JOURNAL OF BACTERIOLOGY, Mar. 1978, p. 1197-1202 0021-9193/78/0133-1197$02.00/0 Copyright X) 1978 American Society for Microbiology
Vol. 133, No. 3 Printed in U.S.A.
Isolation of an Escherichia coli K-12 dnaE Mutation as a Mutator E. BRUCE KONRAD
Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 31 August 1977
Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA. DNA replication in Escherichia coli makes about one error per 109 base pairs replicated (4, 18). One might anticipate that proteins involved in replication could be altered through mutation in such a way as to lower this high level of fidelity. Such "replicative" mutator mutations would be useful in understanding how fidelity is achieved. However, the only mutator mutations presently known that affect an enzyme that participates in DNA replication are temperaturesensitive, conditionally lethal mutants in the dnaE gene (6). This is the structural gene for DNA polymerase III, the E. coli polymerase required for replication (5, 14, 16). These polymerase III mutants have low mutator activities compared with other mutator mutations, which do not lie in genes known to be involved in DNA replication. The proteins altered in these other mutator strains have not been identified (3). MATERIALS AND METHODS Strains. Bacterial strains used in this work were
were similarly assayed on YT plates supplemented with 100 ,tg of rifampin per ml, and viable titers were assayed on YT plates. Plates were incubated at 30°C for 2 days. Viable titers measured in both ways agreed closely with numbers of cells determined with a Petroff-Hauser counter. Thus, the "cell density artifact" discussed by Witkin (21) does not affect these measurements. Partially purified extracts containing DNA polymerase III were prepared according to Wickner et al. (20), up to the ammonium sulfate precipitation and dialysis. (Strains that were poU polB were used.) These extracts were diluted in polymerase III diluent and assayed for polymerase III activity at 300C for 30 min. Reaction mixtures (50 pl) contained 30 mM
tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5), 7 mM MgCl2, 2.5 mM dithiothreitol, 40 uM each dATP, dGTP, and dCTP, 10 ,uM [3H]dTTP (150 cpm/pmol), 2 yM spermidine, 0.4 mg of bovine serum albumin per ml, 1.2 mM nicked salmon sperm DNA, and 5 il of diluted cell extract. Polymerase III diluent (7) was 20 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2), 2 mM dithiothreitol, and 0.5 mg of bovine serum
per ml. Nicked salmon sperm DNA was prederived from E. coli K-12. Their origins and genotypes albumin to Wickner et al. (19). Protein conare given in Table 1. Nomenclature is from Bachmann pared according were determined centrations according to Lowry et al. et al. (1). The generalized transducing phage Plvir was determined of cultures DNA content The (12). was provided by Lee Rosner. Microbiological methods. Cultures were grown according to Burton (2). in LB medium and plated on YT or M63 plates (13). RESULTS Mutagenesis was done using ethyl methane sulfonate (13). Transduction using Plvir and bacterial matings In an attempt to isolate other types of repliwere done according to Miller (13). cative mutator mutations, the following method To obtain relative mutation frequencies, about 200 was developed to allow screening of large numcells were inoculated into each of 10 5-ml LB broth bers of colonies for the mutator trait. A lac Hfr cultures. These cultures were allowed to grow to saturation with aeration at the indicated temperatures. strain (H1023e) (Table 1) was mutagenized and Lac' mutants were titrated by spreading 0.1 ml on plated on lactose tetrazolium indicator plates minimal (M63 medium) plates containing 2% lactose (15) to give 500 colonies per plate. (On these and 2 Ag of glucose per ml. Viable titers were assayed indicator plates, lactose-negative colonies are on the same medium. Rifampin-resistant mutants red, whereas lactose-positive colonies are white.) 1197
1198
J. BACTERIOL.
KONRAD TABLE 1. E. coli K-12 strainsa
Strain
Source or tionconstruc-
Genotype
AB1157 AT980 CD4 E486 H1023e HMS83 HMS126 KL16-99 KS231 KS378 KS484 KS489 KS517
F- thr leu thi argE his proA lac galK mtl xyl ara tsx supE stre Hfr KL16 thi rel-1 dapD Hfr Cavalli metB metD proA rel-1 lac mal tsx F- thi thr leu met dnaE486 thy' dra lac tonA supE strr Hfr P4X metB lac tonA F- poLA polBlO klacZ rha lys thy F- poUl polB100 thy endI rha dnaE1026 Hfr KL16 recAl thi F- pts140 lacYA482 str' F- proA lac galK trp mal mtl metE ara recA56 str' F- lac trp pyrF metB str' F- purA proA thr leu thi lac tonA tsx str' F- polA polB100 lacZ rha lys thy tonA
KS521 KS526 KS532 KS533 KS534
F- metB dapD tonA stre F- recAl lacYA482 tonA strr F- lacYA482 str' F- metD metB recAl str' F- lacYA482 tonA str'
MUilO
F- ptsl40 lacYA482 dnaE9 tonA strr
MU112
F- recAl lacYA482 dnaE9 tonA stre
MU113
F- poUl polB100 dnaE9 lacZ rha lys thy tonA
MU123 MU130 MU137
F- thr met thi dra supE dnaE486 tonA DE5 recAI str' F- lac recAl dnaE9 ara tonA str' F- lacYA482 dnaE9 tonA stre
MU151
F- pts- 140 lacYA482 dnaE486 tonA strr
MU154
F- recAl lacYA482 dnaE486 tonA strr
MU155
F- lacYA482 dnaE486 tonA strr
MU157 MU159 MUB121 F'104/AB2463
F'507/metD metB recAl strr F'507/proA lac galK trp mal mtl metE ara recA56 strr Hfr P4X metB lac tonA dnaE9 F'104/thi thr leu argE his proA recA13 mtl xyl ara galK lac tsx supE
