Mutation Research, 62 ( 1 9 7 9 ) 7 - - 1 7 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
A recA-DEPENDENT MUTATOR OF ESCHERICHIA COLI K12: METHOD OF ISOLATION AND INITIAL CHARACTERIZATION
GERD HOMBRECHER * and WALTER VIELMETTER
Institut fiir Genelik der Universit~'t zu K6ln (West-Deutschland) ( R e c e i v e d 27 N o v e m b e r 1 9 7 8 ) ( R e v i s i o n received 27 M a r c h 1 9 7 9 ) ( A c c e p t e d 5 April 1 9 7 9 )
Summary A number of m u t a t o r strains of E. coli were isolated using histochemical techniques which allow the identification of a single m u t a t o r colony on agar plates with as many as 2000 colonies. Several mutators isolated in this way were found by Pl-mediated transduction to map to the proA--proB region of the E. coli chromosome. The map position of these mutators is very close to that of the conditional mutator, mutD. However, in contrast to mutD, one of these newly isolated mutators was suppressed in a thermosensitive recA strain at 43°C, but not at 30°C. This m u t a t o r m u t a t i o n has been named rout-8. Besides being dependent upon recA, rout-8 is also dependent upon growth in enriched medium for the expression of its m u t a t o r activity. The m u t a t o r activity of mut-8 was f o u n d to be recessive to the wild-type allele.
The appearance of new mutants in a population is an essential part of evolution and there is little d o u b t that the rate of spontaneous mutagenesis is subject to genetic control. A major portion of spontaneous mutagenesis is due to errors in DNA replication, recombination and/or DNA repair. Mutations affecting the spontaneous m u t a t i o n rate have been identified in several organisms and depending on whether they increase or decrease the m u t a t i o n rate they are classed as mutators and antimutators (Drake and Baltz, 1976; Cox, 1976). There are two approaches which can be used to increase our understanding of the processes controlling fidelity of DNA replication and the associated p h e n o m e n a of recombination and DNA repair. One can look at the effect of genes known to be involved in these processes, e.g. polA (Berg, 1971; Kondo, 1973; Vaccaro and Siegel, 1975; Hombrecher, 1978), polC (dnaE) (Hall and * Present address: G. Hombrecher, John Innes I n s t i t u t e , C o l n e y Lane, Norwich (Great Britain).
Brammar, 1973; Siegel, 1973; Sevastopolous and Glaser, 1977), uvrE (mutU) (Siegel, 1973; Mattern and Houtman, 1974). In the case of bacteriophage T4, several of the D N A polymerase mutants have been thoroughly studied and the mechanism by which they affect D N A replication fidelity is understood (Speyer, 1965; Drake and Greening, 1970; Muzyczka et al., 1972). Alternatively, one can directly isolate mutants affecting spontaneous mutation rates and examine these mutants with respect to those processes involved in D N A metabolism. In this study a histochemical staining technique was developed and employed to allow the isolation of mutator mutants of E. coli by the direct examination of colonies derived from mutagenised cultures. Materials and methods Bac teria and phages The bacterial strains used in this study are derivatives of E. coli K12 and are listed in Table 1. Phage P l k c was used for transduction which was performed as described by Lennox (1955). TABLE 1 BACTERIAL STRAINS Strain
Sex
Relevant genotype
S o u r c e , ref.
W1485
F-
p r o t o t r o p h , str r
W. V i e l m e t t e r
m u t a t o r d e r i v a t i v e s o f W 1 4 8 5 ( r n u t a g e n i z e d with M N N G )
this p a p e r
M2 M3 X47S
F-
ara, leu, p r o C , tsx. p u r E , gal, t r p , lys, str r, m e t E , thi, xyl, l a c y
W. V i e l m e t t e r
X2 X3 X5 X6 X7 X8
FFFFFFF-
m u t a t o r d e r i v a t i v e s o f X 4 7 S ( m u t a g e n i z e d with M N N G )
this p a p e r
mut-8
X478-1
F-
derivative o f X 4 7 8 ; proC +, lacY *
this p a p e r
Y1 Y2
F- } F-
m u t a t o r derivatives o f X 4 7 8 - 1 ( m u t a g e n i z e d w i t h M N N G )
this p a p e r
AB1 157 U-2
FHfr
thr, leu, a r g E , his, p r o A , str r l a c I , ara r e g u l a t o r y g e n e s
Broda (1974) K.P. R e i n e r s
KV221 KV388
FF'
A (lac, p r o A , B ) F ' 1 2 8 proA+,B +. lac+/mal, spc, s u p E , A (lac p r o )
W. V i e l m e t t e r W. V i e l m e t t e r
NH4921
F
leu, str r, r e c A 4 4 , tel, thi, a r g E , his, p r o A , thr, ara, galK, lacY, m t l , xyl, tsx, s u p
Hall a n d H o w a r d Flanders (1975)
GHI0 M2-T2
FF-
mut-S, by Pl-transduction from X8 into NH4921 rout, b y P l - t r a n s d u c t i o n f r o m M 2 i n t o A B l 1 5 7
this p a p e r this p a p e r
4358
F-
4359
F-
thi, leu, proC, m e t E , hisF, t h y A , lacZ, ara, m t l , xyl, m a l A , str r, s p e 4358, recA200
L l o y d et al. (1974) L l o y d et al. (1974)
X4
F o r g e n e s y m b o l s s e e B a c h m a n n et al. ( 1 9 7 6 ) .
