Vol. 122, No. 2 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, May 1975, p. 474-484 Copyright @ 1975 American Society for Microbiology

Isolation and Characterization of Mutator Strains of Escherichia coli K-12 RONALD H. HOESS* AND ROBERT K. HERMAN Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 55108 Received for publication 30 December 1974

A selection procedure was devised to select for mutants of Escherichia coli K-12 with enhanced rates of spontaneous frameshift mutation. Three types of mutants were isolated. Two of the mutations apparently represent alleles of previously isolated mutL13 and mutS3. The third type of mutation, represented by two alleles, lies between lysA and thyA, and has been designated mutR. mutR increases the rate of spontaneous frameshift mutation and also the rate of base substitution mutations. The mutator phenotype is recessive. Reversion of a lac amber mutation located on an episome is increased in the presence of the mutator, indicating that mutR can act in trans. No change in sensitivity to ultraviolet irradiation or mitomycin C could be found when mutR34 was compared to the isogenic mutR+ strain. The mutator's activity was little affected by the type of medium in which the strain was grown. Deoxyribonucleoside triphosphate pools were normal in mutR34. Intergenic recombination frequencies were the same in mutR and mutR+ strains, but a two- to threefold increase in intragenic recombination was observed in Hfr x F- crosses when the recipient was mutR34 as compared with mutR+. This increase appeared independent of the distance between the two markers within the gene in which the crossover took place.

Mutations that alter the characteristic spontaneous mutation rate of an organism have been identified in a wide variety of species. They give rise to what are called mutator and antimutator genes, which can be mapped genetically and whose existence suggests that spontaneous mutation rates are genetically controlled. The molecular mechanisms of action of many mutator (or antimutator) genes are not well understood. It is believed that mutator genes may be involved in (i) deoxyribonucleic (DNA) replication, affecting changes either in polymerization or synthesis of substrates for DNA, (ii) DNA repair, (iii) recombination, or a combination of these processes. In bacteriophage T4, for example, certain mutations in the structural gene for the phage DNA polymerase can act as mutators or antimutators (32). In E. coli at least two mutants with altered DNA polymerases, polA (26) and dnaE (18), have been shown to increase the spontaneous mutation rate to a small extent. In addition, a mutant gene that decreases the amount of 6-methyladenine residues in the DNA of E. coli, dam-3, has also been shown to increase the spontaneous mutation rate (29). There are at least six other known mutator genes of E. coli. They all cause dramatic increases in spontaneous mutation rates, and

their enzymatic functions are unknown (9). Two of these mutators may be involved in DNA replication. mutT requires DNA replication for its action (8) and shows a synergistic effect with dnaE, a gene known to be essential for DNA replication (9). The action of mutD is medium dependent (13), and a phosphorylated derivative of thymidine is the apparent effector of mutator activity (H. A. Erlich and E. C. Cox, Genetics [Suppl. ], s20, 1974). Another mutator, mutU, renders the cell sensitive to ultraviolet (UV) light and inviable in combination with polA mutations (42). These features suggest involvement with DNA repair. Less is known about the mode of action of three other mutators: mutH (21), mutL (28), and mutS (10). We have used a simple procedure for isolating mutator strains of E. coli that revert frameshift mutations at a spontaneously high frequency. To date we have identified three classes of mutator genes, two of which appear to be alleles of mutL and mutS. While this work was in progress, Siegel and Kamel (44) showed that the original mutL13 and mutS3 do revert frameshift mutations. In addition, we have found another mutator gene which we have tentatively designated as mutR although it may be allelic with mutHl, which was identified by Hill (21). This 474

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E. COLI MUTATOR GENES

report will mainly concern itself with properties of mutR, its location in the E. coli genome, and evidence suggesting a role for mutR in recombination. MATERIALS AND METHODS Bacterial strains and bacteriophages. Table 1 lists the bacterial strains used and their sources. Plvir and MS2 were gifts from J. Fuchs and P. Kahn, respectively. Media. Liquid minimal medium, minimal agar, and Penassay broth were as described previously (20). Media for P1 transduction including LB broth were those described by Caro and Berg (6). To measure the frequency of streptomycin resistance, cells were plated on Difco nutrient broth plates containing: nutrient broth, 0.8%; NaCl, 0.5%; agar, 1.5%; and streptomycin, 150 gg/ml. When thymine was required 50 Mg/ml was used in both liquid and solid media. Amino acids were added at 20 gg/ml when required. Low-phosphate medium for nucleotide pool measurements was prepared by the method of Edlin and Maaloe (16). Thy- mutants. Strains were made thymine requiring by trimethoprim selection on plates according to Dale and Greenberg (12) with the following exceptions: 7 Asg of trimethoprim per ml was used in the plates, and incubation was at 30 C to reduce the likelihood of isolating temperature-sensitive thymine requirers (2). Transduction. Preparation of P1 lysates and transduction were carried out by the method of Miller (31). To prevent readsorption of phage, saline-citrate buffer (24) was added just prior to plating phage and bacteria. Transductants were selected for and purified on appropriately supplemented Vogel-Bonner plates

(6).