E. Adelberg A. L. Taylor W. Epstein J. Wechsler E. B. Konrad J. Campbell H. Shizuya K. B. Low E. B. Konrad E. B. Konrad E. B. Konrad E. B. Konrad Plvir MUB121 x HMS83 E. B. Konrad KL16-99 x KS231 E. B. Konrad E. B. Konrad Plvir KS231 x MU137 Plvir MUB121 x KS231 x KL16-99 MUllO Plvir MUB121 x HMS83 E. B. Konrad E. B. Konrad Plvir KS231 x MU112 Plvir E486 x KS231 x KL16-99 MU151 Plvir KS231 x MU154 MUB121 x KS533 MU157 x KS378 EMS H1023e K. B. Low
sty, DE5 is the deletion (lac-pro) Xlll of Jacob (10). The F'104 episome includes the region thr-argF (10). The F'507 episome includes thr-proA and carries the mutation dnaE9 (see text). The origin of transfer of HfrP4X lies at 10 min on the E. coli map (10). Transfer occurs in the clockwise direction. Markers trp and pyrF lie at 27 and 28 min, respectively. lacYA482 is an amber mutation. a
Lac' mutants that arise in lac colonies appear as white papillae on the colonies' surface after 3 days of incubation at 30°C. A lac colony that is a mutator will show more papillae than a wildtype lac colony to an extent dependent on the strength of the mutator. I have confirmed this using several known mutator strains (mutT, mutL, mutR, mutU, and mutS) (3). With the use of this isolation method, 1,250 independent mutator strains were obtained. These were screened for temperature sensitivity by streaking on YT plates and testing for growth at 44°C. (One might expect that mutations that
alter a protein essential for DNA replication could be conditionally lethal.) Seventy-six temperature-sensitive mutators were thus recovered. To determine whether both temperature sensitivity and the mutator activity were due to a single mutation, each of these (Hfr) strains was crossed with an F- strain (KS484) carrying markers (pyrF and trp) distal to the point of origin of the donor strain (1, 10, 13). Mating was allowed to proceed for 3 h at 370C, so pyre trp+ recombinants received most or all of the donor chromosome. Sixteen recombinants from each cross were then screened to determine
E. COLI MUTATOR STRAIN
Vol.. 133, 1978
1199
whether the mutator and temperature-sensitive and metD+ str' exconjugants were selected traits segregated together as would be expected (MU157). One of the episomes recovered from if they were due to the same lesion. Only one this cross, F'507, was found to carry other loci mutation, dnaE9, proved to be a temperature- between proA and thr (leu, ara, and dapD) (1) sensitive mutator by this test. as well as metD. To prove that F'507 also carried Reversion studies in which a temperature-re- dnaE9, this episome was introduced into a recA+ sistant revertant of dnaE9 was selected at 44°C dnaE+ strain (AB1157), and it was shown that and then tested for its mutator property also the resulting F' dnaE9/dnaE+ heterogenote indicated that the same lesion was responsible segregated temperature-sensitive F' dnaE9/ for both mutator and temperature-sensitive dnaE9 homogenotes. This was done by streakproperties (Table 2). ing out F'507/AB1157 on YT plates incubated dnaE9 was mapped between 0 and 11 min on at 30°C. About 30% of the resultant colonies the E. coli chromosome, using the rapid map- were found to be temperature sensitive and reping technique of Low (11). Further localization tained the ability to transfer the F'507 episome. to between the tonA and metD loci was achieved dapD tonA proA dneE9 metD using Plvir transduction (13) in two-factor crosses (Fig. 1). This order was confirmed by a three-factor cross (Fig. 2). These results sug" '°.06 .49 .013 gested that dnaE9 might be a dnaE mutation since dnaE is known to lie within this interval .82-4-72
.65
-
(1).
To obtain further genetic evidence that dnaE9 was a dnaE mutation, an in vivo complementation test with a known dnaE mutation (dnaE486) (5, 14, 16) was performed. First, it was shown that both dnaE9 and dnaE486 were recessive to dnaE+. This was done by introducing an episome (F'104) (10) known to carry the region of the chromosome including pro', ara+, and dnaE+ into a recombination-deficient pro dnaE486 strain (MU123) and likewise into a recombination-deficient ara dnaE9 strain (MU130); pro' and ara+ were selected, respectively. The presence of the episome conferred the ability to grow at high temperature (44°C) on both these strains. Thus, the wild-type (dnaE+) allele was dominant to dnaE9 and dnaE486. To determine whether dnaE9 could complement dnaE486, it was necessary to isolate an F' factor carrying dnaE9. This was done using the method of Low (9): an Hfr strain carrying dnaE9 str (MUB121) was crossed with an F- recA metD stre strain (KS533) (Fig. 1),
.14
FIG. 1. Position of the dnaE9 locus on the E. coli map. Cotransduction frequencies were determined by the following Plvir crosses: proA-dnaE9, MUB121 x KS489; metD-proA, metD-tonA, metD-dnaE9, MUB121 x CD4; dapD-dnaE9, dapD-tonA, MUB121 x AT980; dnaE9-tonA, W3110 x MUB121; dapDmetD, CD4 x K5521. In each cross approximately 200 transductants were scored. The order dapD tonA metD given by these data conflicts with the order tonA dapD metD given by Bachmann et al. (1). dnaE9
tonA-
P1 host
metO+
~~~~~~~~~~~~~I
I
Il
tonA+ Genotype dnaE- tonA-
dnaE" tonA+ dnaE- tonA+ dnaE' tonA-
dnaE9+
metD-
No. of Recombinants 39 31 22 2
FIG. 2. Three-point cross between metD, dnaE9, and tonA. These results were obtained from the cross Plvir MUB121 x CD4.
