Molec. gen. Genet. 156, 121-131 (1977) © by Springer-Verlag 1977
Isolation and Characterization of Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light Takesi Kato and Yukiko Shinoura Department of FundamentalRadiology,Facultyof Medicine, Osaka University,Kita-ku, Osaka 530, Japan
Summary. Mutants of E. coli defective in susceptibility to UV-induction of mutations were isolated by direct screening for their UV nonmutable phenotype ( U m u ) . Screening of about 30,000 mutagenized clones of a u v r B - derivative of ABl157 yielded six Umu- strains. The mutants can be classified into three groups by the location of the mutations, u m u A , u m u B and umuC. Mutations u m u A and u m u B are, respectively, mapped close to l e x A and recA genes and mutations at both loci partially reduce UV mutagenesis. The locus of u m u C is between h e m A and p u r B and the mutations at this new locus result in a moderate increase of UV sensitivity. The mutation diminishes UV mutagenesis and UV reactivation of phage ~ without affecting the inducibility of phophage 2 nor the inhibition of cell division following UV irradiation. Related properties of an isogenic strain of a r e c F - mutant are compared with those of umuC
.
Introduction
Ultraviolet light (UV) mutagenesis in Escherichia coli has been thought occur as a result of errors in repair of the damage in DNA produced by UV (Witkin, 1969; Kondo, 1969). Two efficient repair mechanisms known in E. coli are excision repair (Setlow et al., 1964; Boyce et al., 1964) and postreplication repair (Rupp et al., 1968). Postreplication repair has been suggested as a major source of UV mutagenesis since a mutation recA results in total lack of postreplication repair (Smith etal., 1970) and UV mutagenesis (Miura et al., 1968; Witkin, 1969). The misrepair theory gained further support by the existence of a mutation l e x A (or exr) which eliminates UV mutagenesis For offprints contact." Takesi Kato
(Witkin, 1967; Mount et al., 1972) and postreplication repair (Youngs et al., 1973 ; Ganesan et al., 1975). Evidence accumulating, however, shows a complexity of the functions of recA and l e x A genes. UV irradiation results in the followings; prophage induction (Lwoff et al., 1950), inhibition of respiration (Swenson etal., 1974), inhibition of cell division (Green et al., 1969; Inouye, 1971), induction of the inhibitor of Exo V (Marsden et al., 1971), promotion of survival of UV irradiated phage )o (UV reactivation; Weigle, 1953) and induction of mutations as well. Occurrence of all these inducible functions requires the recA + - l e x + gene products, and the apparent coupling of UV mutagenesis with postreplication repair is a facet of the pleiotrophic effects of recA and l e x A mutations. Structural complexity of the two genes is also inferred by the existence of several mutations: t/f (Castellazzi et al., 1972a), zab (Castellazzi et al., 1972b), IexB (Blanco et al., 1975), tsl (Mount et al., 1973), spr (Mount, 1977) and rnm (Volkert et al., 1976). These mutations have been located tightly linked to either recA or l e x A and they cause modification of the pleiotrophic effects of recA and l e x A mutations. Incorporating these observations the current hypothesis of inducible error-prone repair as a cause of UV mutagenesis has been proposed by Radman (1974) and Witkin (1975, 1976). Previously we have shown that mutational block of both known pathways of genetic recombination (Horii et al., 1973) by r e c B - recF mutations does not eliminate UV mutagenesis (Kato et al., 1977) though the strain is recombination deficient and hypersensitive to UV (Rothman et al., 1975). It may be possible to account for the observed UV mutagenesis in the mutant by the hypothetical inducible errorprone repair. In that case, the putative inducible repair system must be relatively inefficient in repairing lethal damage by UV since the recombinational repair pathways which are blocked in the mutant are
122
T. Kato and Y. Shinoura: Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light
Table 1. Bacterial strains Strain
Sex
Genotype a
Source or reference
ABl157
F
Bachmann (1972)