Mutation frequencies. Modified fluctuation tests were carried out by starting a single colony in Penassay broth and allowing the cells to reach stationary phase. The cells were then diluted so that a series of 10 tubes, each containing 1 ml of Penassay broth, was inoculated with about 200 cells per tube and subsequently incubated with shaking for 18 h at 37 C. To assay for spontaneous revertants, 0.1 ml was plated directly on minimal agar, except in the case of Ilv+ revertants where it was first necessary to make a 10-fold dilution before plating. Viable cell count was determined 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 colonies per minimal plate and dividing by the viable cell count. To avoid plates representing "jackpots," colony counts of more than twice the mean were not included. To make valid comparisons between mutator and nonmutator strains it was desirable to have these strains as isogenic as possible except for the mutator character. In all cases when the mutator gene was co-transduced with a selected marker into a recipient strain, a transductant that received only the selected marker and not the mutator gene was saved and used as the isogenic nonmutator strain.

475

UV survival. Overnight cultures were diluted 1:100 in Penassay broth and incubated with shaking until the cultures reached 2 x 108 cells per ml. The cells were then centrifuged and suspended in M63 buffer salts. They were then diluted in M63 buffer salts 1:100, and 2 ml was put in a petri dish (35 by 10 mm) and stirred constantly during irradiation. The UV lamp had previously been calibrated to give a dose of 6.9 ergs/mm2 per s. Irradiated samples were diluted and plated on nutrient broth plates in dim yellow light, and incubated overnight at 37 C. For UV spot tests, a loopful of an overnight culture, spotted on a complete plate, was irradiated for 35 s at a distance of 22 cm with a Mineralight (UVS-12). Mating experiments. Parental cultures growing exponentially in Penassay broth were mixed to give 0.9 x 10s/ml of donors and 2.0 x 108/ml of recipients. Matings were for 90 min at 37 C, after which they were terminated by 30 s of vigorous vortexing. Diluted samples were added to 2 ml of top agar for plating on selective media. Unmated parents were also plated as controls. Mitomycin C sensitivity. Sensitivity to mitomycin C was measured by the method of Marinus and Morris (29). Mutagenesis. An exponentially growing culture was treated with 800 ug of N-methyl-N'-nitro-N-nitrosoguanidine per ml for 45 min at 37 C in tris(hydroxymethyl)aminomethane-maleate buffer (1), pH 6.0. After mutagenesis, cells were collected by centrifugation and washed twice with M63 buffer salts before being resuspended in M63 glucose minimal medium. Pool studies. 32P-labeled deoxyribonucleoside triphosphate pools were measured by the method of Neuhard (33) with the following modifications: 200 ul of 32P-labeled cells was added to 20 ul of cold 4.4 M perchloric acid and allowed to sit on ice for 30 min. The mixture was then centrifuged, and the supernatant neutralized with 19.5 Ml of 6 N KOH plus 0.5 M ethylenediaminetetraacetic acid to raise the pH to about 5.0. This was kept on ice for an additional 30 min and then recentrifuged. To 200 gl of supernatant, 20 ul of deoxyribonucleoside triphosphate standards was added, and 20 Ml of this mixture was then spotted on polyethyleneimine plates (39). Plates were run in two dimensions, and 32P-labeled triphosphates were located by using X-ray film. Spots were cut out and counted in liquid scintillation fluid. RESULTS

Selection for frameshift mutator strains. We devised a simple selection procedure that selects for mutants showing enhanced frequencies of spontaneous reversion of frameshift mutations. We began with strain RH21, which carries seven presumed frameshift mutations, six of which confer amino acid requirements. All seven mutations were induced by and are revertible by ICR-191. An exponentially growing culture of RH21 in glucose minimal medium with the required amino acids was mutagenized

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476

TABLE 1. E. coli K-12 strains

Descriptiona

Sex

Strain

lacZS, F'Iac+ argA21, cysC43, lysA22, str-104 pyrB59, argHl, str-8-9 or -1 7 lacZU131 A(lac pro), F'lacZYA536 purA45, laclacZ36-14(-), str lacZ36-14(+), str thyA-, recA1 argG6, metBl, his-1, leu-6, thyA23, recAl, lac, str-104, F'15 IysA+, thyA+, argA+ lacZL32, recAl lacZ422, trp-8, str lacZ545, trp-8, str lacZ1012, trp-8, str leu-, trp-, lacZU131 leu-, lacZL32, trp-, his-, argA-, LysA-, ilv-, str

F' FFHfrH

A327 AT713 AT2535 CA7049 CSH39 ES4 ICR36-14( -) ICR36-14(+) KL16-99

F'

KL110/F'15

FFFHfr F'