TABLE 2. Mutator activity of dnaE9 and dnaE486 strains' lac+ mutants/cell x 10-' Genotype rif mutants/cell x 10-8 Strain
dnaE
recA
260C"
370C
410C 1.9 1.5 140 180
260C 1.5 0.69 1.9 10
370C
+ 1.9 KS532 1.5,1.1 1.1,1.5 + 1.9 1.8 recA KS526 1.3,1.2 + 23 dnaE9 2.5 MU137 41,31 dnaE9 recA 27 140 MU112 220,110 + 1.0 dnaE486 1.0 50 MU155 211,143 1.7 recA 2.3 50 dnaE486 MU154 147,146 dnaE9 dnaE9 + 2.5 1.5 2.0 2.0 1.5 Revertant Revertant adnaE9 revertant is a spontaneous revertant of dnaE9 (MU137) selected for growth on YT plates at 44°C. Growth temperature. +
1200
J. BACTERIOL.
KONRAD
Finally, the F'507 (MU159) episome was introduced into a recA pro dnaE486 strain (MU123), selecting for transfer of prot. The resulting F' dnaE9/dnaE486 diploid was unable to grow at nonpermissive temperatures. This absence of complementation showed that dnaE9 and dnaE486 probably lay in the same gene. Attempts to demonstrate thermolability in vitro of partially purified DNA polymerase III from a dnaE9 strain deficient in DNA polymerases -I and II (MU113) were not successful (although thermolability of another dnaE mutation [dnaEl026] [5, 17] was confirmed) (Fig. 3). Thus, the following experiment was undertaken to see whether thermolability or temperature-sensitive synthesis of the dnaE9 DNA polymerase Ill could be detected in vivo. Exponentially growing cells of dnaE9 and dnaE4 strains were shifted from the permissive temperature to 44°C (Fig. 4). At intervals after this shift, samples were taken, and the activity of polymerase III and the protein concentration were determined. The results showed that in the dnaE' strain total jolymerase III activity increased roughly in parallel with the optical density of the culture at 595 nm. The specific activity for polymerase III also increased almost twofold, perhaps in response to the more rapid growth 100
dnsE9
w0 E10
dnaE+
dnaE1026 1
2
3
4 5 6 MIN. AT 4e
7
8
FIG. 3. Heat inactivation of DNA polymerase III activity in partially purified extracts. Extracts containing DNA polymerase III were prepared from 3liter samples. These extracts, which contained 35 mg ofproteinper ml, were diluted sixfold into polymerase III diluent and incubated at 42°C. At the indicated timnes, samples were removed, diluted 20-fold, and assayed for polymerase III activity. 1X00% activity was 18.0, 11.6, and 11.4 pmol of dTMP incorporated in 30 min for strains KS517, MU113, and HMS126, respectively. Symbols: 0, wild type (K5517); A, dnaE9 (MU113); 0, dnaE1026 (HMS126).
rate at 440C. With the dnaE9 strain, total polymerase III activity remained constant after the shift, although both the optical density and viable titers increased almost twofold. The specific activity of polymerase III gradually declined about twofold. These results are compatible with either temperature-sensitive synthesis of the mutant polymerase or gradual inactivation of the mutant polymerase in vivo at 44°C. Either alternative would be consistent with the relatively slow shutoff of DNA synthesis in dnaE9 cultures after a shift to nonpermissive temperatures (Fig. 5). Wechsler and Gross (16) have also reported a mutation that maps in the dnaE region (dnaE293) and shows a gradual decline of DNA synthesis at the nonpermissive temperature. Similarly to dnaE9, polymerase III from dnaE293 is not thermolabile in vitro (14). The thermosensitivity of this enzyme in vivo or of its synthesis has not been reported, nor has its mutator character been described. In contrast, other dnaE mutations, such as dnaE489, show a rapid shutoff of DNA synthesis at the nonpermissive temperatures (data not shown) and thermolability of their polymerase III in vitro (14, 16, 17). Although dnaE486 and dnaE9 differ from each other in the response of their DNA synthesis to a shift to nonpermissive temperatures, they both show temperature-dependent mutator activity (Table 2). dnaB(Ts), a mutation that causes an abrupt cessation of DNA replication at nonpermissive temperatures, as well as a number of other agents that block DNA synthesis or insult DNA, induce the "SOS" error-prone repair system and its concomitant mutagenesis. The recA gene product is necessary for this SOS error-prone repair (21). Introducing a recAl allele into dnaE486 and dnaE9 strains did not decrease their frequencies of mutation; in fact, it substantially increased the frequencies of mutation with dnaE9 strains at 26 and 370C. This result suggested that SOS error-prone repair may not be involved in these mutageneses (21).
