Vol. 121, No. 3 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 892-900 Copyright © 1975 American Society for Microbiology
Temperature-Sensitive recA Mutant of Escherichia coli K-12: Deoxyribonucleic Acid Metabolism After Ultraviolet Irradiation JENNIFER D. HALL' AND P. HOWARD-FLANDERS* Department of Molecular Biophysics and Biochemistry* and Department of Therapeutic Radiology, Yale University, New Haven, Connecticut 06520 Received for publication 16 September 1974
A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recAl early and recipients carrying wild-type or mutant alleles. It was found that recA + but not recAl complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA - strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for A. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recA + but not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.
Genetic recombination in Escherichia coli K-12 is blocked in strains carrying a mutation in recA, which maps between cysC and pheA (13). In addition to being recombination deficient, mutants carrying recA - are very ultraviolet light (UV) and X-ray sensitive (5, 7) and are defective in recombinational or post-replication repair (11, 12). Phage X lysogens carrying recAshow virtually no spontaneous or UV-induced phage production (2). Most recA mutants so far isolated exhibit this extreme phenotype, but a heavily mutagenized strain, N203, isolated by H. Ogawa, was temperature sensitive in its ability to form recombinants (H. Ogawa, personal communication). This mutant has been found to carry a temperature-sensitive allele of recA, designated recA44, and possibly a second mutation causing UV sensitivity. The properties of strains carrying the recA44 mutation, transduced away from the mutagenized back'Present address: Department of Biochemical Sciences, Princeton University, Princeton, N.J. 08540.
892
ground, were investigated. As the temperature was raised above 35 C there was a sharp decrease in the ability of these transductants to form recombinants in crosses with Hfr donors. The level of recombinant formation at 42 C was comparable to that observed for other strains carrying recA- (5, 7, 9). However, the sensitivity to UV of recA44 transductants at high temperature was less extreme than that of other recA mutants. Similarly, the depression of prophage induction in X lysogens carrying recA44 was less marked as the temperature was increased. The increase in the molecular weight of DNA synthesized after exposure to UV was inhibited at high temperature. While this work was in progress, a strain carrying another mutation (recA200) with a temperature-sensitive phenotype for recombination was isolated through a different selective procedure (8). MATERIALS AND METHODS Bacterial strains. The strains used are listed in Table 1.
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VOL. 121, 1975
Media. Minimal agar contained: KH2PO4, 2.7 g; Na2HPO4, 4.1 g; 10% MgSO4.7H2O, 1.0 ml; 10% (NH,)2SO4, 10 ml; 1% Ca(NO,)2, 0.5 ml; 0.05% FeSO4 7H2O, 0.5 ml; agar, 13.5 g; and water to 1 liter. Minimal liquid medium contained: NH4Cl, 1 g; Na2HPO4.7H.0, 11 g; KH2PO4, 3 g; glucose, 4 g; MgSO4, 120 mg; CaCl2, 10 mg; thiamine, 0.1 mg; and water to 1 liter. The minimal agar and minimal liquid medium were supplemented with the amino acids and nucleosides required by the individual strains at the following concentrations: 0.01% DL-leucine, 0.01% DL-histidine, 0.02% L-arginine, 0.008% DL-serine, 0.005% DL-cysteine, 0.01% DL-threonine, 0.04% DLproline, and 20 MM thymine. Streptomycin, when included, was added at 200 Mg/ml and nalidixic acid was added at 10 ;sg/ml. K medium contained minimal liquid medium supplemented with 10 g of Casamino Acids and 6 g of glucose per liter. K+1, K+4, and K+100 consisted of K medium supplemented with 1, 4, and 100 Mg of thymidine per ml, respectively. Broth contained 10 g of tryptone (Difco), 5 g of yeast extract, 0.5 g of NaCl, and 120 mg of NaOH per liter of water. YET agar contained broth supplemented with 2% agar. X agar contained 5.0 g of tryptone (Difco), 8.0 g of peptone (Difco), 1.0 g of NaCl, 15 g of agar, and water to 1 liter. Top agar contained 8 g of NaCl and 12 g of agar per liter of water. Buffered saline contained 2.68 g of Na2HPO4.7H20, 1.36 g of KH2PO4, and 5 g of NaCl per liter of water. X buffer contained 6 ml of 1 M tris(hydroxymethyl)aminomethane (pH 7.2), 2.46 g of MgSO4.7H20, and 0.05 g of gelatin per liter of water.
Transduction of P1 bacteriophage. Phage P1 CM cirlO0 (10) was prepared by heat induction of a P1 lysogen of strain N203 with the temperature-sensitive phenotype for recombination and was used to infect recipient cells at 32 C that were incubated at 32 C first and then at 42 C to eliminate lysogenic recipients. Phage P1 grows very poorly on strain N203. Tests on transductants for recombination deficiency. After purification, the transductant cells were inoculated in patches on minimal agar and grown at 42 C. They were replica plated onto selective agar spread with Hfr cells in exponential phase and incubated at 42 C. Quantitative tests on recombination deficiency. The strains to be tested were grown with aeration at either 32 or 42 C in broth to about 2 x 10' cells/ml. Donor cells were grown and brought to the same density at 37 C. Two milliliters of a given recipient culture was placed in a flask at the mating temperature (35 or 42 C) for 10 min and 0.2 ml of the donor culture was added. The mixture was incubated with gentle swirling during the mating period, diluted, plated on selective agar containing streptomycin, and incubated at the appropriate temperature. Prewarmed plates were used for the matings at 42 C. UV irradiations. Irradiations were carried out with a 15-W, low-pressure mercury germicidal lamp giving predominantly 254 nm of light. The dose rate was measured with a General Electric germicidal light meter. The bacterial suspensions were irradiated by placing samples in glass petri dishes in a layer less
TABLE 1. Description of bacterial strains Relevant genetic marker Strain
Sex,
plasmids
Str
r
dra rec uvr thy or
cysC
sensi- Proleu tivity phage
Origin or reference
drm AB259 AB1157 AB2497 KL16
Hfr Hayes FFHfr
+ 6 6 +
S R R S
-
+
.+ + + + 43 +
6 +
R S
-
+
+ +
+ +
+ + + +
+ + + +
+ + -
-
+
+ +
-
-
KL266 MA1079
FHfr
+ Al
N203 NH4915
FF-
A44 + + B45
+
+ -
+ +
-
-
-
R R
-
NH4916
F-
A44 B45
-
-
+
-
R
-
NH4919
F-
+
+
+
-
43
6
R
-
NH4920
F-
A44
+
+
-
+
6
R
-
NH4921
F-
A44
+
+
-
+
6
R
-
F+ FlOl leu+thr- + A44 F-
+ + +
+ +
+
+
+
-
+ + + + +
6
A44 A44
+ + + +
R S R R R
NH4929 NH4932 NH4939 NH4940 NH4941
FF-
-
-
6/+ 6 6 6
-
X -
-
X
(1) (1)
Spontaneous thy-dra/m- mutant of AB1157 Obtained from K. B. Low. It injects thyA+
recA+ his+ Obtained from K. B. Low. maLA suppressed Obtained from B. Low. It carries ser- thi- and injects thyA+ recA+ his+. Obtained from H. Ogawaa uvrB Gal+ (Pro+) recombinant of NH4911 from mating with NH4917a urvB Gal+ (Pro+) recombinant of NH4912 from mating with NH4917 Thy+ cysC- recombinant from mating between AT2427 (1) and AB2497 Rec° cysC+ transductant of NH4919 donor N203 rec- cysC+ transductant of NH4919 donor N203 A lysogen of AB1157 F101 thr- derivative of AB1115. cysC+ recA44 transductant of KL266 cysC+ recA44 transductant of KL266 X lysogen of NH4939
aNH4917 is an F'101 derivative of AB2430 gal+ uvrB45 and was used as donor to transfer gal+ uvrB45 to NH4911 and NH4912. NH4911 is a thyA dra/m derivative of JC1557 argG, his-, met-, gal-, lac-, mtl-, xyl- (1) (parent of N203), and NH4912 is a thyA dra/m derivative of N203.
894
HALL AND HOWARD-FLANDERS
than 3 mm deep and gently swirling the cultures during irradiation. Cell survival after UV irradiation. Logarithmically growing cultures at 32 C were diluted, spread on prewarmed YET agar containing 200 qg of thymine per ml, incubated for 30 min at 32, 39, or 42 C, exposed to UV light and incubated overnight at the same
temperatures.