JC5422 JC3833 JC3836 JC3890 TK50I TK510 TK513 TK516 JC3839 JC8945 JC3931 TK600 TK601 TK603 TK604 DM844 KD48 c TK752 b TK003b $729 AB470
FF FF FF FFFFFF F FF F FF F FF-
KLBMI788 ° CA18 JC158 K10 ° KLI9 KL209 KL16 JE4436 ° KL208 KL98
F ColB* HfrH HfrC Hfr Hfr Hfr Hfr6 Hfr Hfr
thr-1, leu-6, proA2, his-4, argE3, thi-1, lacY1, galK2, ara-14, xyl-5, mtl-1, tsx-33, strA31, supE44 as AB1157 also thyA325 as JC5422 also A[uvrB301, chl , bio , phr , pgi-] as JC3833 also ilv328 as JC3836 also thy +, ilv + as JC3890 also umuC36 as TK501 also trp as TK510 also hemA8, t~T + as TK513 alsopurB-, hem + as JC5422 also ilv325 as JC3839 also uvrA6, thy +, argE + as JC8945 also ilv +, recF143 as JC9845 also trp as TK600 also hemA8, trp + as TK601 also hemA + as TK601 also purB , hem + as AB1157 also lex-1, argE + trpE9829, tyr-, rif-r, m a l A - , uvrA6, supDgal , his-, cysC-, m e t E - , thyA-, argF , phr-, malB argF , uvrA , malB thi-, metB1, hemA8, lacY1, malA1, strA134 proA2, purB15, his-4, thi-1, lacY1, galK2, xyl-1, mtl-1, 2-, supE44 thi , thyA301, bio87, argA-, p h e A - , endAlO1, metE72
b °
serA6, str ~, lacI22, thi-1, relA1, 2relA1, tonA22, T2 a )R malB16, supE44, thi-1, ,~thi-1, relA1, 2purE , gal6, trp ~)R
Bachmann (1972) Kato et al. (1977) Kato et al. (1977) Kato et al. (1977) EMS treatment of JC3890 EMS treatment of TK501 TK510+P1 .$729; Trp + selection TK513+P1 .AB470; Hem + selection Kato et al. (1977) Kato et al. (1977) Kato et al. (1977) EMS treatment of JC8945 TK600 + P 1. $729 ; Trp + selection TK601 + P 1. AB 1157; Hem + selection TK601 +P1 .AB470; Hem + selection Mount et al. (1972)
Guest (1969) Bachmann (1972)
Ishii (1972) Bachmann (1972) Low Low Low Low Low Low
(1972) (1972) (1972) (1972) (1972) (1972)
Genetic symbols are defined by Bachmann, Low and Taylor (1976) These are derived from H/r strain of E. eoli B Strains KD48, KLBM1788 and K10 and JE4436 were obtained through T. Nagata. B. Glickman and H. Ogawa respectively
very effective for repair of lethal damage. An import a n t a p p r o a c h to the p r o b l e m w o u l d be i s o l a t i o n o f mutants
deficient in U V
mutagenesis but resistant
to UV inactivation. T h i s p a p e r will d e s c r i b e t h e i s o l a t i o n a n d c h a r a c terization of the mutants isolated by direct screening for their UV nonmutable phenotype. In addition, we examined related phenotypes of a recF mutant which is d e f e c t i v e in i n d u c t i o n o f p r o p h a g e 2 ( R o b e r t s et al., 1975) a n d p o s t r e p l i c a t i o n r e p a i r ( G a n e s a n 1975), w h i l e it is n o r m a l l y U V m u t a b l e .
etal.,
Materials and Methods Bacterial and Bacteriophage Strains. The strains used are described in Table 1. TK510 and TK600, trp derivatives of TK501 and JC8945, respectively, were isolated by the penicillin screening method from ethyl methanesulfonate (EMS) mutagenized cultures of the parental strains, hemA- strains TK513 and TK601 and
purB strains TK516 and TK604 were constructed by the successive transduction of hemA and purB mutations from donor strains $729 and AB470, respectively, to recipient strains TK510 and TK600 by P1 phage. Bacteriophage ). was used for the experiments of UV-reactivation. General Methods. The methods used for conjugation and transduction were those described by Clark and Margulies (I965) and Willetts, Clark and Low (1969). Media. Lambda broth (2 broth) and semienriched minimal medium (SEM) were described previously (Kato et al., 1977). The buffer 56/2 and Luria broth (L broth) were those described by Willetts, Clark and Low (1969). Minimal medium was Davis minimal salts medium supplemented with glucose (0.4%) and required nutritions. Colicin plates were prepared by the method described Ishii and Kondo (1972). A Col B factor carrying strain CA18 was grown overnight in L broth. 0.1 mls of the overnight culture were plated on L broth agar plates and the plates were incubated for 45 h at 37 C. The cells grown on the plates were killed by chloroform vapor and the plates were overlayed with 5 ml of top agar (2 broth made 1.2% in Difco agar) per plate. Colicin plates were stored at 5C until use.