Chemicals Sources of chemicals were: Nalidixic acid (Nal) and naphthol-AS-MX-phosphate (NAP) (Sigma Chemical Company, St. Louis), fast blue RR salt (FBRR) and 6-bromo-2-naphthyl-fi-D-galactopyranoside (NAG) (Serva, Heidelberg), N-methyl-N-nitroso-N'-nitroguanidine (MNNG) (Koch-Light, Colnbrook), strept o m y c i n (Str) (Bayer, Leverkusen). Antipain was a gift of Bayer (Wuppertal), Giemsa of Merck (Darmstadt).
Media Saline used for dilution was 0.9% NaC1. PB medium contained 1.75% antibiotic medium 3 (Difco) and NB medium 0.8% nutrient broth (Difco) and 0.5% NaC1. TY agar contained 0.8% t r y p t o n e (Difco), 0.5% yeast extract (Difco), 0.5% NaC1 and 1.5% agar (Difco): after autoclaving, MgCl~ was added to a final concentration of 1 mM. Minimal agar (MM) contained 1.5% agar, salt solution was added so that the final concentrations were 0.15% KH2PO4, 0.35% K2HPO4, 0.07% Na-citrate-5~ H20, 0.1% (NH4)~SO4 and in addition 0.1% MgSO4 • 7 H20, 0.2% glucose and 20 pg/ml of the required amino acids and bases. P1 agar contained 1% bacto t r y p t o n e (Difco), 0.5% yeast extract (Difco), 0.75% NaC1 and 1% agar, after autoclaving 0.1% glucose and 2.5 mM CaC12 were added. P1 soft agar had the same ingredients except 0.6% agar only.
Histochemical staining The staining procedures were modifications of methods used by Messer and Vielmetter (1965) and Vielmetter et al. (1968). Giemsa solution mixture contained 40 mg basic fuchsin, 30 mg methylene blue and 17.5 ml Giemsa solution in 1 1 water. This solution was filtered and 5-ml aliquots were poured out per plate for colony-staining. After sufficient staining (10--20 min) the plates were rinsed with water and were observed with a stereomicroscope. NAG solution as an indicator for ~-galactosidase contained 0.2 g NAG in 1 1 0.08 M Tris buffer pH 8. This solution was added to 1.6 mg FBRR per ml NAG solution (which was predissolved in some drops of dimethylsulphoxide). 5 ml of this dye was used immediately after mixing in the way described above for Giemsa staining. NAP solution for alkaline phosphatase contained 1 g NAP in 1 1 Tris 1 M buffer pH 8. The mixture with the azo dye FBRR was prepared as described for NAG.