Incorporation of TdR after UV irradiation. Cultures were grown with aeration at 32 C to about 2 x 108 cells per ml in K medium supplemented with 250 yg of deoxyadenosine per ml, and part of each culture was irradiated. Samples were taken and diluted into the same medium further supplemented with 20 uCi of tritiated thymidine per ml (50 Ci/mmol) and aerated at 32 or 45 C for 2 h. At various times, 0.1-ml aliquots were removed and placed on Whatman 3MM filter disks (25-mm diameter) pretreated with 0.1 ml of 1 N NaOH. The disks were washed in cold 5% trichloroacetic acid and counted. DNA breakdown. The bacteria were grown overnight at 32 C in K+4 medium. Samples were diluted 1:10 into K+1 medium containing [3H ]thymidine (50 Ci/mmol) at 10 oCi/ml and aerated at 32 C for 3 h. The cells were washed and suspended in K medium at five times the original volume and aerated for an additional 60 min at 32 C. Amounts (5 to 10 ml) of the labeled cultures were irradiated and duplicate cultures were incubated at 32 or 45 C for 150 min. At various times, 0.5-ml samples were diluted into 0.5 ml of 10% trichloroacetic acid and held at ice temperature for 1 h. A 0.05-ml amount of 1% bovine serum albumin was added and the samples were spun for 15 min at 10,000 rpm. To measure the acid-soluble radioactivity, duplicate 0.1-ml aliquots were removed and counted in 3 ml of tolulene-Triton X-100-1,4-bis(5-phenyloxazolyl)benzene liquid scintillation fluid. To measure the total radioactivity in the samples, the remaining solutions were heated to 90 C for 30 min. Duplicate 0.1-ml samples were removed and counted as before. Induction of A lysogens by UV light. The lysogenic bacteria to be tested for prophage induction were grown at 32 or 42 C to about 2 x 108 cells/ml. The cells were washed and suspended in A buffer at room temperature. Samples of the suspensions were exposed to 15 or 35 J/m2 of 254-nm UV light. Two-milliliter samples were diluted into 8 ml of prewarmed YET broth and aerated at the required temperature for 2 to 3 h. After adding chloroform, the suspension was centrifuged and the supernatant was assayed for phage by plating on sensitive indicator bacteria. Sedimentation of labeled bacterial DNA in alkaline sucrose gradients. The molecular weight of the DNA in bacteria at various times after exposure to UV irradiation was studied by sedimentation methods. Thymine-requiring bacteria were grown at 32 C in K+4 medium to about 2 x 108 cells/ml. Five-milliliter amounts were exposed to UV light and incubated for 15 min at 32 C. Samples of the control and irradiated cultures were diluted into 2 volumes of K medium supplemented with 40 MCi of ["H ]thymidine per ml and incubated for 10 min at 32 C. Portions of the cultures were washed in K medium and resuspended
J. BACTERIOL.
in twice the volume of prewarmed K + 100 medium. The growth rate in this medium was found to be normal. In temperature shift experiments, the cells were resuspended after labeling in the original volume at 32 C, incubated for the appropriate times, and diluted into twice the volume of fresh medium prewarmed to 45 C. Incubation was then continued at 45 C. One-milliliter aliquots were converted to protoplasts by resuspending them at ice temperature in 0.36 ml of 5% sucrose (wt/vol) in 10 mM tris(hydroxymethyl)aminomethane (pH 8.1). A 0.1-ml amount of 32 mM ethylenediaminetetraacetate and 0.04 ml of 0.1% lysozyme were added. After 5 min, 0.05-ml amounts of the spheroplast mixtures were placed in 0.1 ml of 0.5 M NaOH layered on top of a 5-ml gradient of 5 to 20% (wt/vol) sucrose, 0.7 M NaCl, 1.0 mM ethylenediaminetetraacetate at pH 12.3. Gradients were centrifuged in an SW50.1 rotor for 45 min at 40,000 rpm at 20 C in a Spinco L2B centrifuge. Fractions were collected from the bottom of each centrifuge tube onto filter paper disks, which were washed in 5% trichloroacetic acid and dried as before. Recovery of acid-insoluble counts was generally 70 to 80% for cells incubated at 32 C and 55 to 70% for cells incubated at 45 C. Recovery was not affected by UV irradiation.
RESULTS Genetic analysis of E. coli K-12 strain N203. Strain N203 forms recombinants at 32 but not at 42 C when mated with a suitable Hfr
donor (H. Ogawa, personal communication). This strain was tested for the presence of a temperature-sensitive recA mutation by P1 phage transduction. Since recA is cotransducible with cysC, we tested for cotransduction of the mutant allele in N203. P1 phage grows very poorly on this strain. However, chloramphenicol-resistant lysogens were obtained when it was infected with P1 CM clrlOO (10). The P1 lysogen derived from N203 was induced, and the P1 phage so obtained were used to transduce strain NH4919 cysC- leu- str". Cys+ transductants were tested for the ability to produce Leu+ (StrH) recombinants when mated on selective agar spread with a lawn of the Hfr donor AB259 leu+ Strs. Of the Cys+ transductants produced, 21 of 560 (3.8%) were also rec- by this test. Hence, a recombination-deficient mutant allele from N203, referred to as rec-44, was cotransduced with cysC and might be an allele of recA. Two of these recombination-deficient transductants, designated NH4920 and NH4921, were purified and kept for further examination. Plv phage was found to grow with low yield on the rec-44 transductant NH4920, and progeny phage were used to transduce KL266 cysC-. Two further recombination-deficient transductants, NH4939 and NH4940, were found among the Cys+ transductants.
VOL. 121, 1975
recA MUTANT OF E. COLI K-12
895
TABLE 2. Effect of temperature on yield of Leu+ recombinant colonies for Rec+ and recA44 females mated with F' or Hfr donorsa
(Str") colonies/ml
Recipient strains (Leu- Str5)
Leu+ 35 C
AB259 Hfr H
AB 1157 recA + NH4920 recA44 NH4939 recA44
1.6 x 106 2.4 x 105 1.3 x 105
8.1 x 105 1.1 x 10" 1.0 X 102
100 15 8
100 0.14" 0.01
NH4932 F'
AB1157 recA+ NH4920recA44 NH4939 recA44
2.5 x 106 1.7 x 106 1.5 x 106
2.1 x 106 5.1 x 105 8.0 x 105
100 68 60
100 24 38
Donor strain (Leu+ StrS)
42 C
Percent of wild type 35 C 42 C
a AB259 Hfr H injects leu+ pro+, etc., and NH4932 F' injects F101 leu+. Matings at 35 C were carried out for 30 min and at 42 C for 15 min. The frequency of recombinant formation in strain KL266 was similar to that in strain AB 1157. bThe temperature was 41 to 42 C.
TABLE 3. Zygotic complementation tests for His+ recombinants in females carrying rec-44a Hfr donor (His+ StrP)
KL16 recA + KL16 recA + MA1079 recA I MA1079 recA I
F- recipient (His- Str")
AB1157 recA + NH4920 rec-44 AB1157 recA + NH4920 rec-44
Yield of His+ (StrH) recombinant colonies per ml of mating cells
Percent of recA+ level
35C
40C
35C
40C
4.3 x 105 7.6 x 104 5.0 x 104 5.0 x 109
4.7 x 105 2.4 x 104 7.7 x 104 1.8 x 102
100 18 100
100 5 100
10
0.2
aThe Hfr and F- strains were grown at 35 C and mated at 35 or 40 C for 60 and 45 min, respectively, before being plated and incubated at the same temperature.