T. Kato and Y. Shinoura: Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light
Isolation of Umu Mutants. The first screening of UV nonmutable ( U m u ) strains was done by a replica-plating method against a UV mimetic mutagen 4-Nitroquinoline-l-Oxide (4NQO; Kondo et al., 1968). An exponentially growing culture of JC3890, a uvrBderivative of ABl157, was mixed with an equal volume of 0.3 M EMS disolved in 56/2 buffer and was incubated for 30 min at 37 C. The treated cells were harvested, washed twice with 56/2 by centrifugation before resuspended and divided among 20 tubes of 5 ml L broth. The tubes were incubated overnight at 30 C. About 10% of the treated cells remained viable. After suitable dilution in 56/2, samples from each tube were plated on two minimal medium agar plates. Two hundred colonies representative of the survivors in each tube were patched (6 x 6 mm 2) on fresh minimal agar mediums and the plates were incubated overnight at 30 C. The master plates were replica plated onto 4NQO-SEM plates (SEM containing 1 gg 4NQO) and the plates were incubated for three days at 30 C. Strains which produced less than two to three His + revertants were selected as putative Umu- mutants. Umu + strains exhibited a few tens of His + revertants in the patch of inoculum by this method. Each putative Umn strain was purified and was then subjected to UV irradiation for the second screening. Aliquots of overnight cultures of purified clones of 4NQO nonresponders were patched on SEM plates with a sterile wire loop and the plates were exposed to 0.5 J/m 2 of UV. The plates were incubated for 72 h at 30 C and the mutants which produced significantly small numbers of His + mutants were kept for further tests.
Frequencies of UV Induced Mutations. The method for determining frequency of His + mutations has been described previously (Kato et al., 1977). Detection of the mutation to colicin B resistance (ColB R) was basically that described by Ishii and Kondo (1972). Cells were grown overnight in L broth. The cells were washed twice with, and resuspended in, the minimal salts buffer 56/2 and starved for 1 h at 37 C. The starved overnight cultures were diluted to a concentration of about 2 x 108 cells/ml and were exposed to UV. For the assay of His + mutations, 0.1 mls of the irradiated culture were plated on SEM to count His + mutants. Appropriately diluted samples were also plated for survival. The plates were incubated for 72 h at 37 C and His + and survivors were scored. The frequency of induced mutations (Fmh) was calculated by the formula:
F,,h--
M - M0
N
where M and Mo are the average numbers of mutants scored for UV irradiated and unirradiated cultures, respectively, and N is the average number of survivors. For the mutation ColBR, 1.0 ml samples of the irradiated cultures were transferred to tubes containing 9 ml L broth and were incubated overnight at 37 C to allow expression of mutational events. 0.1 mls of the irradiated overnight cultures were plated on Colicin plates and colonies formed after 48 h incubation were scored as ColBR mutants. Survivors were scored on L broth agar plates. The frequency of induced mutations (Fmc) was calculated as follows : M F,.,. N
M0 NO
where M and Mo are respectively the average numbers of ColBR mutants scored for UV irradiated and unirradiated cultures, N and N o are the average numbers of survivors scored for irradiated and unirradiated cultures, respectively.
UV Reactivation qf2 Phage. Host bacteria were grown in ), broth to exponential phase of about 2 x 108 cells/ml. The cells were
123
harvested by centrifugation, resuspended in the original volume of phosphate buffer (0.15 M pH 7.0) containing 10 2M MgSO4 and were exposed to UV radiation. Preirradiated ). phages were added to the irradiated host bacterial suspensions at a multiplicity of infection of 0.1 and incubated for 15 rain at 37 C to allowed adsorption. The mixtures were plated with 0.1 ml of overnight culture of an indicator bacteria by the standard tops agar layer method and infective centers were scored after 24 h incubation at 37 C. Double mutant AB2480 (recA14 uvrA6) was used indicator bacteria to avoid rescuing unadsorbed phages.
Induction of Prophage 2. Lysogenic bacteria was grown to exponential phase in 2 broth. The cells were harvested by centrifugation, resuspended i n the original volume of phsophate buffer and subjected to UV irradiation. 0.1 mls of the irradiated cells were mixed with 0.2 mls of overnight culture of a rifampicin resistant indicator bacteria KD48 (uvrA rif-r) and the mixtures were plated on 2 broth agar plates with 2.5 ml of top agar. After 2 h of incubation at 37 C, the plates were gently covered with additional 2.5 ml of top agar containing 300 ~tg rifampicin and were incubated overnight at 37 C. Rifampicin was added to minimize the infective centers arising from spontaneously induced prophages during the incubation.