LH164 NG422 NG545 NG1012 RH12 RH21

FFFFHfrH F-

RH22 RH213 RH214 RH218 RH219 RH220 RH221

FFFFFFFt

RH302 RH361 RH362 RH363 RH364 RH365 RH366 RH401 RH402 RH403 RH404 RH405 RH406 RH407 RH408 RH409 RH410 RH411 RH412 RH414

HfrH FFFFFFF-

Source or reference

RH414A RH415 RH416 RH418 RH443 RH451 RH503

Ft F' FFFFFFFF-

Spontaneous leu+ revertant of RH21 leu-, lacZL32, trp-, his-, argA-, ilv-, mutR34, str Same as RH213 except mutR+ Same as RH213 except mutR80 leu-, lacZL32, trp-, ilv-, argA-, mutR34, recAl, str leu-, lacZL32, trp-, ilv-, argA-, mutR80, recAl, str leur, lacZL32; trp-, ilv-, argA-, mutR34, str, F'15 lysA+, thyA+, argA+ Spontaneous spc of RH12 thy- derivative of ICR36-14( -) thy- derivative of ICR36-14(+) lacZ36-14(-), mutR34, str Same as RH363 except mutR+ lacZ36-14(+), mutR34, str Same as RH365 except mutR+ thy- derivative of NG545 thy- derivative of NG422 thy- derivative of NG1012 lacZ545, trp-8, mutR34, str Same as RH404 except mutR+ lacZ422, trp-8, mutR34, str Same as RH406 except mutR+ lacZ1012, trp-8, mutR34, str Same as RH408 except mutR+ thy- derivative of CSH39 A (lac pro), mutR34, F'IacZYA536 Same as RH411 except mutR+ argA21, cysC43, lysA22, lacZU131, leu-, str-104 argA21, cysC43, lysA22, lacZU131, leu-, spc argA21, cysC43, lacZU131, leu-, mutR34, spc Same as RH415 except mutR+ Same as RH415 except mutR80 leu, purA45 leur, pyrB59, argHl, str-8-9, or -17 leu-, lacZL32, mutR34

RH508

F-

leu-, lacZL32, mutR80

FFFFFFFFF'

20 CGSCb CGSCb 20 Cold Spring Harbor CGSC5 35 35 A. J. Clark

CGSCb 20 D. Zipser D. Zipser D. Zipser 22 ICR-191 mutant of RH491 (22)

ORH503_-,RH21c ORH503_RH21c ORH508,RH21c KL16-99 x RH213 KL16-99 x RH218 KL110/F'15 x RH213

tRH503 RH361c

4RH503_RH361c

f RH503_RH362c ORH503--_RH362c

ORH503,RH401c ORH503--+RH401c

kRH503_RH402c ORH503--.RH402c ORH503,RH403c tRH503_RH403c

ORH503-.RH410c

ORH503_RH410c

RH12 x AT713 RH302 x RH414

ORH503_RH414Ac ORH503_-,RH414Ac ORH508_RH414Ac RH12 x ES4 RH12 x AT2535 Mutator selected from RH21 (see text) Mutator selected from RH21 (see text)

RH2130 Fthy- derivative of RH21 a Mutant allele names are given according to the convention proposed by Demerec et al. (14) and used by and Trotter A indicates a deletion, and mutR signifies a new mutator described in text. (45). Taylor 5E. coli Genetic Stock Center, Department of Microbiology, Yale University. Indicates genetic transfer by P1 transduction. --.L

---A

.-.L

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E. COLI MUTATOR GENES

by nitrosoguanidine. Immediately after mutagenesis, survivors were diluted 1:20 in the same medium used prior to mutagenesis, divided among several flasks, and incubated at 37 C for 24 h to provide for segregation of mutations for a minimum of seven generations. Then 10' to 109 cells were transferred from each flask to another flask containing minimal medium supplemented with all but one of the required amino acids. The flasks were incubated until the cells again reached stationary phase, whereupon 10' cells were transferred to medium lacking a second amino acid. This process was repeated until all but one of the required amino acids had been removed from the growth medium. In each transfer step cells were diluted by a factor of 100. Cells from the last step were diluted and plated on glucose minimal agar containing the last required amino acid. Colonies were then tested for mutator activity: samples from overnight cultures in Penassay broth were spread on an agar medium selective for reversion of the last amino acid requirement or for the reversion to Lac+. Nearly every colony tested showed mutator activity. To insure that all mutator strains studied were of independent origin, only one isolate was saved for each starting flask. Mapping the mutators. To map the mutator genes, an F' lac+ plasmid was transferred to the appropriate mutator strain by mating with A327 and selecting for a Lac+StrR recombinant. Each F' mutator strain was mated with RH22, and either Ilv+Leu+, Lys+Leu+, or Arg+Leu+ recombinants were selected. The F' lac+ we have used promotes early transfer of chromosomal markers located on the counterclockwise side of lac. On this basis we were able to divide the mutators into three groups: (i) those that showed mutator activity among 50% of the recombinants when any one of the above markers was selected, (ii) those that had no mutators among the recombinants when Ilv+ was the selected marker but were 50 to 60% mutators when either Lys+ or Arg+ recombinants were selected, and (iii) those that had no mutators among the recombinants when Ilv+ was the selected marker but were nearly 100% mutators when Lys+ or Arg+ was selected. The first group of mutators was further mapped by again using F' lac+ to transfer chromosomal markers from the mutator strain, and mating with strain RH451, which carries mutations in argH and pyrB. When pyrB+ was the selected marker, 53 to 64% of the recombinants were mutators while only 27 to 36% had become argH+. It was already known that mutL13, isolated by Liberfarb and Bryson (28), is co-transducible with purA, which is situated