To test whether rec-44 females were able to accept donor DNA at high temperatures, matings with rec-44 (NH4920 and NH4939) and wild-type (AB1157 and KL266) females, carrying leu- and StrR, were performed with the Strs F' donor NH4932, carrying F101 leu+. The mutant cultures produced 68 and 60% of the wild-type level of Leu+ (Str') colonies at 35 C and 24 and 38% at 42 C (Table 2). Hence, the strain carrying rec-44 showed rather high ability to accept donor DNA at elevated temperature. Any pronounced recombination deficiency observed in this mutant should therefore not be attributed to defective conjugation or chromosome transfer. To test whether recA+ complements the rec44 mutation, Hfr donors carrying either recA+ (KL16) or recAl (MA1079) were mated with the rec-44 (His- StrH) transductant (NH4920) and a related Rec+ strain (AB1157). Both Hfr strains transferred the recA and his genes early during conjugation. Table 3 shows the numbers of His+ (StrR) recombinant colonies produced. At 35 C, the mutant culture gave 18 and 10% of the wild-type level of recombinants in matings with the recA+ and recAl donors, respectively. Hence, the mutant showed only moderate loss
in recombinant formation at this temperature, using the recAl male. However, at 40 C, the mutant female produced only 0.2% of the normal recombinant yield in matings with the recAl donor but 5% of the wild-type yield with the recA+ male. This shows that recA+ but not recA1 complements the mutation responsible for the temperature-sensitive recombination phenotype in the Rec- transductant NH4920. Similar results were obtained in crosses with the original mutant N203 and with the second rec-44 transductant NH4921. The degree to which recA + complements recA- in zygotic temporary partial diploids is known to differ according to the particular allele of recA under test (13) but occurs to a similar extent in females carrying rec-44 and those carrying recA13 (data not shown). Since the entry of recA+ but not recAl caused a substantial increase in recombinant formation in females carrying rec-44, we conclude that rec-44 lies in recA and accordingly designate it recA44. The recA44 transductants NH4920 and NH4921 and also the parental strain NH4919 grew poorly at 42 C, particularly on complete media. However, the original strain N203 and the recA44 transductants NH4939 and NH4940,
896
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HALL AND HOWARD-FLANDERS
well as the parental strain KL266 from which they were derived, all grew well at 43 C on minimal and complete media. Tests on the recombination deficiency of recA44 transductants in crosses involving the transfer of leu+. The Rec- StrR transductants NH4920 and NH4939 carrying leu- recA44 and the related Rec+ leu- strains AB1157 and KL266 were crossed with the Strs Hfr donor AB259, and Leu+ (StrR) colonies were selected. When matings were carried out at 35 C, the mutant recipient produced 15 and 8% of the wild-type level of leu+ recombinants (Table 2). However, when matings were carried out at 42 C, the mutant culture produced Leu+ recombinant colonies at only 0.14 and 0.01% of the wild-type level. Hence, by this test also, the mutant strains appear to be recombination deficient at 42 C but nearly normal at 35 C. Similar results were also obtained for two other recA44 transductants, NH4921 and NH4940. The level of residual recombination observed at 41 to 42 C (0.01 to 0.14%) can be compared with the values (0.01 to 0.02%) obtained with similar experimental procedures for strains carrying recA1 or recA13 (3, 5, 7, 9). Metabolic and physiological response of recA44 transductants to UV irradiation. In view of the greater UV sensitivity and altered DNA metabolism after irradiation reported for strains carrying recA- (3), experiments were carried out to determine how far these characteristics were temperature dependent in strains carrying recA44. The tests described under the following headings were carried out on both the original mutagenized strain N203 and on transductants carrying this mutation. Survival after UV irradiation. The number of colonies formed on agar plates after UV irradiation was compared with the number of colonies on plates that received no irradiation. These surviving fractions are plotted in Fig. 1 as function of the dose. At 32 C, the recA44 strain NH4939 was slightly more sensitive to irradiation than a related recA + strain. This sensitivity increased at 39 C and was more pronounced at 43 C, though less extreme than that associated with recA13. Similar results were obtained in the survival of the original strain N203 after UV irradiation. A second mutation causing a moderate increase in UV sensitivity was detected in strain N203. This mutation was also cotransducible with cysC but did not exhibit a temperature-sensitive phenotype. It was not investigated further. Incorporation of [3H ]TdR after UV irradiation. Log-phase cultures of the recA44 transductant NH4920 and its wild-type parent
_--
T r
109 F
as
I----
108 a
c
"
II\
0
I
I
recA F 43'C 32- C
recA44 32' C
E9 107 n
39' C z 0
X
o6
I0
L)
105 r1 recA/3
43%
_a 0
40 20 ULTRAVIOLET DOSE J/m2
60
FIG. 1. Numbers of colonies formed plotted as a function of ultraviolet dose. Logarithmically growing cultures were diluted, spread on prewarmed YET agar with 20 Ag of thymine per ml, incubated for 30 min at 32, 39, or 43 C, irradiated, and incubated overnight at the same temperatures. The strains and temperatures used were: AB1157 recA+ at 32 C (U) and 43 C (0); NH4939 recA44 at 32 C (0), 39 C (A), and 43 C (0).
irradiated and incubated in the of [3H]TdR at 32 or 45 C (Fig. 2). At 32 C the mutant and wild-type cultures beHTdR into haved similarly and incorporated [3H acid-insoluble material at slightly reduced rates after irradiation as compared with the unirradiated cultures. At 150 min, these irradiated cultures showed 53 and 75% of the incorporation observed for the unirradiated cultures. At 45 C, however, the irradiated wild-type cultures showed normal incorporation, whereas the irradiated mutant culture incorporated [3H TTdR at only 5% of the control culture. This marked HTdR uptake after exposure to reduction in [3H UV light is a characteristic of strains carrying recA- (3, 5). Breakdown of preexisting DNA after UV irradiation. Cultures of the recA44 transductant NH4920 and its wild-type parent AB1157 were labeled with [3H ]TdR and irradiated with 20 J/m 2. The radioactivity remaining acid insoluble was determined during further incubation at 32 or 45 C in unlabeled medium. At 32 C the mutant culture exhibited only slight greater breakdown of its DNA than the wild-type culture (Fig. 3). However, at 45 C both spontaneous and UV-induced breakdown was increased in the mutant strain compared with the wild type, much as reported previously for strains carrying recA- (4, 6).
AB1157
presence
were
E
'~
rec +
rec+
cE w
aw
5-
-j
9 w
cr
a.
897
recA MUTANT OF E. COLI K-12
VOL. 121, 1975
v ~~~~~~~u
Q 0 - 32°c C 320
(c)
4d) ~~450C
in medium lacking the label. and incubated for various times at 32 or 45 C. The DNA from cells exposed to dose of 10 J/m2 (Fig. 4a and d) sedimented more slowly than the DNA from unirradiated cells (Fig. 4a). Upon further incubation of irradiated cultures at 32 C for 30 min (Fig. 4b) or 90 min (Fig. 4c), the DNA increased in molecular weight. By 90 min a large proportion of the DNA from both cultures had sedimented at the position of DNA from unirradiated cells. When irradiated cultures were incubated at 45 C after labeling, the DNA sedimented as shown in Fig. 4e and f. The recA + strain showed a significant proportion of fast-sedimenting DNA by 45 min. However, most of the DNA from the recA44 culture remained slow sediW
-j
100
45" C
320 C
D1
60
Z -UV
rc
rec+ rec+ +UV N
recA44
rec++UV I
60 ~~"N,-o ~recA44,+ UV recA44
H~ recA44 ~ ~weeiraiae ~~ORcwtAzr4()or2 NH92 'Uv 20 k 0
2
1
recA44,+UV
3
2
3 0
a. HOURS
0
Incorporation of [3H]TdR irtto cold acidinsoluble material plotted versus time after addition of the label. Log-phase cultures of AB1157 recA and FIG.
+
NH4920 recA44
were
irradiated with
zero
(0)
or
25
(0) JIm2 of UV light. ['H]TdR was then added and duplicate samples were incubated at 32 or 45 C.
Similar results were obtained with the second recA44 transductant NH4921 and with the original mutagenized strain N203 as regards both DNA synthesis and DNA breakdown after UV irradiation (not shown). Induction of X lysogens. The UV induction of wild-type (NH4929) and recA44 (NH4941) A lysogens was investigated at 32 or 42 C (Table 4). Both spontaneous and UV-induced phage production was reduced in the mutant lysogen carrying recA44 as compared with wild type, the effect being more marked at 42 than at 32 C. However, the block in phage production was much less complete than that observed in lysogens carrying recAl or recA13 (1). Alkaline sucrose sedimentation of celiular DNA after irradiation. To examine the effect of the recA44 mutation on post-replication repair in UV-irradiated cells, the DNA from cultures of NH4915 uvrB- recA+ and NH4916 uvrB- recA44 was analyzed in alkaline sucrose gradients. Log-phase cultures were irradiated and labeled at 32 C with [3H]TdR for 10 min. Part of the irradiated cultures was resuspended
2
2
0
HOURS AFTER IRRADIATION
2.
FIG. 3. Percentage of acid-insoluble AH radioactivity plotted versus time after UV irradiation. Cultures were labeled with [3H]TdR at 32 C, washed and resuspended in nonradioactive medium, exposed to 0 or 20 J/m2 of UVlight, and incubated at 32 (a) or 45 C (b). The acid-soluble and total radioactivity of the cultures was measured, and the fraction of acidinsoluble counts was determined. The strains used were AB1157 recA+ before (0) and after (a) irradiation, and NH4920 recA44 before (0) and after (0) irradiation. TABLE 4. Induction of A lysogens carrying recA+ or recA44a rec allele
recA+
recA44
Incubation temp
(C)
UV dose (J/m2)
32
0
42
35 0 35
32
,42
0 35 0 15 35
Phage/ml
1.5 X 1.6 x 3.0 x 2.4 x
104 108 105 108
5x 6x 3x 3x
105 107 104 105
3x
10'
a Log-phase cultures of NH4929 recA+ and NH4941 recA44 were irradiated with 254 nm of light, incubated in broth at the required temperature for 2.5 h, and assayed for free phage.