UV Irradiation. Bacterial suspensions of about 2 x 108 cells per ml in 56/2 buffer were irradiated in a glass petri dish at 75 cm from two 15 W germicidal lamps. The fluence rate at 75 cm was 0.9 J/m2/s. Lower fluence rates of 0.067 and 0.0067 j/m2/s for UV sensitive strains were attained by a diaphragm placed between the lamps and subject and by a motor-drived rotating sector in combination with the diaphragm, respectively.
Results Isolation o f U m u - M u t a n t s S c r e e n i n g o f a b o u t 30,000 JC3890 by the 4NQO-SEM
mutagenized clones of replica plating method
y i e l d e d 53 i s o l a t e s w h i c h d i d n o t p r o d u c e H i s + m u tants on 4NQO-SEM plate. There were several types o f m u t a t i o n s w h i c h c o u l d r e s u l t in t h i s p h e n o t y p e : 1) a s e c o n d site m u t a t i o n in t h e his o p e r o n , 2) m u t a t i o n s in g e n e s c o d e d f o r m e m b r a n e p e r m e a b i l i t y o f 4 N Q O o r g e n e s i n v o l v e d in a c t i v a t i o n o f 4 N Q O . T o eliminate those unwanted mutants, a second screening w a s d o n e w i t h 53 4 N Q O n o n r e s p o n d o r s f o r t h e i r a b i l ity t o i n d u c e H i s + a n d A r g ÷ r e v e r s i o n s b y U V . T h e rationale for the second screening relied on the fact t h a t t h e m u t a t i o n s his-4 a n d argE3 o f t h e p a r e n t a l s t r a i n J C 3 8 9 0 a r e ochre n o n s e n s e m u t a t i o n s s i n c e a majority of UV-induced His+ and Arg + phenotypic r e v e r s i o n s a r e d u e t o a n ochre s u p p r e s s o r m u t a t i o n o c c u r e d in supE44 r e g i o n . T h u s a l a r g e f r a c t i o n s o f the UV-induced His + revertants are also reverted to Arg ÷ . These mutants are also characterized by having l o s t t h e amber s u p p r e s s i n g a c t i v i t y o f supE44 ( K a t o a n d Clark, u n p u b l i s h e d results). O n e can t h e r e f o r e e x p e c t t h a t , I) t h o s e m u t a n t s in w h i c h 4 N Q O u p t a k e o r m e t a b o l i s m is a f f e c t e d w o u l d p r o d u c e b o t h A r g + a n d H i s + r e v e r t a n t s b y U V , II) t h o s e m u t a n t s w h i c h
124
T. Kato and Y. Shinoura: Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light
have a second his mutation would produce Arg ÷ but not His + revertants by UV and III) those mutants which are UV nonmutable would produce neither Arg ÷ nor His ÷ revertants. The results of the second screening are presented in Table 2. Thirteen out of 53 isolates showed the type III response and were kept for further characterization. The remaining were either type I or type II and were discarded. Finally, dose-frequency kinetics of UV-induced mutations for two independent characters His ÷ and ColB R were carried out with the type-III mutants and the results are presented in Table 3. Six out of 13 mutants exhibited a more or less frequency of induced mutations to both His ÷ and ColB R and were designated as Umu mutants. The frequencies of induced mutations in the remaining strains were comparable to those of the parental strain JC3890. These strains were also highly sensitive to UV and that could account for the detection of these strains as UV nonrespondors in the second screening. Recombination proficiency and UV sensitivity were then examined and the six U m u - mutants were placed into three groups; group C, recombination proficient, moderately sensitive to UV and no UV mutability (TK501, TK502 and TK503), group B, slightly recombination
deficient, highly sensitive to UV and reduced mutability (TK504 and TK505); group A, recombination proficient, moderately sensitive to UV and reduced UV mutability (TK506).
