatures. Rates are given as mutations per cell per generation. t Stimulation factor: SG902 mutation rate divided by corresponding value for DG17, i.e., a measure of mutator activity.

Temperature, 'C 25 30 34 37

tants of these strains were obtained and their mutation frequencies were measured. It was also speculated that perhaps some reversions might result in mutator levels falling below wild-type mutator activity, thus creating an antimutator effect. The results for ten independent temperature-resistant revertants (Ts+) are listed in Table 2. As can be seen, mutator activities were reduced drastically to approximately wild-type levels, indicating that the original increase in mutation frequencies was due to action of the defective dnaE gene. It is conceivable, albeit highly improbable, that the powerful mutator action of some strains in Table 1 may be caused by some unknown mutator (mut) function also present in the SG genome. However, because the high mutator activity is not present in the Ts+ revertants, there would have to be some interaction between dnaE and mut products in the putative dnaE mut double mutant that disappears in the dnaE + mut revertant, i.e. dnaE + product suppresses the function of mut. The opposite has been reported (19), namely that DNA synthesis was suppressed in a mutTl dnaE293 double mutant (mut + dnaE293 was not a mutator). No antimutator action was detected in temperature-resistant revertants of strains SG345, SG902, and SG1424 (Table 2). Temperature Dependence of Mutation Rates. The mutagenic behavior of strains SG902 (Table 1) and DG17 (wild-type) was studied at four different temperatures by measuring rates of mutation to ampicillin and rifampicin resistance (AmpS RifR). AmpR, Rifs Mutation rates (m) were computed using the formula m = ln2[(R2 R1)/(N2 N1)], in which RI and R2 are the number of drug-resistant colonies -

-

(mutant clones) counted on membranes transferred to ampicillin or rifampicin plates at times 1 and 2; N1 and N2 are the corresponding total population sizes as determined by viable counts (20, 21). The results are summarized in Table 3. Mutation rates are

SG902 10-6 C

0

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dn0 10-9

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tan

G92nE7 DG17 n

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Genetics: Sevastopoulos and Glaser

Proc. Nati. Acad. Sci. USA 74 (1977) We acknowledge the capable technical assistance of Mrs. Ruth Ford. This investigation was supported by U.S. Public Health Service Research Grants GM13244, GM19439, GM22021, RR00961, and Training Grant 5-TO1-GM00829 to C.G.S., from the National Institute of General Medical Sciences.

= 10-5 _

1.

2.

ization of mutator strains of E. colh K-12," J. Bacteriol. 122, 474-484. 3. Speyer, J. F. (1965) "Mutagenic DNA polymerase," Biochem. Biophys. Res. Commun. 21, 6-8. 4. Treffers, H. P., Spinelli, V. & Belser, N. 0. (1954) "A factor (or

10'6 C.,

E107;

mutator gene)

DG17

5.

0

6.

7.

lo-8

25

Green, M. M. (1970) "The genetics of a mutator gene in Drosophila melanogaster," Mutat. Res. 10, 353-363. Hoess, R. H. & Herman, R. K. (1975), "Isolation and character-

30

35

40

Temperature, 0C FIG. 2. Effect of temperature on rate of spontaneous mutation to rifampicin resistance for strains SG902dnaE672 and DG17

8.

expressed as mutations per cell per generation, and the values listed represent averages from three separate experiments for both strains (SG902, DG17) at each of four temperatures. It can be seen from these results that SG902 remains a powerful mutator in the 25°-370 temperature range. It is interesting to note that stimulation factors (a measure of mutator activity) are rather independent of temperature for the mutation to ampicillin resistance but appear to increase with temperature in the case of mutation to rifampicin resistance. Plots of mutation rates versus temperature, using the results in Table 3, are shown in Figs. 1 and 2. The curves suggest that the temperature dependence can be described by the Arrhenius equation, m = A exp [-Ea/kT], in which m is the mutation rate, k is the Boltzmann constant, Ea is the activation energy of the mutagenic reaction, T is the absolute temperature, and A is a proportionality factor. The activation energies for the Amps 1- AmpR, Rifs -- RifR mutation reactions in strains SG902 and DG17 can be calculated by replotting the data in Table 3 as log m versus I/T and determining the slope of that linear curve. § In doing so, it was found (curves and calculations not shown) that Amps AmpR activation energies were 2.2 eV (SG902) and 2.0 eV (DG17), while those for the Rifs -- RifR reaction amounted to 2.4 eV (SG902) and 0.8 eV (DG17) (1 eV = 1.6 X 10-'9 J). Similar values of activation energy have been reported for spontaneous or induced mutations in bacteriophage T4 (9, 22), and for spontaneous mutations in E. coli strains with polA or recA defects (9). The differences in activation energies may imply that different mechanisms or pathways of mutation are involved in the two classes of mutation reactions and in the two strains that we observed.