898
J. BACTICRIOL.
HALL AND HOWARD-FLANDERS
0
06 I-
0~
z 2
0 CL,
z n
7
13
7 13 FRACTION NUMBER
1
7
13
FIG. 4. Distributions of radioactivity in the fractions collected from alkaline sucrose gradients of [9H]thymidine-labeled DNA from NH4915 uvr- recA + and NH4916 uvr- recA44. Unirradiated cells (U, recA +; 0, recA44) were labeled for 10 min with [3H]TdR. Cells exposed to 10 J/m' of UV light (@, recA+; 0, recA44) were incubated for 15 min in nonradioactive medium at 32 C, incubated for 10 min with [3H]TdR at 32 C, washed, resuspended in twice the volume of nonradioactive growth medium, and incubated for various times at 32 or 45 C. (a) Pulse-labeled irradiated and unirradiated cultures; (b) and (c) the same irradiated cultures as in (a) incubated an additional 30 or 90 min at 32 C in cold medium; (d) pulse-labeled irradiated cultures; (e and f) the same irradiated cultures as in (d) incubated an additional 30 or 45 min at 45 C in cold medium. Essentially similar results (not shown) were obtained in comparable experiments on the recA44 transductant NH4920.
menting and did not change even after 90 min at 45 C (data not shown). Therefore, the increase in molecular weight found in the wildtype strain at both 32 and 45 C occurred in the recA44 strain at 32 but not at 45 C. Evidently recA+ is required for the post-replication increase in molecular weight in UV-irradiated cells. Similar data were obtained with the recA44 transductant NH4920 but are not shown since NH4920 is uvr+ Experiments were performed to determine whether the failure of the DNA to increase m molecular weight seen in the recA44 strain at 45 C would still occur if the culture was incubated for 40 min at 32 C before shifting to 45 C. Cells were grown, irradiated, and labeled as before. Figure 5 shows the sedimentation patterns of DNA from recA + and recA44 strains after incubation in nonradioactive medium at 32 C for 20 or 40 min and after identical incubation times at 32 C followed by additional 45-min incubations at 45 C. DNA from both strains showed a similar shift toward higher molecular weights during incubations at 32 C. After a subsequent incubation at 45 C, DNA from the recA+ strain sedimented at a position
approaching that observed with DNA from unirradiated cells. DNA from the recA44 strain, however, did not significantly change its sedimentation position from that observed before the temperature shift. Hence, the continued presence of a functional recA product was required for the full increase in molecular weight.
DISCUSSION The mutation in a strain of E. coli K-12 N203, with a temperature-sensitive recombination phenotype, is cotransducible with cysC at a frequency of 4%, a value to be compared with 4 to 7% previously reported for recA (13). This mutation was tested for complementation by known recA alleles in temporary zygotic partial diploids (Table 3). In crosses at 42 C with Hfr donors that transfer recA early during mating, mutant recipients produce recombinants at 5% of the wild-type level with a recA + donor but at only 0.2% of normal with a recAl donor. Hence, recA+ but not recAl complements this mutation, which is accordingly accepted as being in recA and designated recA44. Strains to which the mutation recA44 has
VOL. 121, 1975
recA MUTANT OF E. COLI K-12
899
The results show the phenotype of recA44 to be less extreme than that of recAl or recA13 (3, 7). While recombinant formation is depressed to verv low levels in recA44 at restrictive temperatures and approaches those for recA13 under Additional Additional 4 2 45 min 45 sC 45 mins -C comparable conditions, the phenotype for other functions is intermediate. Cells carrying recA44 show greater levels of survival and X prophage induction after UV irradiation than is the case 3 for cells carrying recAl or recA13 (2, 7). The properties of cells carrying recA44 and those carrying the independently isolated temperature-dependent allele recA200 (8) are similar, the latter showing slightly greater UV sensitiv(c) re recA44 w(d) a 40 mins Ca. ity at 43 C in isogenic strains. These results do not establish the rate of 0t4 change of phenotype after a change in temperature, nor whether the active product controlled by the recA gene renatures or has to be synthe7 7 1 sized again after reduction from restrictive to permissive temperatures. The recovery of the RecA+ phenotype is observed only through the Additional mini 45p 45 processes it controls, all of which take time to nonra iociegothmdim(). h aecl complete. In summary, a mutation transferred by coI transduction with cysC from strain N203 shows FIG. 5. Distributions of radioactivity in the fracno complementation with recAl and is desigtions collected from alkaline gradients of nated recA44. Strains carrying recA44 are re[3'H]thymidine-labeled DNA from NH4915 uvrcombination deficient at 42 C but almost norrecA+ and NH4916 uvr- recA44. Cultures posed 10rJIM2 of light, incubated for 15 min in mal at 32 C. At 42 C they exhibit many of the nonradioactive medium 32 C, and incubated for 10 characteristics of the phenotype that has been min with [H]TdR and then for 20 or 40 mi at 32 C in reported for strains carrying recA. rect
6
)
I L.
+rec
3
20 mins 32- C 40 mins 32 4C
*2
recA44
20 mini
a.
32 C
32 C
z
z
2 0
/)
ditional
5
C
mins
45* C
7
13
FRACTION
7
13
NUMBER
sucrose
were
ex-
to
at
nonradioactive growth medium (0). The same cultures were diluted 1:2 into fresh medium and incubated an additional 45 mn at 45 C (0).
been transferred by transduction exhibit temperature-sensitive phenotype. At 40 to 45 C, these strains behave like recA mutants and a
show reduced mation in Hfr
frequencies of recombinant formatings, reduced colony forma-
synthesis after UV irradiation and abnormally high levels of DNA breakdown. X lysogens carrying recA44 show reduced level of lysogenic induction and release of X phage after UV irradiation (2).
tion, and DNA
a
Strains carrying recA44 also show
capacity
for
post-replication repair
irradiation. At 45 C they are less the molecular weight of the
crease
a
reduced
after
able to
UV
in-
DNA mole-
synthesized after irradiation to normal(Fig. 4f and 5d). These data confirm the need for recA + for the post-replication increase in DNA molecular weight after UV irradiation (11, 12) and show that, even after incubation at 32 C for 40 min, the subsequent shift to 45 C prevents molecular weight increase (Fig. 5d). cules
sized molecules
ACKNOWLEDGMENTS We are grateful to H. Ogawa for the gift of the mutant strain N203. This work was carried out in partial fulfillment of the requirements for the doctorate of philosophy at Yale University supported by a Public Health Service predoctoral fellowship held by J.D.H. This work was also supported by Public Health Service grants CA 06519 (from the National Cancer Institute) and AM K 69397 (from the National Institute of Arthritis, Metabolism and Digestive Diseases). We are indebted to Eva Bardwell, who constructed and tested strains NH4939 and NH4940.
LITERATURE CITED 1. Bachman, B. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Brooks, K., and A. J. Clark. 1967. Behavior of A bacteriophage in a recombination deficient strain of Escherichia coli. J. Virol. 1:283-293. 3. Clark, A. J. 1967. The beginning of a genetic analysis of recombination proficiency. J. Cell Physiol. 70 (Suppl. 1):165-180. 4. Clark, A. J., M. Chamberlin, R. P. Boyce, and P. Howard-Flanders. 1966. Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K-12. J. Mol. Biol. 19:442-454. 5. Clark, A. J., and A. D. Margulies. 1965. Isolation and characterization of recombination deficient mutants of Escherichia coli K-12. Genetics 53:451-459. 6. Howard-Flanders, P., and R. P. Boyce. 1966. DNA repair and genetic recombination: studies on mutants of E.
900 7.
8.
9. 10.
HALL AND HOWARD-FLANDERS
coli defective in these processes. Radiat. Res. Suppl. 6:156-184. Howard-Flanders, P., and L. Theriot. 1966. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics 53:1137-1150. Lloyd, R., B. Low, N. Godson, and E. Birge. 1974. Isolation and characterization of a mutant of Escherichia coli K-12 with a temperature-sensitive RecAphenotype. J. Bacteriol. 120:407-415. Low, K. B. 1968. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K-12. Proc. Nat. Acad. Sci. U.S.A. 60:160-167. Rosner, J. L. 1972. Formation, induction and curing of
11.
J. BACTERIOL.
bacteriophage P1 lysogens. Virology 48:679-689. Rupp, D., and P. Howard-Flanders. 1968. Discontinuities
in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J. Mol. Biol. 31:291-304. 12. Smith, K. C., and D. H. C. Meun. 1970. Repair of radiation-induced damage in Escherichia coli. I. Effects of the rec mutations on post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51:459-472. 13. Willetts, N. S., A. J. Clark, and B. Low. 1969. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J. Bacteriol. 97:244-249.
JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 892-900 Copyright © 1975 American Society for Microbiology
Temperature-Sensitive recA Mutant of Escherichia coli K-12: Deoxyribonucleic Acid Metabolism After Ultraviolet Irradiation JENNIFER D. HALL' AND P. HOWARD-FLANDERS* Department of Molecular Biophysics and Biochemistry* and Department of Therapeutic Radiology, Yale University, New Haven, Connecticut 06520 Received for publication 16 September 1974
A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene. To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recAl early and recipients carrying wild-type or mutant alleles. It was found that recA + but not recAl complements this mutation in zygotic temporary partial diploids. The mutant allele was accordingly designated recA44. A transductant carrying recA44 behaved normally at low temperatures but more like recA - strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for A. Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C. Hence, recA+ is required for this molecular weight increase. Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C. This material increased in molecular weight in recA + but not in recA44 cells during subsequent incubation at 45 C. Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process.
Genetic recombination in Escherichia coli K-12 is blocked in strains carrying a mutation in recA, which maps between cysC and pheA (13). In addition to being recombination deficient, mutants carrying recA - are very ultraviolet light (UV) and X-ray sensitive (5, 7) and are defective in recombinational or post-replication repair (11, 12). Phage X lysogens carrying recAshow virtually no spontaneous or UV-induced phage production (2). Most recA mutants so far isolated exhibit this extreme phenotype, but a heavily mutagenized strain, N203, isolated by H. Ogawa, was temperature sensitive in its ability to form recombinants (H. Ogawa, personal communication). This mutant has been found to carry a temperature-sensitive allele of recA, designated recA44, and possibly a second mutation causing UV sensitivity. The properties of strains carrying the recA44 mutation, transduced away from the mutagenized back'Present address: Department of Biochemical Sciences, Princeton University, Princeton, N.J. 08540.
892
ground, were investigated. As the temperature was raised above 35 C there was a sharp decrease in the ability of these transductants to form recombinants in crosses with Hfr donors. The level of recombinant formation at 42 C was comparable to that observed for other strains carrying recA- (5, 7, 9). However, the sensitivity to UV of recA44 transductants at high temperature was less extreme than that of other recA mutants. Similarly, the depression of prophage induction in X lysogens carrying recA44 was less marked as the temperature was increased. The increase in the molecular weight of DNA synthesized after exposure to UV was inhibited at high temperature. While this work was in progress, a strain carrying another mutation (recA200) with a temperature-sensitive phenotype for recombination was isolated through a different selective procedure (8). MATERIALS AND METHODS Bacterial strains. The strains used are listed in Table 1.
893
recA MUTANT 0 F E. COLI K-12
VOL. 121, 1975
Media. Minimal agar contained: KH2PO4, 2.7 g; Na2HPO4, 4.1 g; 10% MgSO4.7H2O, 1.0 ml; 10% (NH,)2SO4, 10 ml; 1% Ca(NO,)2, 0.5 ml; 0.05% FeSO4 7H2O, 0.5 ml; agar, 13.5 g; and water to 1 liter. Minimal liquid medium contained: NH4Cl, 1 g; Na2HPO4.7H.0, 11 g; KH2PO4, 3 g; glucose, 4 g; MgSO4, 120 mg; CaCl2, 10 mg; thiamine, 0.1 mg; and water to 1 liter. The minimal agar and minimal liquid medium were supplemented with the amino acids and nucleosides required by the individual strains at the following concentrations: 0.01% DL-leucine, 0.01% DL-histidine, 0.02% L-arginine, 0.008% DL-serine, 0.005% DL-cysteine, 0.01% DL-threonine, 0.04% DLproline, and 20 MM thymine. Streptomycin, when included, was added at 200 Mg/ml and nalidixic acid was added at 10 ;sg/ml. K medium contained minimal liquid medium supplemented with 10 g of Casamino Acids and 6 g of glucose per liter. K+1, K+4, and K+100 consisted of K medium supplemented with 1, 4, and 100 Mg of thymidine per ml, respectively. Broth contained 10 g of tryptone (Difco), 5 g of yeast extract, 0.5 g of NaCl, and 120 mg of NaOH per liter of water. YET agar contained broth supplemented with 2% agar. X agar contained 5.0 g of tryptone (Difco), 8.0 g of peptone (Difco), 1.0 g of NaCl, 15 g of agar, and water to 1 liter. Top agar contained 8 g of NaCl and 12 g of agar per liter of water. Buffered saline contained 2.68 g of Na2HPO4.7H20, 1.36 g of KH2PO4, and 5 g of NaCl per liter of water. X buffer contained 6 ml of 1 M tris(hydroxymethyl)aminomethane (pH 7.2), 2.46 g of MgSO4.7H20, and 0.05 g of gelatin per liter of water.
Transduction of P1 bacteriophage. Phage P1 CM cirlO0 (10) was prepared by heat induction of a P1 lysogen of strain N203 with the temperature-sensitive phenotype for recombination and was used to infect recipient cells at 32 C that were incubated at 32 C first and then at 42 C to eliminate lysogenic recipients. Phage P1 grows very poorly on strain N203. Tests on transductants for recombination deficiency. After purification, the transductant cells were inoculated in patches on minimal agar and grown at 42 C. They were replica plated onto selective agar spread with Hfr cells in exponential phase and incubated at 42 C. Quantitative tests on recombination deficiency. The strains to be tested were grown with aeration at either 32 or 42 C in broth to about 2 x 10' cells/ml. Donor cells were grown and brought to the same density at 37 C. Two milliliters of a given recipient culture was placed in a flask at the mating temperature (35 or 42 C) for 10 min and 0.2 ml of the donor culture was added. The mixture was incubated with gentle swirling during the mating period, diluted, plated on selective agar containing streptomycin, and incubated at the appropriate temperature. Prewarmed plates were used for the matings at 42 C. UV irradiations. Irradiations were carried out with a 15-W, low-pressure mercury germicidal lamp giving predominantly 254 nm of light. The dose rate was measured with a General Electric germicidal light meter. The bacterial suspensions were irradiated by placing samples in glass petri dishes in a layer less
TABLE 1. Description of bacterial strains Relevant genetic marker Strain
Sex,
plasmids
Str
r
dra rec uvr thy or
cysC
sensi- Proleu tivity phage
Origin or reference
drm AB259 AB1157 AB2497 KL16
Hfr Hayes FFHfr
+ 6 6 +
S R R S
-
+
.+ + + + 43 +
6 +
R S
-
+
+ +
+ +
+ + + +
+ + + +
+ + -
-
+
+ +
-
-
KL266 MA1079
FHfr
+ Al
N203 NH4915
FF-
A44 + + B45
+
+ -
+ +
-
-
-
R R
-
NH4916
F-
A44 B45
-
-
+
-
R
-
NH4919
F-
+
+
+
-
43
6
R
-
NH4920
F-
A44
+
+
-
+
6
R
-
NH4921
F-
A44
+
+
-
+
6
R
-
F+ FlOl leu+thr- + A44 F-
+ + +
+ +
+
+
+
-
+ + + + +
6
A44 A44
+ + + +
R S R R R
NH4929 NH4932 NH4939 NH4940 NH4941
FF-
-
-
6/+ 6 6 6
-
X -
-
X
(1) (1)
Spontaneous thy-dra/m- mutant of AB1157 Obtained from K. B. Low. It injects thyA+
recA+ his+ Obtained from K. B. Low. maLA suppressed Obtained from B. Low. It carries ser- thi- and injects thyA+ recA+ his+. Obtained from H. Ogawaa uvrB Gal+ (Pro+) recombinant of NH4911 from mating with NH4917a urvB Gal+ (Pro+) recombinant of NH4912 from mating with NH4917 Thy+ cysC- recombinant from mating between AT2427 (1) and AB2497 Rec° cysC+ transductant of NH4919 donor N203 rec- cysC+ transductant of NH4919 donor N203 A lysogen of AB1157 F101 thr- derivative of AB1115. cysC+ recA44 transductant of KL266 cysC+ recA44 transductant of KL266 X lysogen of NH4939
aNH4917 is an F'101 derivative of AB2430 gal+ uvrB45 and was used as donor to transfer gal+ uvrB45 to NH4911 and NH4912. NH4911 is a thyA dra/m derivative of JC1557 argG, his-, met-, gal-, lac-, mtl-, xyl- (1) (parent of N203), and NH4912 is a thyA dra/m derivative of N203.
894
HALL AND HOWARD-FLANDERS
than 3 mm deep and gently swirling the cultures during irradiation. Cell survival after UV irradiation. Logarithmically growing cultures at 32 C were diluted, spread on prewarmed YET agar containing 200 qg of thymine per ml, incubated for 30 min at 32, 39, or 42 C, exposed to UV light and incubated overnight at the same
temperatures.