DISCUSSION An extensive search has yielded only one temperature-sensitive, conditionally lethal mutator mutation. This has proved to be a dnaE mutation (dnaE9) by both genetic and enzymological criteria. Cultures of dnaE9 showed a slow shutoff of DNA synthesis upon shift to a nonpermissive temperature. This slow shutoff might reflect either temperature-sensitive synthesis of polymerase III or slow inactivation of the enzyme in vivo under nonpermissive conditions. dnaE9 showed mutator activity which was not dependent on the recA gene product and in this respect is like dnaE486, a polymerase III
E. COLI MUTATOR STRAIN
VOL. 133, 1978
c1.1
10'tlilO
~~~~~~~~~~~~~10io0~
0
5*
C3
3)X|00 440
.01
0
1201
200
100
300 1440 300 0 TIME (min)
100
1
200
10
300
FIG. 4. In vivo inactivation of DNA polymerase III from a dnaE9 strain (MU113) grown at the nonpermissive temperature. Cells were grown at 30°C and then shifted to 44°C. At the times after the shift indicated by arrows, 1.5-liter samples were taken. DNA polymerase III was partially purified and assayed at 30°C as in Fig. 3. Total polymerase activity is the total activity per extract made from each 1.5-liter culture sample. The increase in viable titer in these experiments approximately paralleled that of the optical density at 595 nm (OD506 (data not shown). (a) A dnaE9 strain (MU113); (b) a wild-type strain (KS517).
100.0
100.0
X
6
0I
0 O___
O
10.0-/80
10.0
-
*0.0~~~~~~~~~~~~
100
200
300
400 500
100 200
300
Time (min)
FIG. 5. DNA content of cultures shifted to a nonpermissive temperature. Exponentially growing cells were shifted from 30 to 44°C at the indicated time, and their DNA content was determined. Cells were kept at an optical density at 595 nm (OD50 of between 0.2 and 0.5 by progressive dilution. (a) A dnaE9 strain (MU127); (b) a wild-type strain (KS534).
1202
KONRAD
mutation which, contrary to dnaE9, shows a rapid cessation of DNA synthesis on shift to a nonpermissive temperature. This finding suggests that the mutator activities of dnaE9 and dnaE486 are not due directly to the SOS errorprone repair system. Alternatively, the alterations of DNA polymerase III in these strains might increase mutation frequencies directly by decreasing the accuracy of nucleotide selection during incorporation and/or decreasing the removal of incorrect nucleotides, presumably by the 3' to 5' exonuclease activity associated with the polymerase (8), or indirectly through some error-prone repair system that is not recA dependent. ACKNOWLEDGMNENTS I am grateful to Nancy G. Nossal of this laboratory for useful discussions and assistance with assays for DNA polymerase activity. I also think the E. coli Genetic Stock Center (Department of Human Genetics, Yale University School of Medicine, New Haven, Conn.) for providing some of the strains used in this work.
LITERATURE CITED B. Bachmann, 1. J., K. B. Low, and C. L. Taylor. 1976. Recalibrated linkage map for Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 2. Burton, K. 1956. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemistry 62:315-323. 3. Cox, E. C. 1976. Bacterial mutator genes and the control of spontaneous mutation. Annu. Rev. Genet. 10:135-156. 4. Drake, J. W. 1970. The molecular basis of mutation. Holden-Day, San Francisco. 5. Gefter, M. L., Y. Hirota, T. Kornberg, J. A. Wechsler, and C. Bernoux. 1971. Analysis of DNA polymerases II and III in mutants of Escherichia coli thermosensitive for DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 68:3150-3153. 6. Hall, R. M., and W. J. Brammer. 1973. Increased spontaneous mutation rates in mutants of Escherichia coli with altered DNA polymerase III. Mol. Gen. Genet. 121:271-276. 7. livingston, D. M., D. C. Hinkle, and C. C. Richardson.
J. BACTERIOL. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties. J. Biol. Chem. 250:461-469. 8. Livingston, D. M., and C. C. Richardson. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated activities. J. Biol. Chem. 260:470-478. 9. Low, B. 1968. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 60:160-167. 10. Low, K. B. 1972. Escherichia coli K-12 F-prime factors, old and new. Bacteriol. Rev. 36:587-607. 11. Low, K. B. 1973. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J. Bacteriol. 113:798-812. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Richardson, C. C., J. L. Campbell, J. W. Chase, D. V. Hinkle, D. M. Livingston, H. L Mulcahy, and H. Shizuya. 1973. DNA polymerases of Escherichia coli, p. 65-69. In R. D. Wells and R. B. Inman (ed.), DNA synthesis in vitro. University Park Press, Baltimore. 15. Signer, E. R., J. R. Beckwith, and S. Brenner. 1965. Mapping of suppressor loci in Escherichia coli. J. Mol. Biol. 14:153-160. 16. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. 17. Wechsler, J. A., V. Nuesslein, B. Otto, A. Klein, F. Bonhoeffer, R. Herrmann, L Gloger, and H. Shaller. 1973. Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication. J. Bacteriol. 113:1381-1388. 18. Whitfield, Jr., H. J., R. C. Martin, and B. N. Ames. 1966. Classification of aminotransferase (C gene) mutants in the histidine operon. J. Mol. Biol. 21:335-355. 19. Wickner, R. B., B. Ginsberg, I. Berkower, and G. Hurwitz. 1972. Deoxyribonucleic acid polymerase II of Escherichia coli. I. The purification and characterization of the enzyme. J. Biol. Chem. 247:489-497. 20. Wickner, S., M. Wright, I. Berkower, and J. Hurwitz. 1974. Isolation of dna gene products of Escherichia coli, p. 195-215. In R. B. Wickner (ed.), DNA replication. Marcel Dekker, Inc., New York. 21. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible deoxyribonucleic acid repair in Escherichia coli. Bacteriol. Rev. 40:869-907.