Genetic Loci of Mutations to UV Nonmutability To locate the mutations confering UV nonmutable phenotype a variety of conjugational and transductional crosses were performed. For the preliminary assignment of location of those mutations, the set of Hfr strains shown in Figure 1 was used. The conjugational crosses showed that one locus (urnuA of TK506) was located around the malB gene, another (umuB of TK504 and TK505) was placed between
[
Kkl9
~pyrD
2
Table 2. Classification of the initially isolated 4NQO nonrespondors by their UV mutability Type
No. of mutants isolated
4NQO
UV
His ÷
His +
Arg ÷
17 23 i3
_a --
_}_a --
@a + -
e
Isolation and Characterization of Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light Takesi Kato and Yukiko Shinoura Department of FundamentalRadiology,Facultyof Medicine, Osaka University,Kita-ku, Osaka 530, Japan
Summary. Mutants of E. coli defective in susceptibility to UV-induction of mutations were isolated by direct screening for their UV nonmutable phenotype ( U m u ) . Screening of about 30,000 mutagenized clones of a u v r B - derivative of ABl157 yielded six Umu- strains. The mutants can be classified into three groups by the location of the mutations, u m u A , u m u B and umuC. Mutations u m u A and u m u B are, respectively, mapped close to l e x A and recA genes and mutations at both loci partially reduce UV mutagenesis. The locus of u m u C is between h e m A and p u r B and the mutations at this new locus result in a moderate increase of UV sensitivity. The mutation diminishes UV mutagenesis and UV reactivation of phage ~ without affecting the inducibility of phophage 2 nor the inhibition of cell division following UV irradiation. Related properties of an isogenic strain of a r e c F - mutant are compared with those of umuC
.
Introduction
Ultraviolet light (UV) mutagenesis in Escherichia coli has been thought occur as a result of errors in repair of the damage in DNA produced by UV (Witkin, 1969; Kondo, 1969). Two efficient repair mechanisms known in E. coli are excision repair (Setlow et al., 1964; Boyce et al., 1964) and postreplication repair (Rupp et al., 1968). Postreplication repair has been suggested as a major source of UV mutagenesis since a mutation recA results in total lack of postreplication repair (Smith etal., 1970) and UV mutagenesis (Miura et al., 1968; Witkin, 1969). The misrepair theory gained further support by the existence of a mutation l e x A (or exr) which eliminates UV mutagenesis For offprints contact." Takesi Kato
(Witkin, 1967; Mount et al., 1972) and postreplication repair (Youngs et al., 1973 ; Ganesan et al., 1975). Evidence accumulating, however, shows a complexity of the functions of recA and l e x A genes. UV irradiation results in the followings; prophage induction (Lwoff et al., 1950), inhibition of respiration (Swenson etal., 1974), inhibition of cell division (Green et al., 1969; Inouye, 1971), induction of the inhibitor of Exo V (Marsden et al., 1971), promotion of survival of UV irradiated phage )o (UV reactivation; Weigle, 1953) and induction of mutations as well. Occurrence of all these inducible functions requires the recA + - l e x + gene products, and the apparent coupling of UV mutagenesis with postreplication repair is a facet of the pleiotrophic effects of recA and l e x A mutations. Structural complexity of the two genes is also inferred by the existence of several mutations: t/f (Castellazzi et al., 1972a), zab (Castellazzi et al., 1972b), IexB (Blanco et al., 1975), tsl (Mount et al., 1973), spr (Mount, 1977) and rnm (Volkert et al., 1976). These mutations have been located tightly linked to either recA or l e x A and they cause modification of the pleiotrophic effects of recA and l e x A mutations. Incorporating these observations the current hypothesis of inducible error-prone repair as a cause of UV mutagenesis has been proposed by Radman (1974) and Witkin (1975, 1976). Previously we have shown that mutational block of both known pathways of genetic recombination (Horii et al., 1973) by r e c B - recF mutations does not eliminate UV mutagenesis (Kato et al., 1977) though the strain is recombination deficient and hypersensitive to UV (Rothman et al., 1975). It may be possible to account for the observed UV mutagenesis in the mutant by the hypothetical inducible errorprone repair. In that case, the putative inducible repair system must be relatively inefficient in repairing lethal damage by UV since the recombinational repair pathways which are blocked in the mutant are