Incorporation of TdR after UV irradiation. Cultures were grown with aeration at 32 C to about 2 x 108 cells per ml in K medium supplemented with 250 yg of deoxyadenosine per ml, and part of each culture was irradiated. Samples were taken and diluted into the same medium further supplemented with 20 uCi of tritiated thymidine per ml (50 Ci/mmol) and aerated at 32 or 45 C for 2 h. At various times, 0.1-ml aliquots were removed and placed on Whatman 3MM filter disks (25-mm diameter) pretreated with 0.1 ml of 1 N NaOH. The disks were washed in cold 5% trichloroacetic acid and counted. DNA breakdown. The bacteria were grown overnight at 32 C in K+4 medium. Samples were diluted 1:10 into K+1 medium containing [3H ]thymidine (50 Ci/mmol) at 10 oCi/ml and aerated at 32 C for 3 h. The cells were washed and suspended in K medium at five times the original volume and aerated for an additional 60 min at 32 C. Amounts (5 to 10 ml) of the labeled cultures were irradiated and duplicate cultures were incubated at 32 or 45 C for 150 min. At various times, 0.5-ml samples were diluted into 0.5 ml of 10% trichloroacetic acid and held at ice temperature for 1 h. A 0.05-ml amount of 1% bovine serum albumin was added and the samples were spun for 15 min at 10,000 rpm. To measure the acid-soluble radioactivity, duplicate 0.1-ml aliquots were removed and counted in 3 ml of tolulene-Triton X-100-1,4-bis(5-phenyloxazolyl)benzene liquid scintillation fluid. To measure the total radioactivity in the samples, the remaining solutions were heated to 90 C for 30 min. Duplicate 0.1-ml samples were removed and counted as before. Induction of A lysogens by UV light. The lysogenic bacteria to be tested for prophage induction were grown at 32 or 42 C to about 2 x 108 cells/ml. The cells were washed and suspended in A buffer at room temperature. Samples of the suspensions were exposed to 15 or 35 J/m2 of 254-nm UV light. Two-milliliter samples were diluted into 8 ml of prewarmed YET broth and aerated at the required temperature for 2 to 3 h. After adding chloroform, the suspension was centrifuged and the supernatant was assayed for phage by plating on sensitive indicator bacteria. Sedimentation of labeled bacterial DNA in alkaline sucrose gradients. The molecular weight of the DNA in bacteria at various times after exposure to UV irradiation was studied by sedimentation methods. Thymine-requiring bacteria were grown at 32 C in K+4 medium to about 2 x 108 cells/ml. Five-milliliter amounts were exposed to UV light and incubated for 15 min at 32 C. Samples of the control and irradiated cultures were diluted into 2 volumes of K medium supplemented with 40 MCi of ["H ]thymidine per ml and incubated for 10 min at 32 C. Portions of the cultures were washed in K medium and resuspended
J. BACTERIOL.
in twice the volume of prewarmed K + 100 medium. The growth rate in this medium was found to be normal. In temperature shift experiments, the cells were resuspended after labeling in the original volume at 32 C, incubated for the appropriate times, and diluted into twice the volume of fresh medium prewarmed to 45 C. Incubation was then continued at 45 C. One-milliliter aliquots were converted to protoplasts by resuspending them at ice temperature in 0.36 ml of 5% sucrose (wt/vol) in 10 mM tris(hydroxymethyl)aminomethane (pH 8.1). A 0.1-ml amount of 32 mM ethylenediaminetetraacetate and 0.04 ml of 0.1% lysozyme were added. After 5 min, 0.05-ml amounts of the spheroplast mixtures were placed in 0.1 ml of 0.5 M NaOH layered on top of a 5-ml gradient of 5 to 20% (wt/vol) sucrose, 0.7 M NaCl, 1.0 mM ethylenediaminetetraacetate at pH 12.3. Gradients were centrifuged in an SW50.1 rotor for 45 min at 40,000 rpm at 20 C in a Spinco L2B centrifuge. Fractions were collected from the bottom of each centrifuge tube onto filter paper disks, which were washed in 5% trichloroacetic acid and dried as before. Recovery of acid-insoluble counts was generally 70 to 80% for cells incubated at 32 C and 55 to 70% for cells incubated at 45 C. Recovery was not affected by UV irradiation.
RESULTS Genetic analysis of E. coli K-12 strain N203. Strain N203 forms recombinants at 32 but not at 42 C when mated with a suitable Hfr
donor (H. Ogawa, personal communication). This strain was tested for the presence of a temperature-sensitive recA mutation by P1 phage transduction. Since recA is cotransducible with cysC, we tested for cotransduction of the mutant allele in N203. P1 phage grows very poorly on this strain. However, chloramphenicol-resistant lysogens were obtained when it was infected with P1 CM clrlOO (10). The P1 lysogen derived from N203 was induced, and the P1 phage so obtained were used to transduce strain NH4919 cysC- leu- str". Cys+ transductants were tested for the ability to produce Leu+ (StrH) recombinants when mated on selective agar spread with a lawn of the Hfr donor AB259 leu+ Strs. Of the Cys+ transductants produced, 21 of 560 (3.8%) were also rec- by this test. Hence, a recombination-deficient mutant allele from N203, referred to as rec-44, was cotransduced with cysC and might be an allele of recA. Two of these recombination-deficient transductants, designated NH4920 and NH4921, were purified and kept for further examination. Plv phage was found to grow with low yield on the rec-44 transductant NH4920, and progeny phage were used to transduce KL266 cysC-. Two further recombination-deficient transductants, NH4939 and NH4940, were found among the Cys+ transductants.
VOL. 121, 1975
recA MUTANT OF E. COLI K-12
895
TABLE 2. Effect of temperature on yield of Leu+ recombinant colonies for Rec+ and recA44 females mated with F' or Hfr donorsa
(Str") colonies/ml
Recipient strains (Leu- Str5)
Leu+ 35 C
AB259 Hfr H
AB 1157 recA + NH4920 recA44 NH4939 recA44
1.6 x 106 2.4 x 105 1.3 x 105
8.1 x 105 1.1 x 10" 1.0 X 102
100 15 8
100 0.14" 0.01
NH4932 F'
AB1157 recA+ NH4920recA44 NH4939 recA44
2.5 x 106 1.7 x 106 1.5 x 106
2.1 x 106 5.1 x 105 8.0 x 105
100 68 60
100 24 38
Donor strain (Leu+ StrS)
42 C
Percent of wild type 35 C 42 C
a AB259 Hfr H injects leu+ pro+, etc., and NH4932 F' injects F101 leu+. Matings at 35 C were carried out for 30 min and at 42 C for 15 min. The frequency of recombinant formation in strain KL266 was similar to that in strain AB 1157. bThe temperature was 41 to 42 C.
TABLE 3. Zygotic complementation tests for His+ recombinants in females carrying rec-44a Hfr donor (His+ StrP)
KL16 recA + KL16 recA + MA1079 recA I MA1079 recA I
F- recipient (His- Str")
AB1157 recA + NH4920 rec-44 AB1157 recA + NH4920 rec-44
Yield of His+ (StrH) recombinant colonies per ml of mating cells
Percent of recA+ level
35C
40C
35C
40C
4.3 x 105 7.6 x 104 5.0 x 104 5.0 x 109
4.7 x 105 2.4 x 104 7.7 x 104 1.8 x 102
100 18 100
100 5 100
10
0.2
aThe Hfr and F- strains were grown at 35 C and mated at 35 or 40 C for 60 and 45 min, respectively, before being plated and incubated at the same temperature.
To test whether rec-44 females were able to accept donor DNA at high temperatures, matings with rec-44 (NH4920 and NH4939) and wild-type (AB1157 and KL266) females, carrying leu- and StrR, were performed with the Strs F' donor NH4932, carrying F101 leu+. The mutant cultures produced 68 and 60% of the wild-type level of Leu+ (Str') colonies at 35 C and 24 and 38% at 42 C (Table 2). Hence, the strain carrying rec-44 showed rather high ability to accept donor DNA at elevated temperature. Any pronounced recombination deficiency observed in this mutant should therefore not be attributed to defective conjugation or chromosome transfer. To test whether recA+ complements the rec44 mutation, Hfr donors carrying either recA+ (KL16) or recAl (MA1079) were mated with the rec-44 (His- StrH) transductant (NH4920) and a related Rec+ strain (AB1157). Both Hfr strains transferred the recA and his genes early during conjugation. Table 3 shows the numbers of His+ (StrR) recombinant colonies produced. At 35 C, the mutant culture gave 18 and 10% of the wild-type level of recombinants in matings with the recA+ and recAl donors, respectively. Hence, the mutant showed only moderate loss
in recombinant formation at this temperature, using the recAl male. However, at 40 C, the mutant female produced only 0.2% of the normal recombinant yield in matings with the recAl donor but 5% of the wild-type yield with the recA+ male. This shows that recA+ but not recA1 complements the mutation responsible for the temperature-sensitive recombination phenotype in the Rec- transductant NH4920. Similar results were obtained in crosses with the original mutant N203 and with the second rec-44 transductant NH4921. The degree to which recA + complements recA- in zygotic temporary partial diploids is known to differ according to the particular allele of recA under test (13) but occurs to a similar extent in females carrying rec-44 and those carrying recA13 (data not shown). Since the entry of recA+ but not recAl caused a substantial increase in recombinant formation in females carrying rec-44, we conclude that rec-44 lies in recA and accordingly designate it recA44. The recA44 transductants NH4920 and NH4921 and also the parental strain NH4919 grew poorly at 42 C, particularly on complete media. However, the original strain N203 and the recA44 transductants NH4939 and NH4940,