Vol. 133, No. 3 Printed in U.S.A.
Isolation of an Escherichia coli K-12 dnaE Mutation as a Mutator E. BRUCE KONRAD
Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received for publication 31 August 1977
Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA. DNA replication in Escherichia coli makes about one error per 109 base pairs replicated (4, 18). One might anticipate that proteins involved in replication could be altered through mutation in such a way as to lower this high level of fidelity. Such "replicative" mutator mutations would be useful in understanding how fidelity is achieved. However, the only mutator mutations presently known that affect an enzyme that participates in DNA replication are temperaturesensitive, conditionally lethal mutants in the dnaE gene (6). This is the structural gene for DNA polymerase III, the E. coli polymerase required for replication (5, 14, 16). These polymerase III mutants have low mutator activities compared with other mutator mutations, which do not lie in genes known to be involved in DNA replication. The proteins altered in these other mutator strains have not been identified (3). MATERIALS AND METHODS Strains. Bacterial strains used in this work were
were similarly assayed on YT plates supplemented with 100 ,tg of rifampin per ml, and viable titers were assayed on YT plates. Plates were incubated at 30°C for 2 days. Viable titers measured in both ways agreed closely with numbers of cells determined with a Petroff-Hauser counter. Thus, the "cell density artifact" discussed by Witkin (21) does not affect these measurements. Partially purified extracts containing DNA polymerase III were prepared according to Wickner et al. (20), up to the ammonium sulfate precipitation and dialysis. (Strains that were poU polB were used.) These extracts were diluted in polymerase III diluent and assayed for polymerase III activity at 300C for 30 min. Reaction mixtures (50 pl) contained 30 mM
tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.5), 7 mM MgCl2, 2.5 mM dithiothreitol, 40 uM each dATP, dGTP, and dCTP, 10 ,uM [3H]dTTP (150 cpm/pmol), 2 yM spermidine, 0.4 mg of bovine serum albumin per ml, 1.2 mM nicked salmon sperm DNA, and 5 il of diluted cell extract. Polymerase III diluent (7) was 20 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.2), 2 mM dithiothreitol, and 0.5 mg of bovine serum
per ml. Nicked salmon sperm DNA was prederived from E. coli K-12. Their origins and genotypes albumin to Wickner et al. (19). Protein conare given in Table 1. Nomenclature is from Bachmann pared according were determined centrations according to Lowry et al. et al. (1). The generalized transducing phage Plvir was determined of cultures DNA content The (12). was provided by Lee Rosner. Microbiological methods. Cultures were grown according to Burton (2). in LB medium and plated on YT or M63 plates (13). RESULTS Mutagenesis was done using ethyl methane sulfonate (13). Transduction using Plvir and bacterial matings In an attempt to isolate other types of repliwere done according to Miller (13). cative mutator mutations, the following method To obtain relative mutation frequencies, about 200 was developed to allow screening of large numcells were inoculated into each of 10 5-ml LB broth bers of colonies for the mutator trait. A lac Hfr cultures. These cultures were allowed to grow to saturation with aeration at the indicated temperatures. strain (H1023e) (Table 1) was mutagenized and Lac' mutants were titrated by spreading 0.1 ml on plated on lactose tetrazolium indicator plates minimal (M63 medium) plates containing 2% lactose (15) to give 500 colonies per plate. (On these and 2 Ag of glucose per ml. Viable titers were assayed indicator plates, lactose-negative colonies are on the same medium. Rifampin-resistant mutants red, whereas lactose-positive colonies are white.) 1197
1198
J. BACTERIOL.
KONRAD TABLE 1. E. coli K-12 strainsa
Strain
Source or tionconstruc-
Genotype
AB1157 AT980 CD4 E486 H1023e HMS83 HMS126 KL16-99 KS231 KS378 KS484 KS489 KS517
F- thr leu thi argE his proA lac galK mtl xyl ara tsx supE stre Hfr KL16 thi rel-1 dapD Hfr Cavalli metB metD proA rel-1 lac mal tsx F- thi thr leu met dnaE486 thy' dra lac tonA supE strr Hfr P4X metB lac tonA F- poLA polBlO klacZ rha lys thy F- poUl polB100 thy endI rha dnaE1026 Hfr KL16 recAl thi F- pts140 lacYA482 str' F- proA lac galK trp mal mtl metE ara recA56 str' F- lac trp pyrF metB str' F- purA proA thr leu thi lac tonA tsx str' F- polA polB100 lacZ rha lys thy tonA
KS521 KS526 KS532 KS533 KS534
F- metB dapD tonA stre F- recAl lacYA482 tonA strr F- lacYA482 str' F- metD metB recAl str' F- lacYA482 tonA str'
MUilO
F- ptsl40 lacYA482 dnaE9 tonA strr
MU112
F- recAl lacYA482 dnaE9 tonA stre
MU113
F- poUl polB100 dnaE9 lacZ rha lys thy tonA
MU123 MU130 MU137
F- thr met thi dra supE dnaE486 tonA DE5 recAI str' F- lac recAl dnaE9 ara tonA str' F- lacYA482 dnaE9 tonA stre
MU151
F- pts- 140 lacYA482 dnaE486 tonA strr
MU154
F- recAl lacYA482 dnaE486 tonA strr
MU155
F- lacYA482 dnaE486 tonA strr
MU157 MU159 MUB121 F'104/AB2463
F'507/metD metB recAl strr F'507/proA lac galK trp mal mtl metE ara recA56 strr Hfr P4X metB lac tonA dnaE9 F'104/thi thr leu argE his proA recA13 mtl xyl ara galK lac tsx supE