477

about midway between pyrB and argH. Indeed, we found this group of mutators to be 88 to 92% co-transducible with purA+ when transduced into RH443, and for reasons mentioned later we feel that these are alleles of mutL13. It has been reported by Siegel and Bryson (43) that another mutator gene, mutS3, is co-transducible with cysC. Using P1 lysates of the second group of mutators and transducing RH414 to cysC+, we found that this group co-transduced with a frequency of 11 to 58%. We therefore believe that these are probably all alleles of mutS3. The third group of mutators we found was co-transducible with both lysA (57 to 86%) and argA (37 to 48%). Since lysA and argA are only 20%o co-transducible, the third group of mutators must lie between them. mutS3 has already been mapped in relation to nearby markers (10), and both argA and cysC lie between our third group of mutators and mutS3. Clearly then, our third group is not allelic to mutS3. Two representatives of this third group have been shown to be allelic (see below) and we have designated them as mutR34 and mutR80. Thus far 11 mutators have been mapped. Of these, three are of the group one class (mutL), five are of the group two class (mutS), and three are of the third class (mutR). Ordering mutR with respect to nearby markers. To map mutR34 and mutR80 more precisely, four-factor transductional analysis was carried out. P1 lysates were grown on RH503 and RH508, two of our mutator strains whose mutator genes map between lysA and argA. Strain RH2130, which is lysA -, thyA-, and argA-, was used as the recipient in transduction. lysA+ transductants were selected and analyzed for co-inheritance of thyA+, argA+, and the mutator gene. argA+ transductants and thyA+ transductants were selected also and analyzed in analogous fashion. Mutator activity was assayed by measuring the frequency of reversion to Leu+ of an overnight culture from each transductant (Table 2). Two possible orders were considered: one would have mutR to the lysA side of thyA, and the other would have mutR to the argA side of thyA. Note that if the second order were correct than among the transductants selected as lysA+ the mutR+thyA+argA- class would require only two crossovers, whereas the mutR- thyA -argA - class would require four, two of which would be in the same two regions required by the former class. Table 2, however, shows that there were only 5 transductants in the former class and 25 in the latter. Similarly, if the second order were correct, then among the transductants selected

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478

TABLE 2. Analysis of four-point transductional cross involving IysA, mutR, thyA, and argA argA

thyA

mutR

lysA I

1

Selected

marker

4

3

2

Uneetdmre" markera Unselected

Regions of

5

No. of

required

trans-

1,2

75

1,3

25 57 29

crossovers ductants0

IysA+ mutR- thyA+ argA+ O +

+ + + O O O

0 0 + + 0

0 0 0 + +

+ 0 +

+ + 0

thyA+ IysA+ mutR- argA+ 0 0 O 0 O + + O O +

+ 0

+ +

0 0

0 +

+

+

+

+ 0 +

1,4 1,5 1,3,4,5 1,2,3,5 1, 2, 4, 5 1,2,3,4 3,4 2,4 1,4 3,5 2,5 1,5

1,2,3,4 1,2,3,5

argA+ lysA+ mutR- thyA+ O O 0 + + +

O +

O 0

0

+

+ +

+ + 0

+

+ 0 0 0

0

+

4,5 3,5 2,5 1,5 1,3,4,5 1,2,4,5 2,3,4,5 1,2,3,5

0 0 1 5

32 21 99 4

9 29 2 1

80 16 14 85 0 4

3 1

+, Marker from donor; 0, marker from recipient. 'Numbers represent combined data with P1 lysates of RH503 and RI1508. The data were pooled since both mutator alleles gave very similar results. a

as argA+ the IysA-mutR-thyA- class would require only two crossovers, whereas the IysA-mutR+thyA+ class would require four, two of which would be in the same two regions required by the former class. Table 2 shows that there were only 3 transductants in the former class and 16 in the latter. We conclude that the correct order is IysA-mutR-thyA-argA. We have extended Wu's (47) model for analysis of three-point transductional data to four-point transductional data. Using the data of Table 2 we obtain the following distances (in minutes) of the E. coli map, 0.26, 0.13, and 0.43 between lysA, mutR, thyA, and argA, respectively, assuming the value of 2.16 min as the average length of the transducing particle (23). Action of mutR. When transduced back into RH21, mutR34 and mutR80 increased the spon-

J. BACrE:IOL.

taneous reversion of the six remaining presumed frameshift mutations (Table 3). The frequency of reversion varied considerably depending on the marker tested, ranging from an 8-fold increase for the reversion to Trp+ to a 2,000-fold increase for reversion to Lac+. Since these markers were only presumed to be frameshift mutations, reversion of two lacZ mutations that have been better characterized as frameshifts (35) were tested. Thymine-requiring derivatives of strains carrying lacZ36-14(+) or kacZ3614(-) were isolated and then transduced to Thy+ by a P1 lysate of RH503. Transductants were purified and tested for mutator activity by noting increased reversion to Lac+. In addition, one thyA+mutR+ transductant was saved in each case to serve as an isogenic nonmutator control. The frequency of reversion to Lac+ in RH365 [lacZ36-14(+) mutR34J was stimulated approximately 70-fold above that of RH366, the isogenic mutR+ strain. RH363 [lacZ36-14(-) mutR34] only showed a frequency of reversion 19-fold above that of the isogenic mutR+ strain, RH364. To make certain that we indeed transduced mutR34 into RH363, RH12 was mated with RH363, and also RH364 as a control, and Lac+StrR recombinants were selected. Among the recombinants were those that had become Leu-, having inherited this trait from the male parent. One Leu-Lac+StrR recombinant was saved from each mating and tested for reversion to Leu+. As expected, the Leu- reverted at a much higher frequency in the strain derived TABLE 3. Mutation frequencies in mutR and mutR+ strains Original mutation leu-