122
T. Kato and Y. Shinoura: Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light
Table 1. Bacterial strains Strain
Sex
Genotype a
Source or reference
ABl157
F
Bachmann (1972)
JC5422 JC3833 JC3836 JC3890 TK50I TK510 TK513 TK516 JC3839 JC8945 JC3931 TK600 TK601 TK603 TK604 DM844 KD48 c TK752 b TK003b $729 AB470
FF FF FF FFFFFF F FF F FF F FF-
KLBMI788 ° CA18 JC158 K10 ° KLI9 KL209 KL16 JE4436 ° KL208 KL98
F ColB* HfrH HfrC Hfr Hfr Hfr Hfr6 Hfr Hfr
thr-1, leu-6, proA2, his-4, argE3, thi-1, lacY1, galK2, ara-14, xyl-5, mtl-1, tsx-33, strA31, supE44 as AB1157 also thyA325 as JC5422 also A[uvrB301, chl , bio , phr , pgi-] as JC3833 also ilv328 as JC3836 also thy +, ilv + as JC3890 also umuC36 as TK501 also trp as TK510 also hemA8, t~T + as TK513 alsopurB-, hem + as JC5422 also ilv325 as JC3839 also uvrA6, thy +, argE + as JC8945 also ilv +, recF143 as JC9845 also trp as TK600 also hemA8, trp + as TK601 also hemA + as TK601 also purB , hem + as AB1157 also lex-1, argE + trpE9829, tyr-, rif-r, m a l A - , uvrA6, supDgal , his-, cysC-, m e t E - , thyA-, argF , phr-, malB argF , uvrA , malB thi-, metB1, hemA8, lacY1, malA1, strA134 proA2, purB15, his-4, thi-1, lacY1, galK2, xyl-1, mtl-1, 2-, supE44 thi , thyA301, bio87, argA-, p h e A - , endAlO1, metE72
b °
serA6, str ~, lacI22, thi-1, relA1, 2relA1, tonA22, T2 a )R malB16, supE44, thi-1, ,~thi-1, relA1, 2purE , gal6, trp ~)R
Bachmann (1972) Kato et al. (1977) Kato et al. (1977) Kato et al. (1977) EMS treatment of JC3890 EMS treatment of TK501 TK510+P1 .$729; Trp + selection TK513+P1 .AB470; Hem + selection Kato et al. (1977) Kato et al. (1977) Kato et al. (1977) EMS treatment of JC8945 TK600 + P 1. $729 ; Trp + selection TK601 + P 1. AB 1157; Hem + selection TK601 +P1 .AB470; Hem + selection Mount et al. (1972)
Guest (1969) Bachmann (1972)
Ishii (1972) Bachmann (1972) Low Low Low Low Low Low
(1972) (1972) (1972) (1972) (1972) (1972)
Genetic symbols are defined by Bachmann, Low and Taylor (1976) These are derived from H/r strain of E. eoli B Strains KD48, KLBM1788 and K10 and JE4436 were obtained through T. Nagata. B. Glickman and H. Ogawa respectively
very effective for repair of lethal damage. An import a n t a p p r o a c h to the p r o b l e m w o u l d be i s o l a t i o n o f mutants
deficient in U V
mutagenesis but resistant
to UV inactivation. T h i s p a p e r will d e s c r i b e t h e i s o l a t i o n a n d c h a r a c terization of the mutants isolated by direct screening for their UV nonmutable phenotype. In addition, we examined related phenotypes of a recF mutant which is d e f e c t i v e in i n d u c t i o n o f p r o p h a g e 2 ( R o b e r t s et al., 1975) a n d p o s t r e p l i c a t i o n r e p a i r ( G a n e s a n 1975), w h i l e it is n o r m a l l y U V m u t a b l e .
etal.,
Materials and Methods Bacterial and Bacteriophage Strains. The strains used are described in Table 1. TK510 and TK600, trp derivatives of TK501 and JC8945, respectively, were isolated by the penicillin screening method from ethyl methanesulfonate (EMS) mutagenized cultures of the parental strains, hemA- strains TK513 and TK601 and
purB strains TK516 and TK604 were constructed by the successive transduction of hemA and purB mutations from donor strains $729 and AB470, respectively, to recipient strains TK510 and TK600 by P1 phage. Bacteriophage ). was used for the experiments of UV-reactivation. General Methods. The methods used for conjugation and transduction were those described by Clark and Margulies (I965) and Willetts, Clark and Low (1969). Media. Lambda broth (2 broth) and semienriched minimal medium (SEM) were described previously (Kato et al., 1977). The buffer 56/2 and Luria broth (L broth) were those described by Willetts, Clark and Low (1969). Minimal medium was Davis minimal salts medium supplemented with glucose (0.4%) and required nutritions. Colicin plates were prepared by the method described Ishii and Kondo (1972). A Col B factor carrying strain CA18 was grown overnight in L broth. 0.1 mls of the overnight culture were plated on L broth agar plates and the plates were incubated for 45 h at 37 C. The cells grown on the plates were killed by chloroform vapor and the plates were overlayed with 5 ml of top agar (2 broth made 1.2% in Difco agar) per plate. Colicin plates were stored at 5C until use.