E. Adelberg A. L. Taylor W. Epstein J. Wechsler E. B. Konrad J. Campbell H. Shizuya K. B. Low E. B. Konrad E. B. Konrad E. B. Konrad E. B. Konrad Plvir MUB121 x HMS83 E. B. Konrad KL16-99 x KS231 E. B. Konrad E. B. Konrad Plvir KS231 x MU137 Plvir MUB121 x KS231 x KL16-99 MUllO Plvir MUB121 x HMS83 E. B. Konrad E. B. Konrad Plvir KS231 x MU112 Plvir E486 x KS231 x KL16-99 MU151 Plvir KS231 x MU154 MUB121 x KS533 MU157 x KS378 EMS H1023e K. B. Low
sty, DE5 is the deletion (lac-pro) Xlll of Jacob (10). The F'104 episome includes the region thr-argF (10). The F'507 episome includes thr-proA and carries the mutation dnaE9 (see text). The origin of transfer of HfrP4X lies at 10 min on the E. coli map (10). Transfer occurs in the clockwise direction. Markers trp and pyrF lie at 27 and 28 min, respectively. lacYA482 is an amber mutation. a
Lac' mutants that arise in lac colonies appear as white papillae on the colonies' surface after 3 days of incubation at 30°C. A lac colony that is a mutator will show more papillae than a wildtype lac colony to an extent dependent on the strength of the mutator. I have confirmed this using several known mutator strains (mutT, mutL, mutR, mutU, and mutS) (3). With the use of this isolation method, 1,250 independent mutator strains were obtained. These were screened for temperature sensitivity by streaking on YT plates and testing for growth at 44°C. (One might expect that mutations that
alter a protein essential for DNA replication could be conditionally lethal.) Seventy-six temperature-sensitive mutators were thus recovered. To determine whether both temperature sensitivity and the mutator activity were due to a single mutation, each of these (Hfr) strains was crossed with an F- strain (KS484) carrying markers (pyrF and trp) distal to the point of origin of the donor strain (1, 10, 13). Mating was allowed to proceed for 3 h at 370C, so pyre trp+ recombinants received most or all of the donor chromosome. Sixteen recombinants from each cross were then screened to determine
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1199
whether the mutator and temperature-sensitive and metD+ str' exconjugants were selected traits segregated together as would be expected (MU157). One of the episomes recovered from if they were due to the same lesion. Only one this cross, F'507, was found to carry other loci mutation, dnaE9, proved to be a temperature- between proA and thr (leu, ara, and dapD) (1) sensitive mutator by this test. as well as metD. To prove that F'507 also carried Reversion studies in which a temperature-re- dnaE9, this episome was introduced into a recA+ sistant revertant of dnaE9 was selected at 44°C dnaE+ strain (AB1157), and it was shown that and then tested for its mutator property also the resulting F' dnaE9/dnaE+ heterogenote indicated that the same lesion was responsible segregated temperature-sensitive F' dnaE9/ for both mutator and temperature-sensitive dnaE9 homogenotes. This was done by streakproperties (Table 2). ing out F'507/AB1157 on YT plates incubated dnaE9 was mapped between 0 and 11 min on at 30°C. About 30% of the resultant colonies the E. coli chromosome, using the rapid map- were found to be temperature sensitive and reping technique of Low (11). Further localization tained the ability to transfer the F'507 episome. to between the tonA and metD loci was achieved dapD tonA proA dneE9 metD using Plvir transduction (13) in two-factor crosses (Fig. 1). This order was confirmed by a three-factor cross (Fig. 2). These results sug" '°.06 .49 .013 gested that dnaE9 might be a dnaE mutation since dnaE is known to lie within this interval .82-4-72
.65
-
(1).
To obtain further genetic evidence that dnaE9 was a dnaE mutation, an in vivo complementation test with a known dnaE mutation (dnaE486) (5, 14, 16) was performed. First, it was shown that both dnaE9 and dnaE486 were recessive to dnaE+. This was done by introducing an episome (F'104) (10) known to carry the region of the chromosome including pro', ara+, and dnaE+ into a recombination-deficient pro dnaE486 strain (MU123) and likewise into a recombination-deficient ara dnaE9 strain (MU130); pro' and ara+ were selected, respectively. The presence of the episome conferred the ability to grow at high temperature (44°C) on both these strains. Thus, the wild-type (dnaE+) allele was dominant to dnaE9 and dnaE486. To determine whether dnaE9 could complement dnaE486, it was necessary to isolate an F' factor carrying dnaE9. This was done using the method of Low (9): an Hfr strain carrying dnaE9 str (MUB121) was crossed with an F- recA metD stre strain (KS533) (Fig. 1),
.14
FIG. 1. Position of the dnaE9 locus on the E. coli map. Cotransduction frequencies were determined by the following Plvir crosses: proA-dnaE9, MUB121 x KS489; metD-proA, metD-tonA, metD-dnaE9, MUB121 x CD4; dapD-dnaE9, dapD-tonA, MUB121 x AT980; dnaE9-tonA, W3110 x MUB121; dapDmetD, CD4 x K5521. In each cross approximately 200 transductants were scored. The order dapD tonA metD given by these data conflicts with the order tonA dapD metD given by Bachmann et al. (1). dnaE9
tonA-
P1 host
metO+
~~~~~~~~~~~~~I
I
Il
tonA+ Genotype dnaE- tonA-
dnaE" tonA+ dnaE- tonA+ dnaE' tonA-
dnaE9+
metD-
No. of Recombinants 39 31 22 2
FIG. 2. Three-point cross between metD, dnaE9, and tonA. These results were obtained from the cross Plvir MUB121 x CD4.