9.

dna+.

§ The Arrhenius equation can be written as log m = (-E,/2.3kT) + log A; the plot of log m against 1/T is a straight line, and the activation energy E0 is calculated from its slope.

10. 11.

12.

influencing mutation rates in Escherichia coil,"

Proc. Natl. Acad. Sci. USA 40, 1064-1071. Von Borstel, R. C., Cain, K. T. & Steinberg, C. M. (1971) "Inheritance of spontaneous mutability in yeast," Genetics 60, 17-27. Cox, E. C. (1976) "Bacterial mutator genes and the control of spontaneous mutation," Annu. Rev. Genet. 10, 135-156. Drake, J. W., Allen, E. F., Forsberg, S. A., Preparata, BR. M. & Greening, E. 0. (1969) "Spontaneous mutation: Genetic control of mutation rates in bacteriophage T4," Nature 221, 11281132. Muzyczka, N., Poland, R. L. & Bessman, M. J. (1972) "Studies on the biochemical basis of spontaneous mutation. I. A comparison of the DNA polymerases of mutator, antimutator and wild-type strains of bacteriophage T4," J. Biol. Chem. 247, 7116-7122. Kondo, S. (1973) "Evidence that mutations are induced by errors in repair and replication," Genetics (Suppl.) 73, 109-122. Hall, R. M. & Brammar, W. J. (1973) "Increased spontaneous mutation rates in mutants of E. coli with altered DNA polymerase III," Mol. Gen. Genet. 121, 271-276. Cox, E. C. (1970) "Mutator gene action and the replication of bacteriophage DNA," J. Mol. Biol. 50, 129-135. Sevastopoulos, C. G., Wehr, C. T. & Glaser, D. A. (1977) "Large-scale automated isolation of Escherichia coli mutants with thermosensitive DNA replication," Proc. Natl. Acad. Sci. USA

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13. Wehr, C. T., Waskell, L. & Glaser, D. A. (1975) "Characteristics of cold-sensitive mutants of Escherlchia coli K-12 defective in DNA replication," J. Bacteriol. 121, 99-107. 14. Luria, S. E., Adams, J. N. & Ting, R. C. (1960) "Transduction of lactose utilizing ability among strains of E. coli and S. dysenteriae and the properties of the transducing phage particles," Virology

12,348-390.

15. Miller, J. H. (1972) Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), p. 224. 16. Newcombe, H. B. (1949) "Origin of bacterial variants," Nature 17. 18.

164, 150-152.

Matney, T. S. (1955) "New uses of membrane filters. I. The determination of the spontaneous mutation rate of Escherichia coli to streptomycin resistance," J. Bacteriol. 69, 101-102. Cox, E. C. & Yanofsky, C. (1969) "Mutator gene studies in

Escherichia coli," J. Bacteriol. 100, 390-397. 19. Cox, E. C. (1973) "Mutator gene studies in Escherichia coli: The mutT gene," Genetics (Suppl.) 73, 67-80. 20. Beale, G. H. (1948) "A method for the measurement of mutation rate from phage sensitivity of phage resistance in E. coil," J. Gen. Microbiol. 2, 131-136. 21. Newcombe, H. B. (1948) "Delayed phenotypic expression of spontaneous mutations in E. coli," Genetics 33, 447-452. 22. Ishiwa, H., Yan, Y. & Kondo, S. (1964) "Temperature-dependence of lethal and mutagenic actions of HNO2 on phage T4," Biochim. Biophys. Acta 91, 160-163.

Mutator action by Escherichia coli strains carrying dnaE mutations.

Proc. Natl. Acad. Sci. USA Vol. 74, No. 9, pp. 3947-3950, September 1977 Genetics Mutator action by Escherichia coli strains carrying dnaE mutations...
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