T. Kato and Y. Shinoura: Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light
Isolation of Umu Mutants. The first screening of UV nonmutable ( U m u ) strains was done by a replica-plating method against a UV mimetic mutagen 4-Nitroquinoline-l-Oxide (4NQO; Kondo et al., 1968). An exponentially growing culture of JC3890, a uvrBderivative of ABl157, was mixed with an equal volume of 0.3 M EMS disolved in 56/2 buffer and was incubated for 30 min at 37 C. The treated cells were harvested, washed twice with 56/2 by centrifugation before resuspended and divided among 20 tubes of 5 ml L broth. The tubes were incubated overnight at 30 C. About 10% of the treated cells remained viable. After suitable dilution in 56/2, samples from each tube were plated on two minimal medium agar plates. Two hundred colonies representative of the survivors in each tube were patched (6 x 6 mm 2) on fresh minimal agar mediums and the plates were incubated overnight at 30 C. The master plates were replica plated onto 4NQO-SEM plates (SEM containing 1 gg 4NQO) and the plates were incubated for three days at 30 C. Strains which produced less than two to three His + revertants were selected as putative Umu- mutants. Umu + strains exhibited a few tens of His + revertants in the patch of inoculum by this method. Each putative Umn strain was purified and was then subjected to UV irradiation for the second screening. Aliquots of overnight cultures of purified clones of 4NQO nonresponders were patched on SEM plates with a sterile wire loop and the plates were exposed to 0.5 J/m 2 of UV. The plates were incubated for 72 h at 30 C and the mutants which produced significantly small numbers of His + mutants were kept for further tests.
Frequencies of UV Induced Mutations. The method for determining frequency of His + mutations has been described previously (Kato et al., 1977). Detection of the mutation to colicin B resistance (ColB R) was basically that described by Ishii and Kondo (1972). Cells were grown overnight in L broth. The cells were washed twice with, and resuspended in, the minimal salts buffer 56/2 and starved for 1 h at 37 C. The starved overnight cultures were diluted to a concentration of about 2 x 108 cells/ml and were exposed to UV. For the assay of His + mutations, 0.1 mls of the irradiated culture were plated on SEM to count His + mutants. Appropriately diluted samples were also plated for survival. The plates were incubated for 72 h at 37 C and His + and survivors were scored. The frequency of induced mutations (Fmh) was calculated by the formula:
F,,h--
M - M0
N
where M and Mo are the average numbers of mutants scored for UV irradiated and unirradiated cultures, respectively, and N is the average number of survivors. For the mutation ColBR, 1.0 ml samples of the irradiated cultures were transferred to tubes containing 9 ml L broth and were incubated overnight at 37 C to allow expression of mutational events. 0.1 mls of the irradiated overnight cultures were plated on Colicin plates and colonies formed after 48 h incubation were scored as ColBR mutants. Survivors were scored on L broth agar plates. The frequency of induced mutations (Fmc) was calculated as follows : M F,.,. N
M0 NO
where M and Mo are respectively the average numbers of ColBR mutants scored for UV irradiated and unirradiated cultures, N and N o are the average numbers of survivors scored for irradiated and unirradiated cultures, respectively.
UV Reactivation qf2 Phage. Host bacteria were grown in ), broth to exponential phase of about 2 x 108 cells/ml. The cells were
123
harvested by centrifugation, resuspended in the original volume of phosphate buffer (0.15 M pH 7.0) containing 10 2M MgSO4 and were exposed to UV radiation. Preirradiated ). phages were added to the irradiated host bacterial suspensions at a multiplicity of infection of 0.1 and incubated for 15 rain at 37 C to allowed adsorption. The mixtures were plated with 0.1 ml of overnight culture of an indicator bacteria by the standard tops agar layer method and infective centers were scored after 24 h incubation at 37 C. Double mutant AB2480 (recA14 uvrA6) was used indicator bacteria to avoid rescuing unadsorbed phages.
Induction of Prophage 2. Lysogenic bacteria was grown to exponential phase in 2 broth. The cells were harvested by centrifugation, resuspended i n the original volume of phsophate buffer and subjected to UV irradiation. 0.1 mls of the irradiated cells were mixed with 0.2 mls of overnight culture of a rifampicin resistant indicator bacteria KD48 (uvrA rif-r) and the mixtures were plated on 2 broth agar plates with 2.5 ml of top agar. After 2 h of incubation at 37 C, the plates were gently covered with additional 2.5 ml of top agar containing 300 ~tg rifampicin and were incubated overnight at 37 C. Rifampicin was added to minimize the infective centers arising from spontaneously induced prophages during the incubation.