Mutator

Reversion

mutR/ mutR+

RH213 mutR34 RH214 mutR+ RH219 mutR34

2.00 x 10-' 7.00 x 10-' 2.86 x 10-'

290

Strain

gene

frequency

410

(recAl) RH213 RH214 RH213 hisRH218 RH214 RH213 trp RH218 RH214 ilvRH213 RH218 RH214 RH213 lacZL32 RH218 RH214 RH415 strA+ RH416 lacZ36-14(+) RH363 RH364 lacZ36-14(-) RH365 RH366

argA

mutR34 mutR+ mutR34 mutR80 mutR+ mutR34 mutR80 mutR+ mutR34 mutR80 mutR+ mutR34 mutR80 mutR+ mutR34 mutR+ mutR34 mutR+ mutR34 mutR+

5.23 x 10-7 2.59 x 106' 3.47 x 10-6 2.50 x 10-' 1.65 x 10-' 2.12 x 10-' 1.82 x 10-f 2.35 x 10-' 9.78 x 106.52 x 10-' 3.75 x 103.39 x 10-' 2.82 x 10-' 1.62 x 10-' 2.96 x 10-' 3.86 x 10-10 3.71 x 10' 5.18 x 10-' 2.95 x 10-' 1.54 x 10-'

200

2,100 1,500 9.0 7.7

260 170

2,100 1,700 770 72 19

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E. COLI MUTATOR GENES

from RH363 than in the one derived from RH364. When mutR34 and mutR80 were transduced into RH414A, which is Strs, a marked increase in the mutation frequency to StrR was observed (Table 3). Breckenridge and Gorini (5) have shown that mutations to Str' only occur by base substitution, so we conclude that mutR has not only frameshift reversion activity but base substitution activity as well. To determine whether these two activities might be separable, five other mutR34 and mutR80 transductants were tested. All showed both activities. Of the other two groups of mutators isolated, all showed increased base substitution activity, as determined by mutation to StrR. This is consistent with the supposition that these mutators are alleles of mutL and mutS, since the original mutL and mutS strains were isolated as base substitution mutators (28, 43). It is also worth noting that the mutator activity of mutR does not require the presence of a recA + gene: strain RH219, for example, shows normal mutator activity (Table 3). Dominance test. F'15 covers the region from lysA through argA. KL110/F'15 was mated with RH213, and argA+metB+ recombinants were selected. Eight of the recombinants were purified and tested for sensitivity to the malespecific phage MS2 and mutator activity by measuring frequency of reversion to Leu+. All eight were sensitive to MS2, indicating the presence of the episome, but none showed mutator activity. One of these merodiploid strains was grown in acridine orange for 24 h (20) and spread on MacConkey-lactose agar. After 2 to 3 days of incubation on MacConkeylactose plates, colonies with mutator activity showed a large number of red papillae, whereas wild-type colonies showed no red papillae. After acridine orange treatment of the merodiploid, many colonies exhibiting mutator activity appeared. Five of these were tested with MS2 and, in contrast to five colonies that showed no papillae (and proved to be resistant to MS2), all were sensitive. A fluctuation test was performed on a cured colony (Table 4), and full mutator activity was restored. We conclude that mutR34 is recessive to its wild-type allele. Complementation test between mutR34 and mutR80. Colonies of RH221 on MacConkey-lactose agar were incubated 2 to 3 days at 37 C. Since most of the colonies are phenotypically nonmutator because of the presence of the episome, the colonies remain totally white. However, should either the episome be lost or should homogenate formation have taken place to give rise to mutR34/F'15 mutR34, one would expect to find red papillae. A number of colo-

TABLE 4. Mutation frequencies and MS2 sensitivity in merodiploids and cured strains Strain

Genotype

RH213 RH221

mutR34

RH221a

mutR+ mutR34

Frequency of MS2 reversion sensito Leu+

tivity

2.00 x 10-6

-

1.07 x 10-8 2.40 x 10-6

+

mutR34/F'15

aStrain RH221 cured with acridine orange.