896
J. BACTERIOL.
HALL AND HOWARD-FLANDERS
well as the parental strain KL266 from which they were derived, all grew well at 43 C on minimal and complete media. Tests on the recombination deficiency of recA44 transductants in crosses involving the transfer of leu+. The Rec- StrR transductants NH4920 and NH4939 carrying leu- recA44 and the related Rec+ leu- strains AB1157 and KL266 were crossed with the Strs Hfr donor AB259, and Leu+ (StrR) colonies were selected. When matings were carried out at 35 C, the mutant recipient produced 15 and 8% of the wild-type level of leu+ recombinants (Table 2). However, when matings were carried out at 42 C, the mutant culture produced Leu+ recombinant colonies at only 0.14 and 0.01% of the wild-type level. Hence, by this test also, the mutant strains appear to be recombination deficient at 42 C but nearly normal at 35 C. Similar results were also obtained for two other recA44 transductants, NH4921 and NH4940. The level of residual recombination observed at 41 to 42 C (0.01 to 0.14%) can be compared with the values (0.01 to 0.02%) obtained with similar experimental procedures for strains carrying recA1 or recA13 (3, 5, 7, 9). Metabolic and physiological response of recA44 transductants to UV irradiation. In view of the greater UV sensitivity and altered DNA metabolism after irradiation reported for strains carrying recA- (3), experiments were carried out to determine how far these characteristics were temperature dependent in strains carrying recA44. The tests described under the following headings were carried out on both the original mutagenized strain N203 and on transductants carrying this mutation. Survival after UV irradiation. The number of colonies formed on agar plates after UV irradiation was compared with the number of colonies on plates that received no irradiation. These surviving fractions are plotted in Fig. 1 as function of the dose. At 32 C, the recA44 strain NH4939 was slightly more sensitive to irradiation than a related recA + strain. This sensitivity increased at 39 C and was more pronounced at 43 C, though less extreme than that associated with recA13. Similar results were obtained in the survival of the original strain N203 after UV irradiation. A second mutation causing a moderate increase in UV sensitivity was detected in strain N203. This mutation was also cotransducible with cysC but did not exhibit a temperature-sensitive phenotype. It was not investigated further. Incorporation of [3H ]TdR after UV irradiation. Log-phase cultures of the recA44 transductant NH4920 and its wild-type parent
_--
T r
109 F
as
I----
108 a
c
"
II\
0
I
I
recA F 43'C 32- C
recA44 32' C
E9 107 n
39' C z 0
X
o6
I0
L)
105 r1 recA/3
43%
_a 0
40 20 ULTRAVIOLET DOSE J/m2
60
FIG. 1. Numbers of colonies formed plotted as a function of ultraviolet dose. Logarithmically growing cultures were diluted, spread on prewarmed YET agar with 20 Ag of thymine per ml, incubated for 30 min at 32, 39, or 43 C, irradiated, and incubated overnight at the same temperatures. The strains and temperatures used were: AB1157 recA+ at 32 C (U) and 43 C (0); NH4939 recA44 at 32 C (0), 39 C (A), and 43 C (0).
irradiated and incubated in the of [3H]TdR at 32 or 45 C (Fig. 2). At 32 C the mutant and wild-type cultures beHTdR into haved similarly and incorporated [3H acid-insoluble material at slightly reduced rates after irradiation as compared with the unirradiated cultures. At 150 min, these irradiated cultures showed 53 and 75% of the incorporation observed for the unirradiated cultures. At 45 C, however, the irradiated wild-type cultures showed normal incorporation, whereas the irradiated mutant culture incorporated [3H TTdR at only 5% of the control culture. This marked HTdR uptake after exposure to reduction in [3H UV light is a characteristic of strains carrying recA- (3, 5). Breakdown of preexisting DNA after UV irradiation. Cultures of the recA44 transductant NH4920 and its wild-type parent AB1157 were labeled with [3H ]TdR and irradiated with 20 J/m 2. The radioactivity remaining acid insoluble was determined during further incubation at 32 or 45 C in unlabeled medium. At 32 C the mutant culture exhibited only slight greater breakdown of its DNA than the wild-type culture (Fig. 3). However, at 45 C both spontaneous and UV-induced breakdown was increased in the mutant strain compared with the wild type, much as reported previously for strains carrying recA- (4, 6).
AB1157
presence
were
E
'~
rec +
rec+
cE w
aw
5-
-j
9 w
cr
a.
897
recA MUTANT OF E. COLI K-12
VOL. 121, 1975
v ~~~~~~~u
Q 0 - 32°c C 320
(c)
4d) ~~450C
in medium lacking the label. and incubated for various times at 32 or 45 C. The DNA from cells exposed to dose of 10 J/m2 (Fig. 4a and d) sedimented more slowly than the DNA from unirradiated cells (Fig. 4a). Upon further incubation of irradiated cultures at 32 C for 30 min (Fig. 4b) or 90 min (Fig. 4c), the DNA increased in molecular weight. By 90 min a large proportion of the DNA from both cultures had sedimented at the position of DNA from unirradiated cells. When irradiated cultures were incubated at 45 C after labeling, the DNA sedimented as shown in Fig. 4e and f. The recA + strain showed a significant proportion of fast-sedimenting DNA by 45 min. However, most of the DNA from the recA44 culture remained slow sediW
-j
100
45" C
320 C
D1
60
Z -UV
rc
rec+ rec+ +UV N
recA44
rec++UV I
60 ~~"N,-o ~recA44,+ UV recA44
H~ recA44 ~ ~weeiraiae ~~ORcwtAzr4()or2 NH92 'Uv 20 k 0
2
1
recA44,+UV
3
2
3 0
a. HOURS
0
Incorporation of [3H]TdR irtto cold acidinsoluble material plotted versus time after addition of the label. Log-phase cultures of AB1157 recA and FIG.
+
NH4920 recA44
were
irradiated with
zero
(0)
or
25
(0) JIm2 of UV light. ['H]TdR was then added and duplicate samples were incubated at 32 or 45 C.
Similar results were obtained with the second recA44 transductant NH4921 and with the original mutagenized strain N203 as regards both DNA synthesis and DNA breakdown after UV irradiation (not shown). Induction of X lysogens. The UV induction of wild-type (NH4929) and recA44 (NH4941) A lysogens was investigated at 32 or 42 C (Table 4). Both spontaneous and UV-induced phage production was reduced in the mutant lysogen carrying recA44 as compared with wild type, the effect being more marked at 42 than at 32 C. However, the block in phage production was much less complete than that observed in lysogens carrying recAl or recA13 (1). Alkaline sucrose sedimentation of celiular DNA after irradiation. To examine the effect of the recA44 mutation on post-replication repair in UV-irradiated cells, the DNA from cultures of NH4915 uvrB- recA+ and NH4916 uvrB- recA44 was analyzed in alkaline sucrose gradients. Log-phase cultures were irradiated and labeled at 32 C with [3H]TdR for 10 min. Part of the irradiated cultures was resuspended
2
2
0
HOURS AFTER IRRADIATION
2.
FIG. 3. Percentage of acid-insoluble AH radioactivity plotted versus time after UV irradiation. Cultures were labeled with [3H]TdR at 32 C, washed and resuspended in nonradioactive medium, exposed to 0 or 20 J/m2 of UVlight, and incubated at 32 (a) or 45 C (b). The acid-soluble and total radioactivity of the cultures was measured, and the fraction of acidinsoluble counts was determined. The strains used were AB1157 recA+ before (0) and after (a) irradiation, and NH4920 recA44 before (0) and after (0) irradiation. TABLE 4. Induction of A lysogens carrying recA+ or recA44a rec allele
recA+
recA44
Incubation temp
(C)
UV dose (J/m2)
32
0
42
35 0 35
32
,42
0 35 0 15 35
Phage/ml
1.5 X 1.6 x 3.0 x 2.4 x
104 108 105 108
5x 6x 3x 3x
105 107 104 105
3x
10'
a Log-phase cultures of NH4929 recA+ and NH4941 recA44 were irradiated with 254 nm of light, incubated in broth at the required temperature for 2.5 h, and assayed for free phage.