TABLE 2. Mutator activity of dnaE9 and dnaE486 strains' lac+ mutants/cell x 10-' Genotype rif mutants/cell x 10-8 Strain
dnaE
recA
260C"
370C
410C 1.9 1.5 140 180
260C 1.5 0.69 1.9 10
370C
+ 1.9 KS532 1.5,1.1 1.1,1.5 + 1.9 1.8 recA KS526 1.3,1.2 + 23 dnaE9 2.5 MU137 41,31 dnaE9 recA 27 140 MU112 220,110 + 1.0 dnaE486 1.0 50 MU155 211,143 1.7 recA 2.3 50 dnaE486 MU154 147,146 dnaE9 dnaE9 + 2.5 1.5 2.0 2.0 1.5 Revertant Revertant adnaE9 revertant is a spontaneous revertant of dnaE9 (MU137) selected for growth on YT plates at 44°C. Growth temperature. +
1200
J. BACTERIOL.
KONRAD
Finally, the F'507 (MU159) episome was introduced into a recA pro dnaE486 strain (MU123), selecting for transfer of prot. The resulting F' dnaE9/dnaE486 diploid was unable to grow at nonpermissive temperatures. This absence of complementation showed that dnaE9 and dnaE486 probably lay in the same gene. Attempts to demonstrate thermolability in vitro of partially purified DNA polymerase III from a dnaE9 strain deficient in DNA polymerases -I and II (MU113) were not successful (although thermolability of another dnaE mutation [dnaEl026] [5, 17] was confirmed) (Fig. 3). Thus, the following experiment was undertaken to see whether thermolability or temperature-sensitive synthesis of the dnaE9 DNA polymerase Ill could be detected in vivo. Exponentially growing cells of dnaE9 and dnaE4 strains were shifted from the permissive temperature to 44°C (Fig. 4). At intervals after this shift, samples were taken, and the activity of polymerase III and the protein concentration were determined. The results showed that in the dnaE' strain total jolymerase III activity increased roughly in parallel with the optical density of the culture at 595 nm. The specific activity for polymerase III also increased almost twofold, perhaps in response to the more rapid growth 100
dnsE9
w0 E10
dnaE+
dnaE1026 1
2
3
4 5 6 MIN. AT 4e
7
8
FIG. 3. Heat inactivation of DNA polymerase III activity in partially purified extracts. Extracts containing DNA polymerase III were prepared from 3liter samples. These extracts, which contained 35 mg ofproteinper ml, were diluted sixfold into polymerase III diluent and incubated at 42°C. At the indicated timnes, samples were removed, diluted 20-fold, and assayed for polymerase III activity. 1X00% activity was 18.0, 11.6, and 11.4 pmol of dTMP incorporated in 30 min for strains KS517, MU113, and HMS126, respectively. Symbols: 0, wild type (K5517); A, dnaE9 (MU113); 0, dnaE1026 (HMS126).
rate at 440C. With the dnaE9 strain, total polymerase III activity remained constant after the shift, although both the optical density and viable titers increased almost twofold. The specific activity of polymerase III gradually declined about twofold. These results are compatible with either temperature-sensitive synthesis of the mutant polymerase or gradual inactivation of the mutant polymerase in vivo at 44°C. Either alternative would be consistent with the relatively slow shutoff of DNA synthesis in dnaE9 cultures after a shift to nonpermissive temperatures (Fig. 5). Wechsler and Gross (16) have also reported a mutation that maps in the dnaE region (dnaE293) and shows a gradual decline of DNA synthesis at the nonpermissive temperature. Similarly to dnaE9, polymerase III from dnaE293 is not thermolabile in vitro (14). The thermosensitivity of this enzyme in vivo or of its synthesis has not been reported, nor has its mutator character been described. In contrast, other dnaE mutations, such as dnaE489, show a rapid shutoff of DNA synthesis at the nonpermissive temperatures (data not shown) and thermolability of their polymerase III in vitro (14, 16, 17). Although dnaE486 and dnaE9 differ from each other in the response of their DNA synthesis to a shift to nonpermissive temperatures, they both show temperature-dependent mutator activity (Table 2). dnaB(Ts), a mutation that causes an abrupt cessation of DNA replication at nonpermissive temperatures, as well as a number of other agents that block DNA synthesis or insult DNA, induce the "SOS" error-prone repair system and its concomitant mutagenesis. The recA gene product is necessary for this SOS error-prone repair (21). Introducing a recAl allele into dnaE486 and dnaE9 strains did not decrease their frequencies of mutation; in fact, it substantially increased the frequencies of mutation with dnaE9 strains at 26 and 370C. This result suggested that SOS error-prone repair may not be involved in these mutageneses (21).