nies with red papillae were found and tested to see whether they still retained the episome. One of those still containing the episome was mated with RH220 (mutR80 argA recAl), and Arg+His+ recombinants were selected. Because the recipient in this mating is a mutator, we wanted to be certain that the Arg+His+ recombinants were a result of episomal transfer and not just a mutator-induced Arg+ revertant. A number of recombinants were purified and tested for UV sensitivity (presence of recA) and sensitivity to MS2 (presence of the episome). Those that were sensitive in both tests were grown overnight and then plated to test for their ability to revert Leu-. All showed a high frequency of reversion to Leu+, and we conclude from this that mutR34 and mutR80 do not complement each other and are therefore allelic. Action of mutR34 in trans. We wanted to determine whether mutR can increase the reversion frequency of genes in trans. CSH39 has a lac-pro deletion on the chromosome and carries an F'lac episome with an amber mutation YA536 in the lacZ gene. Mutator and nonmutator strains were constructed in the usual manner by obtaining a thymine-requiring derivative of CSH39 and then transducing to Thy+ with a P1 lysate of RH503. To determine whether the mutator had been co-transduced with thyA+, overnight cultures of purified transductants were tested for the frequency of Lac+ reversion and mutation to StrR. A fluctuation test was performed on the mutator and nonmutator derived from CSH39. At the termination of the test, all tubes were assayed for Lac+ revertants on selective plates and, in addition, tested for MS2 sensitivity to determine whether the episome had been retained. All tubes showed sensitivity to MS2. The reversion frequency of F'lacZYA536 in the mutator strain was nearly 100-fold higher than in the nonmutator strain. To determine whether the reversion events really occurred on the episome, 16 of the Lac+ revertants were spotmated with LH164 (lacZL32 recAl), and Lac+StrR recombinants were selected. Fifteen of

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480

the 16 Lac+ revertants tested gave recombinants when mated with LH164. This result shows that mutR34 did indeed promote mutations on the episome. It also suggests that a small fraction of the revertants induced by the mutator are suppressor mutations not located on the episome. Is mutR involved in DNA repair? It has been postulated that deficiencies in DNA repair might result in an increased spontaneous mutation rate. Since at least one mutator, mutU, apparently plays a role in repair of UV damage (41), we decided to investigate whether mutR34 might be involved in some type of repair mechanism. RH213 (mutR34) and the isogenic control RH214 (mutR+) were UV irradiated as described aboye. We found no evidence to suggest that RH213 was more sensitive or resistant to UV irradiation than the control RH214 (Fig. 1). Similarly, when these two strains were exposed to 5 gg of mitomycin C for a period of 40 min, we found no difference in sensitivity of these

strains to the drug at a 10-3 survival level (data not shown). Is mutR a conditional mutator? Degnen and Cox (13) have recently reported finding a conditional mutator, mutD, whose mutator activity is much greater when the cells are grown in LB medium than in minimal medium, apparently owing primarily to the stimulatory effect of thymidine. Since matR is closely linked with thyA, the gene for thymidylate synthetase, we decided to investigate the possibility that mutR34 might be conditional. From an overnight culture of RH213 grown in M63 glucose plus required amino acids and started from a single colony, approximately 50 cells were added to each of 10 tubes. Five of the tubes contained 1 ml each of LED and the other five contained 1 ml of M63 glucose plus required amino acids. The tubes were incubated at 37 C until they reached saturation. This required 18 h in LB and 33 h in minimal medium. Cells were centrifuged and washed twice with buffer before being plated on glucose-minimal agar coptaining no leucine. The results indicate (Table 5) that there is little difference in the frequency of reversion to Leu+ between cells grown in rich medium as compared with minimal medium. Pool studies on mutR. We have considered the possibility that mutator activity would be correlated with an alteration -of the deoxyribonucleoside pools. It has been shown, for example, that mutations are induced by thymine starvation (15). RH213 and RH214 were grown in low-phosphate glucose-minimal medium with the required amino acids. Exponentially growing cells were labeled with- [32P orthophosphate for several generations before extraction of the deoxyribonucleoside pools. Figure 2 shows the four principal deoxyribonucleoside triphosphate pools for mutR34 and mutR+. We found no significant differences between the mutator and nonmutator strains. A repeat experiment confirmed these results. Is mutR involved in recombination? Results of crosses between CA7049 and either RH213 (mutR34) (and in one case RH218) or the Mutation frequencies after growth in rich and minimal medium

T

UV Doe (sec) FIG. 1. UV survival of strain RH213 (mutR34) (0) and its isogenic mutR+ strain RH214 (A).

Strain

Genotype

Medium

RH214

mutR+

LB

RH213

mutR34

M63 LB M63

Frequency of reversion

toLeu+

3.00 x 10-' 1.88 x 10-' 1.26 x 103.71 x 10-"

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E. COLI MUTATOR GENES

TABLE 6. Conjugation and intragenic recombination frequencies in mutR and mutR+ strains 50 Conjugation Intragenic

Cross

40[ 0

0-

donor cells

Uu+StrH

0.245 0.182 x = 0.214

3.15 2.81 x = 2.98

CA7049 x RH213 (leu- lacZL32 mutR34 str)

0.223 0.217 0.194 0.205 0.180

20.2 6.89 8.72 7.49 12.0

CA7049 x RH218 (leu- lacZL32 mutR80str)

0.204 x = 0.204

8.95 x = 10.7

CA7049 (lacZU131) x RH214 (leu lacZL32str)

301 x

0-4

frequency frequency Leu+StrR/ Lac+StrH/

201

10

dOTP dATP dCTP dTTP FIG. 2. Nucleoside triphosphate pools of strain RH213 (mutR34) (open bars) and its isogenic mutR+ strain RH214 (solid bars).