898
J. BACTICRIOL.
HALL AND HOWARD-FLANDERS
0
06 I-
0~
z 2
0 CL,
z n
7
13
7 13 FRACTION NUMBER
1
7
13
FIG. 4. Distributions of radioactivity in the fractions collected from alkaline sucrose gradients of [9H]thymidine-labeled DNA from NH4915 uvr- recA + and NH4916 uvr- recA44. Unirradiated cells (U, recA +; 0, recA44) were labeled for 10 min with [3H]TdR. Cells exposed to 10 J/m' of UV light (@, recA+; 0, recA44) were incubated for 15 min in nonradioactive medium at 32 C, incubated for 10 min with [3H]TdR at 32 C, washed, resuspended in twice the volume of nonradioactive growth medium, and incubated for various times at 32 or 45 C. (a) Pulse-labeled irradiated and unirradiated cultures; (b) and (c) the same irradiated cultures as in (a) incubated an additional 30 or 90 min at 32 C in cold medium; (d) pulse-labeled irradiated cultures; (e and f) the same irradiated cultures as in (d) incubated an additional 30 or 45 min at 45 C in cold medium. Essentially similar results (not shown) were obtained in comparable experiments on the recA44 transductant NH4920.
menting and did not change even after 90 min at 45 C (data not shown). Therefore, the increase in molecular weight found in the wildtype strain at both 32 and 45 C occurred in the recA44 strain at 32 but not at 45 C. Evidently recA+ is required for the post-replication increase in molecular weight in UV-irradiated cells. Similar data were obtained with the recA44 transductant NH4920 but are not shown since NH4920 is uvr+ Experiments were performed to determine whether the failure of the DNA to increase m molecular weight seen in the recA44 strain at 45 C would still occur if the culture was incubated for 40 min at 32 C before shifting to 45 C. Cells were grown, irradiated, and labeled as before. Figure 5 shows the sedimentation patterns of DNA from recA + and recA44 strains after incubation in nonradioactive medium at 32 C for 20 or 40 min and after identical incubation times at 32 C followed by additional 45-min incubations at 45 C. DNA from both strains showed a similar shift toward higher molecular weights during incubations at 32 C. After a subsequent incubation at 45 C, DNA from the recA+ strain sedimented at a position
approaching that observed with DNA from unirradiated cells. DNA from the recA44 strain, however, did not significantly change its sedimentation position from that observed before the temperature shift. Hence, the continued presence of a functional recA product was required for the full increase in molecular weight.
DISCUSSION The mutation in a strain of E. coli K-12 N203, with a temperature-sensitive recombination phenotype, is cotransducible with cysC at a frequency of 4%, a value to be compared with 4 to 7% previously reported for recA (13). This mutation was tested for complementation by known recA alleles in temporary zygotic partial diploids (Table 3). In crosses at 42 C with Hfr donors that transfer recA early during mating, mutant recipients produce recombinants at 5% of the wild-type level with a recA + donor but at only 0.2% of normal with a recAl donor. Hence, recA+ but not recAl complements this mutation, which is accordingly accepted as being in recA and designated recA44. Strains to which the mutation recA44 has
VOL. 121, 1975
recA MUTANT OF E. COLI K-12
899
The results show the phenotype of recA44 to be less extreme than that of recAl or recA13 (3, 7). While recombinant formation is depressed to verv low levels in recA44 at restrictive temperatures and approaches those for recA13 under Additional Additional 4 2 45 min 45 sC 45 mins -C comparable conditions, the phenotype for other functions is intermediate. Cells carrying recA44 show greater levels of survival and X prophage induction after UV irradiation than is the case 3 for cells carrying recAl or recA13 (2, 7). The properties of cells carrying recA44 and those carrying the independently isolated temperature-dependent allele recA200 (8) are similar, the latter showing slightly greater UV sensitiv(c) re recA44 w(d) a 40 mins Ca. ity at 43 C in isogenic strains. These results do not establish the rate of 0t4 change of phenotype after a change in temperature, nor whether the active product controlled by the recA gene renatures or has to be synthe7 7 1 sized again after reduction from restrictive to permissive temperatures. The recovery of the RecA+ phenotype is observed only through the Additional mini 45p 45 processes it controls, all of which take time to nonra iociegothmdim(). h aecl complete. In summary, a mutation transferred by coI transduction with cysC from strain N203 shows FIG. 5. Distributions of radioactivity in the fracno complementation with recAl and is desigtions collected from alkaline gradients of nated recA44. Strains carrying recA44 are re[3'H]thymidine-labeled DNA from NH4915 uvrcombination deficient at 42 C but almost norrecA+ and NH4916 uvr- recA44. Cultures posed 10rJIM2 of light, incubated for 15 min in mal at 32 C. At 42 C they exhibit many of the nonradioactive medium 32 C, and incubated for 10 characteristics of the phenotype that has been min with [H]TdR and then for 20 or 40 mi at 32 C in reported for strains carrying recA. rect
6
)
I L.
+rec
3
20 mins 32- C 40 mins 32 4C
*2
recA44
20 mini
a.
32 C
32 C
z
z
2 0
/)
ditional
5
C
mins
45* C
7
13
FRACTION
7
13
NUMBER
sucrose
were
ex-
to
at
nonradioactive growth medium (0). The same cultures were diluted 1:2 into fresh medium and incubated an additional 45 mn at 45 C (0).
been transferred by transduction exhibit temperature-sensitive phenotype. At 40 to 45 C, these strains behave like recA mutants and a
show reduced mation in Hfr
frequencies of recombinant formatings, reduced colony forma-
synthesis after UV irradiation and abnormally high levels of DNA breakdown. X lysogens carrying recA44 show reduced level of lysogenic induction and release of X phage after UV irradiation (2).
tion, and DNA
a
Strains carrying recA44 also show
capacity
for
post-replication repair
irradiation. At 45 C they are less the molecular weight of the
crease
a
reduced
after
able to
UV
in-
DNA mole-
synthesized after irradiation to normal(Fig. 4f and 5d). These data confirm the need for recA + for the post-replication increase in DNA molecular weight after UV irradiation (11, 12) and show that, even after incubation at 32 C for 40 min, the subsequent shift to 45 C prevents molecular weight increase (Fig. 5d). cules
sized molecules
ACKNOWLEDGMENTS We are grateful to H. Ogawa for the gift of the mutant strain N203. This work was carried out in partial fulfillment of the requirements for the doctorate of philosophy at Yale University supported by a Public Health Service predoctoral fellowship held by J.D.H. This work was also supported by Public Health Service grants CA 06519 (from the National Cancer Institute) and AM K 69397 (from the National Institute of Arthritis, Metabolism and Digestive Diseases). We are indebted to Eva Bardwell, who constructed and tested strains NH4939 and NH4940.
LITERATURE CITED 1. Bachman, B. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Brooks, K., and A. J. Clark. 1967. Behavior of A bacteriophage in a recombination deficient strain of Escherichia coli. J. Virol. 1:283-293. 3. Clark, A. J. 1967. The beginning of a genetic analysis of recombination proficiency. J. Cell Physiol. 70 (Suppl. 1):165-180. 4. Clark, A. J., M. Chamberlin, R. P. Boyce, and P. Howard-Flanders. 1966. Abnormal metabolic response to ultraviolet light of a recombination deficient mutant of Escherichia coli K-12. J. Mol. Biol. 19:442-454. 5. Clark, A. J., and A. D. Margulies. 1965. Isolation and characterization of recombination deficient mutants of Escherichia coli K-12. Genetics 53:451-459. 6. Howard-Flanders, P., and R. P. Boyce. 1966. DNA repair and genetic recombination: studies on mutants of E.
900 7.
8.
9. 10.
HALL AND HOWARD-FLANDERS
coli defective in these processes. Radiat. Res. Suppl. 6:156-184. Howard-Flanders, P., and L. Theriot. 1966. Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics 53:1137-1150. Lloyd, R., B. Low, N. Godson, and E. Birge. 1974. Isolation and characterization of a mutant of Escherichia coli K-12 with a temperature-sensitive RecAphenotype. J. Bacteriol. 120:407-415. Low, K. B. 1968. Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K-12. Proc. Nat. Acad. Sci. U.S.A. 60:160-167. Rosner, J. L. 1972. Formation, induction and curing of
11.
J. BACTERIOL.
bacteriophage P1 lysogens. Virology 48:679-689. Rupp, D., and P. Howard-Flanders. 1968. Discontinuities
in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J. Mol. Biol. 31:291-304. 12. Smith, K. C., and D. H. C. Meun. 1970. Repair of radiation-induced damage in Escherichia coli. I. Effects of the rec mutations on post-replication repair of damage due to ultraviolet radiation. J. Mol. Biol. 51:459-472. 13. Willetts, N. S., A. J. Clark, and B. Low. 1969. Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J. Bacteriol. 97:244-249.