UV Irradiation. Bacterial suspensions of about 2 x 108 cells per ml in 56/2 buffer were irradiated in a glass petri dish at 75 cm from two 15 W germicidal lamps. The fluence rate at 75 cm was 0.9 J/m2/s. Lower fluence rates of 0.067 and 0.0067 j/m2/s for UV sensitive strains were attained by a diaphragm placed between the lamps and subject and by a motor-drived rotating sector in combination with the diaphragm, respectively.
Results Isolation o f U m u - M u t a n t s S c r e e n i n g o f a b o u t 30,000 JC3890 by the 4NQO-SEM
mutagenized clones of replica plating method
y i e l d e d 53 i s o l a t e s w h i c h d i d n o t p r o d u c e H i s + m u tants on 4NQO-SEM plate. There were several types o f m u t a t i o n s w h i c h c o u l d r e s u l t in t h i s p h e n o t y p e : 1) a s e c o n d site m u t a t i o n in t h e his o p e r o n , 2) m u t a t i o n s in g e n e s c o d e d f o r m e m b r a n e p e r m e a b i l i t y o f 4 N Q O o r g e n e s i n v o l v e d in a c t i v a t i o n o f 4 N Q O . T o eliminate those unwanted mutants, a second screening w a s d o n e w i t h 53 4 N Q O n o n r e s p o n d o r s f o r t h e i r a b i l ity t o i n d u c e H i s + a n d A r g ÷ r e v e r s i o n s b y U V . T h e rationale for the second screening relied on the fact t h a t t h e m u t a t i o n s his-4 a n d argE3 o f t h e p a r e n t a l s t r a i n J C 3 8 9 0 a r e ochre n o n s e n s e m u t a t i o n s s i n c e a majority of UV-induced His+ and Arg + phenotypic r e v e r s i o n s a r e d u e t o a n ochre s u p p r e s s o r m u t a t i o n o c c u r e d in supE44 r e g i o n . T h u s a l a r g e f r a c t i o n s o f the UV-induced His + revertants are also reverted to Arg ÷ . These mutants are also characterized by having l o s t t h e amber s u p p r e s s i n g a c t i v i t y o f supE44 ( K a t o a n d Clark, u n p u b l i s h e d results). O n e can t h e r e f o r e e x p e c t t h a t , I) t h o s e m u t a n t s in w h i c h 4 N Q O u p t a k e o r m e t a b o l i s m is a f f e c t e d w o u l d p r o d u c e b o t h A r g + a n d H i s + r e v e r t a n t s b y U V , II) t h o s e m u t a n t s w h i c h
124
T. Kato and Y. Shinoura: Mutants of Escherichia coli Deficient in Induction of Mutations by Ultraviolet Light
have a second his mutation would produce Arg ÷ but not His + revertants by UV and III) those mutants which are UV nonmutable would produce neither Arg ÷ nor His ÷ revertants. The results of the second screening are presented in Table 2. Thirteen out of 53 isolates showed the type III response and were kept for further characterization. The remaining were either type I or type II and were discarded. Finally, dose-frequency kinetics of UV-induced mutations for two independent characters His ÷ and ColB R were carried out with the type-III mutants and the results are presented in Table 3. Six out of 13 mutants exhibited a more or less frequency of induced mutations to both His ÷ and ColB R and were designated as Umu mutants. The frequencies of induced mutations in the remaining strains were comparable to those of the parental strain JC3890. These strains were also highly sensitive to UV and that could account for the detection of these strains as UV nonrespondors in the second screening. Recombination proficiency and UV sensitivity were then examined and the six U m u - mutants were placed into three groups; group C, recombination proficient, moderately sensitive to UV and no UV mutability (TK501, TK502 and TK503), group B, slightly recombination
deficient, highly sensitive to UV and reduced mutability (TK504 and TK505); group A, recombination proficient, moderately sensitive to UV and reduced UV mutability (TK506).
Genetic Loci of Mutations to UV Nonmutability To locate the mutations confering UV nonmutable phenotype a variety of conjugational and transductional crosses were performed. For the preliminary assignment of location of those mutations, the set of Hfr strains shown in Figure 1 was used. The conjugational crosses showed that one locus (urnuA of TK506) was located around the malB gene, another (umuB of TK504 and TK505) was placed between
[
Kkl9
~pyrD
2
Table 2. Classification of the initially isolated 4NQO nonrespondors by their UV mutability Type
No. of mutants isolated
4NQO
UV
His ÷
His +
Arg ÷
17 23 i3
_a --
_}_a --
@a + -
e