DISCUSSION An extensive search has yielded only one temperature-sensitive, conditionally lethal mutator mutation. This has proved to be a dnaE mutation (dnaE9) by both genetic and enzymological criteria. Cultures of dnaE9 showed a slow shutoff of DNA synthesis upon shift to a nonpermissive temperature. This slow shutoff might reflect either temperature-sensitive synthesis of polymerase III or slow inactivation of the enzyme in vivo under nonpermissive conditions. dnaE9 showed mutator activity which was not dependent on the recA gene product and in this respect is like dnaE486, a polymerase III
E. COLI MUTATOR STRAIN
VOL. 133, 1978
c1.1
10'tlilO
~~~~~~~~~~~~~10io0~
0
5*
C3
3)X|00 440
.01
0
1201
200
100
300 1440 300 0 TIME (min)
100
1
200
10
300
FIG. 4. In vivo inactivation of DNA polymerase III from a dnaE9 strain (MU113) grown at the nonpermissive temperature. Cells were grown at 30°C and then shifted to 44°C. At the times after the shift indicated by arrows, 1.5-liter samples were taken. DNA polymerase III was partially purified and assayed at 30°C as in Fig. 3. Total polymerase activity is the total activity per extract made from each 1.5-liter culture sample. The increase in viable titer in these experiments approximately paralleled that of the optical density at 595 nm (OD506 (data not shown). (a) A dnaE9 strain (MU113); (b) a wild-type strain (KS517).
100.0
100.0
X
6
0I
0 O___
O
10.0-/80
10.0
-
*0.0~~~~~~~~~~~~
100
200
300
400 500
100 200
300
Time (min)
FIG. 5. DNA content of cultures shifted to a nonpermissive temperature. Exponentially growing cells were shifted from 30 to 44°C at the indicated time, and their DNA content was determined. Cells were kept at an optical density at 595 nm (OD50 of between 0.2 and 0.5 by progressive dilution. (a) A dnaE9 strain (MU127); (b) a wild-type strain (KS534).
1202
KONRAD
mutation which, contrary to dnaE9, shows a rapid cessation of DNA synthesis on shift to a nonpermissive temperature. This finding suggests that the mutator activities of dnaE9 and dnaE486 are not due directly to the SOS errorprone repair system. Alternatively, the alterations of DNA polymerase III in these strains might increase mutation frequencies directly by decreasing the accuracy of nucleotide selection during incorporation and/or decreasing the removal of incorrect nucleotides, presumably by the 3' to 5' exonuclease activity associated with the polymerase (8), or indirectly through some error-prone repair system that is not recA dependent. ACKNOWLEDGMNENTS I am grateful to Nancy G. Nossal of this laboratory for useful discussions and assistance with assays for DNA polymerase activity. I also think the E. coli Genetic Stock Center (Department of Human Genetics, Yale University School of Medicine, New Haven, Conn.) for providing some of the strains used in this work.
LITERATURE CITED B. Bachmann, 1. J., K. B. Low, and C. L. Taylor. 1976. Recalibrated linkage map for Escherichia coli K-12. Bacteriol. Rev. 40:116-167. 2. Burton, K. 1956. A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochemistry 62:315-323. 3. Cox, E. C. 1976. Bacterial mutator genes and the control of spontaneous mutation. Annu. Rev. Genet. 10:135-156. 4. Drake, J. W. 1970. The molecular basis of mutation. Holden-Day, San Francisco. 5. Gefter, M. L., Y. Hirota, T. Kornberg, J. A. Wechsler, and C. Bernoux. 1971. Analysis of DNA polymerases II and III in mutants of Escherichia coli thermosensitive for DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 68:3150-3153. 6. Hall, R. M., and W. J. Brammer. 1973. Increased spontaneous mutation rates in mutants of Escherichia coli with altered DNA polymerase III. Mol. Gen. Genet. 121:271-276. 7. livingston, D. M., D. C. Hinkle, and C. C. Richardson.
J. BACTERIOL. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties. J. Biol. Chem. 250:461-469. 8. Livingston, D. M., and C. C. Richardson. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. Characterization of associated activities. J. Biol. Chem. 260:470-478. 9. Low, B. 1968. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc. Natl. Acad. Sci. U.S.A. 60:160-167. 10. Low, K. B. 1972. Escherichia coli K-12 F-prime factors, old and new. Bacteriol. Rev. 36:587-607. 11. Low, K. B. 1973. Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K-12. J. Bacteriol. 113:798-812. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Richardson, C. C., J. L. Campbell, J. W. Chase, D. V. Hinkle, D. M. Livingston, H. L Mulcahy, and H. Shizuya. 1973. DNA polymerases of Escherichia coli, p. 65-69. In R. D. Wells and R. B. Inman (ed.), DNA synthesis in vitro. University Park Press, Baltimore. 15. Signer, E. R., J. R. Beckwith, and S. Brenner. 1965. Mapping of suppressor loci in Escherichia coli. J. Mol. Biol. 14:153-160. 16. Wechsler, J. A., and J. D. Gross. 1971. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol. Gen. Genet. 113:273-284. 17. Wechsler, J. A., V. Nuesslein, B. Otto, A. Klein, F. Bonhoeffer, R. Herrmann, L Gloger, and H. Shaller. 1973. Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication. J. Bacteriol. 113:1381-1388. 18. Whitfield, Jr., H. J., R. C. Martin, and B. N. Ames. 1966. Classification of aminotransferase (C gene) mutants in the histidine operon. J. Mol. Biol. 21:335-355. 19. Wickner, R. B., B. Ginsberg, I. Berkower, and G. Hurwitz. 1972. Deoxyribonucleic acid polymerase II of Escherichia coli. I. The purification and characterization of the enzyme. J. Biol. Chem. 247:489-497. 20. Wickner, S., M. Wright, I. Berkower, and J. Hurwitz. 1974. Isolation of dna gene products of Escherichia coli, p. 195-215. In R. B. Wickner (ed.), DNA replication. Marcel Dekker, Inc., New York. 21. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible deoxyribonucleic acid repair in Escherichia coli. Bacteriol. Rev. 40:869-907.