isogenic RH214 (mutR+) are listed in Table 6. The first column shows the frequencies of appearance of Leu+StrR recombinants. Because these frequencies were the same in both mutator and nonmutator strains, they were used as a basis for comparing intragenic recombination. This was done to account for variations in donor-recipient ratios and efficiencies of mating in various experiments. To measure intragenic recombination we utilized the same parents as before but selected for Lac+StrR recombinants. CA7049 carries lacZU131, a nonsense mutation, and RH213 and RH214 carry lacZL32, a frameshift mutation. In these experiments we have expressed intragenic recombination frequency as a ratio of the number of Lac+StrR recombinants divided by the number of Leu+StrR recombinants. The computed values are given in the last column of Table 6. When the recipient was mutR34, there was a threefold higher number of intragenic recombinants than when the recipient was an isogenic mutR+ strain. This higher number cannot be accounted for by spontaneous reversion of lacZL32 mutation in the recipient since that frequency is about 100 times lower than the recombination frequency.

We then asked whether this phenomenon of increased intragenic recombination would occur if the recipient's lacZ mutation was a nonsense rather than a frameshift mutation and whether distance between the two lacZ mutations has any effect. To answer these questions, strains with three lacZ nonsense mutations previously mapped (36, 37) were made thymine requiring and then transduced to Thy+ to construct isogenic mutR34 and mutR+ strains. The results of a series of matings with these strains as recipients are summarized in Table 7. In these experiments the number of intragenic (Lac+StrR) recombinants was normalized by the number of Trp+StrR recombinants. It can be seen that, when the recipient contains a lacZ nonsense mutation (rather than a frameshift) mutR34 promotes about the same increase in intragenic recombination as before. Somewhat surprisingly we found that the same factor increase is obtained regardless of the distance between the two lacZ mutations. For instance, lacZ545 maps within the same deletion segment as lacZU131 (where the Z gene is divided into 16 segments), while lacZ1012 is 11 segments away from lacZU131, yet they show about the same increase (2.3- and 2.2-fold, respectively) in intragenic recombination with lacZU131 when they are present with mutR34.

DISCUSSION We have devised a selection procedure that allows us to select for mutator strains that exhibit increased frequencies of spontaneous reversion of frameshift mutations. Thus far we have identified two of the mutator genes known to affect frameshift mutations, mutL and mutS (44), as well as another mutator gene, mutR, which may be allelic with mutHl (21; see

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482

TABLE 7. Intragenic recombination frequencies in mutR34 and mutR+ strains carrying lacZ nonsense mutations

Cross Cross

|

Lac+StrR/ '~~~fr-P+St-rIt

Cross

Lac+StrY

TistrR

CA7049 x RH405 (lacZ545 trp-8 mutR+ str)

0.205 0.264 0.360 0.360 s = 0.297

CA7049 x RH404 (lacZ545 trp-8 mutR34 str)

0.461 0.607 0.778 0.926 x = 0.693

CA7049 x RH407 (lacZ422 trp-8 mutR+ str)

0.229 0.140 0.221 0.221 I = 0.202

CA7049 x RH406 (lacZ422 trp-8 mutR34 str)

0.388 0.295 0.426 0.427 x = 0.384

CA7049 x RH409 (lacZ1012 trp-8 mutR+ str)

0.087 0.141 0.172 0.172 s = 0.143

CA7049 x RH408 (lacZ1012 trp-8 mutR34 str)

0.301 0.312 0.358 0.295 x = 0.317

below). Two other mutators, mutD (17) and mutU (44), also revert frameshift mutations, and it might be asked why we did not find these among our mutators, although we have mapped only 11 of our mutator genes thus far. For mutD the most reasonable answer is that no thymidine was present in the minimal medium in which the selection was carried out and, since mutD is a conditional mutator dependent largely on an exogenous supply of thymidine for its mutator activity, it would be at a distinct disadvantage when competing with other mutators. There is no clear reason why we did not find alleles of mutU. It should be pointed out, however, that our selection procedure depends on the mutators exhibiting good growth in minimal medium in order for them to be selected. In addition, mutators such as polA (26), which only increase the spontaneous rate by a small amount, were probably lost in our selection procedure because virtually every flask contained one or more "strong" mutators that were more strongly selected for. One might circumvent this problem by starting with a smaller number of cells per flask after mutagenesis so that some flasks would not contain any strong mutator cells. Later, when we carried out our selection procedure again, we were able to isolate the three types of mutators after only two of the required amino acids had been removed. Because strong mutators were isolated after only two cycles of enrichment, it should be possible to isolate the weaker mutators after a few more cycles of enrichment. We expect that each enrichment step increases the ratio of mutator cells to wild type by a factor equal to the mutator activity, assuming that mutator and wild-type cells grow at the same rate;

obviously, weak mutators would thus require more enrichment steps than strong mutators. It should also be noted that the number of cells transferred at each enrichment step is crucial, since the mutator clone is liable to be lost if too few cells are transferred. This would be particularly true in the case of weak mutators which would generate fewer revertants. Our P1 transduction data place mutR between lysA and thyA. This is distinct from the other known mutator in this region, mutS, which maps approximately 2.7 min away, at 52 min, on the E. coli map (45). Hill has found a mutator, mutHI, that maps in the lysA-cysC region (21). Although it has often been referred to as a possible allele of mutS3, mutHI might very well be an allele of mutR, particularly if one notes the close linkage with lysA from Hill's original mapping data (21). Moreover, Siegel and Kamel (44) have reported, as a personal communication from Hill (now deceased), that mutHI conferred frameshift mutator activity. Shimada et al. (40) have found that one of the secondary attachment sites for phage lambda, when its normal attachment site has been deleted, is between lysA and thyA. Chung and Greenberg (7) observed that, after induction of lambda which has been integrated at this site, among the survivors one could find deletions to the lysA side of lambda, but not to the thyA side. They concluded that there was an essential gene located between lambda and thyA. Although we have not mapped our mutator with respect to lambda at this secondary attachment site, it appears that mutR is more closely linked to thyA than to lysA. It would be interesting to determine whether mutR is an essential gene. Although we know that polA (27) and dnaE (46)

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E. COLI MUTATOR GENES

are essential genes, it has not been determined whether any of the strong mutators are essential. We have shown that mutR increases both base substitution mutations and frameshift mutations. Base substitution activity has been measured by using mutation to streptomycin resistance. It is not known whether mutR induces transitions or transversions or both. The reversion frequency of different frameshift mutations varies considerably. Siegel and Kamel (44) found that with mutU4 and mut-25, which is probably an allele of mutL13, there was a much higher frequency of reversion of lacZ3616(+) than of lacZ36-16(-). We obtained a similar result: reversion promoted by mutR34 was higher in the case of lacZ36-14(+) than lacZ36-

14(-). The possibility that mutR is a conditional mutator was investigated. Growing a mutR34 strain in minimal medium produces a reversion frequency roughly the same as when the strain is grown in rich medium, so mutR34 does not appear to be a conditional mutator. When the deoxyribonucleoside pools were examined, no difference could be found between mutR34 and mutR+. One might ask how much of a change in pool size is necessary for increasing the spontaneous rate of mutations. Evidence obtained from work on thymine auxotrophs show that there is a considerable change in pool size (ratio of deoxycytosine 5'-triphosphate to deoxythymidine 5'-triphosphate becomes approximately 20-fold higher than in a Thy+ strain) under optimal growth conditions (34). Under these conditions the spontaneous mutation rate shows no appreciable change (15), so it seems unlikely that the increased mutation rate of mutR34 can be accounted for by changes in pool size, however small. Our technique cannot rule out the possibility that an unusual base is produced and subsequently incorporated into the DNA of mutR strains. This idea was once proposed for mutL in Salmonella typhimurium (25), a mutator closely linked to purA, but has since been ruled unlikely (E. C. Siegel and J. J. Ivers, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, G173, p. 48). No evidence was found to suggest that mutR is involved in repair of damage produced by UV irradiation or mitomycin C, and no deficiency could be found in recombination. Isolation of mutants that have increased frequencies of intragenic recombination and increased spontaneous mutation rates has been reported (K. B. Low, Genetics [Suppl.], s163, 1973) in E. coli. Balbinder (3) has reported that the frequency of recovery of certain transductant classes in S. typhimurium is modified by the presence of a

mutator gene. We used a series of mutR34 strains with various lacZ mutations as recipients and found that, when they were crossed with an Hfr carrying a lacZ nonsense mutation, intragenic recombination was increased two- to threefold above the frequency obtained with isogenic mutR+ strains. This increase occurred whether the recipient lac mutation was a frameshift or nonsense mutation; moreover, mutR34 promoted the same factor increase in intragenic recombination whether the markers were very close together or rather far apart within the Z gene. Konrad and Lehman (27) have recently reported finding a "hyper-recombination" mutant which is greatly reduced in the 5'- to 3'-exonuclease activity of DNA polymerase I. This mutant shows high intragenic recombination between a lac mutation on the chromosome and a lac mutation on a lysogenized k80 dlac. These authors also noted briefly that a number of other mutants that accumulate "Okazaki fragments" (including a DNA ligase mutant) exhibit the hyper-recombination phenotype. It will be of interest to see how these mutants behave with respect to intragenic recombination as it occurs in conjugation. Exonucleases clearly play an important role in recombination (38). Our own results indicate that the increase in spontaneous mutation frequency does not require the presence of a functional recA+ gene. Crawford and Preiss (11) have proposed that intragenic recombination in E. coli is usually a result of conversion-like events rather than conservative breakage and reunion within the gene. It might prove interesting to study in more detail the nature of the intragenic events promoted by mutR. A transductional analysis like that of Crawford and Preiss, employing nonselected markers closely flanking the intragenic pair of markers, could be conducted, and the reciprocality of recombination between episomal and chromosomal markers could also be investigated (4, 19, 30). Finally, it would be of interest to determine whether other mutators, particularly mutL and mutS, which so far have shown no distinguishable traits other than increased mutation rate, also effect intragenic recombination. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AM 11321 from the National Institute of Arthritis and Metabolic Diseases. One of us (R.H.H.) was supported by Public Health Service training grant GM 01156 from the National Institute of General Medical Sciences. We wish to thank J. Fuchs for excellent advice on the pool studies, D. Fan for critical comments on the manuscript, B. Bachmann and D. Zipser for strains, and H. J. Creech for ICR-191.

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J. BACTERIOL.

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