Isolation procedure For mutagenesis an exponentially growing culture (5 × 107 cells/ml) in PB was incubated with N-methyl-N-nitroso-N'-nitroguanidine (MNNG) (concentration 40 pg/ml) for 4 min. After a 500-fold dilution cells were grown to allow segregation and expression of the mutations for about 5--6 generations and then plated in a special way to screen for m u t a t o r mutants. The cells were embedded in a double top layer of soft agar. The first layer, containing 1.5 ml of 0.45% soft agar plus 2 × 103 cells, was poured onto a TY agar plate. After solidifying, 1.5 ml of soft agar was added w i t h o u t bacteria as a top layer to prevent colonies from being washed away during the staining procedure. Plates were kept open for 1 h to dry. After about 16 h of incubation at 37°C lens-like microcolonies had formed. For the first screening of mutators we used Giemsa
10
staining because at least 3 different phenotypes could be seen. These genetically uncharacterized mutants absorbed alkaline dyes out of the solution mixture to a greater extent than wild-type cells. Mutant colonies were red, blue or violet and by subculturing such coloured colonies one can demonstrate that the staining reaction is determined genetically. This staining m e t h o d is highly sensitive so that different coloured m u t a n t sectors can be detected in a single colony of the prospective m u t a t o r strain. In order to show that the isolated strains had general m u t a t o r activity we used indicators for known gene functions (NAG, for ~-galactosidase constitutive and NAP for alkaline phosphatase constitutive mutants). For illustration see pictures of stained m u t a t o r colonies (Hombrecher et al., 1972). Results
I. The isolation o f mutator strains In order to isolate m u t a t o r mutants of E. coli we developed a technique based upon a combination of histochemical stains as has been described in Materials and Methods. Techniques were developed by which a single m u t a t o r colony on an agar plate could be quickly and reliably identified among a background of 2000 wild-type colonies. The scanning of more than 2.7 million colonies derived from MNNG-treated bacterial cultures following Giemsa staining resulted in the isolation of some 120 suspected m u t a t o r colonies which were subjected to restreaking and purification followed by staining with Giemsa, NAG for lacI, and NAP for p h o R and p h o S to test forward mutagenesis. This second screening left 24 strains which were classed as having m u t a t o r phenotypes based upon the appearance of predominantly sectored colonies following the staining procedures. The remaining 96 either did n o t show strong m u t a t o r activity or were not general mutators. Mutants were called strong mutators if more than 10% of sectored colonies could be found after staining with NAP corresponds to a m u t a t i o n frequency higher than 10 -4 mutants/plated cell. The m u t a t o r isolates were further examined for their spontaneous mutation frequency for a variety of mutational sites. The results for two of the isolated mutators (Table 2) are typical of those obtained for the mutators tested. From TABLE 2 MUTATION Strain
FREQUENCIES
FOR WILD-TYPE AND MUTATOR
STRAINS
Characteristics examined pho
+ ~
l a c ÷ --* l a c l
hal +-~ naIA
leu-6
~
Leu +
__b _ b 7.6 X 10 -3 ( 5 8 5 0 ) 1.3 X 10 -6
2 1 1.4 1.0
6.7 × 10 -7 (51) 1 . 3 × 1 0 -8 _c _c
his-4
~
His +
phoR,S
XS rout a 10-3 (1000) X 4 7 8 rout + 1 X 10 -6 M 2 - T 2 rout 2.7 × 10 -3 A B l 1 5 7 rout ÷ _ c
× × X X
1 0 -5 ( 2 0 0 0 ) 1 0 -8 10 -6 ( 1 4 0 ) 1 0 -8
M u t a t i o n f r e q u e n c i e s were m e a s u r e d in PB m e d i u m a f t e r o v e r - n i g h t g r o w t h . a Estimate made from densely populated agar plates. b S t r a i n s e x a m i n e d are l a c Y . c Not determined. T h e n u m b e r s i n b r a c k e t s a r e t h e relative m u t a t i o n f r e q u e n c i e s c o m p a r e d t o t h e w i l d - t y p e s t r a i n .
__ c _ c 2 × 10 -7 (40) 5 X 10 -9
11
TABLE EFFECT Culture
1 2 3 4 5
3 OF MEDIUM
ON THE MUTATION
Initial frequency
Frequency in minimal medium
1.8 1.2 1.7 7.1 --
2.1 2.8 1.1 1.3 1.4
× × X X
10 -8 10 -8 10 -8 10 -8
The results given are for mutation
X X X X X
FREQUENCIES
10 -8 10 -8 10 -8 10 -8 10 -8
OF STRAIN
M2-T2
Frequency in
Frequency
minimal medium + Thd
in NB
2.7 6.8 5.5 4.8 --
1.1 1.5 1.8 1.1 1.5
X X X X
10 -8 10 -8 10 -8 10 -8
X X X X X
10 -6 10 -6 10 -6 10 -6 10 -6
Factor MM/NB
52 54 164 85 107
t o nal r e s i s t a n c e .
these results and those reported above, it can be concluded that these strains are indeed generalized mutators. This technique is also useful for identifying weak mutators as for example a forward m u t a t i o n frequency of 10 -s mutants/cell gives approximately 1% of sectored colonies.
II. Mapping o f the mutator strains Mapping of the m u t a t o r genes proved to be difficult for at least two reasons: first an effect of growth medium was observed so t h a t the staining techniques could n o t be used on minimal medium plates following conjugation and secondly only 12 of the 24 m u t a n t s were able to produce sufficiently high titres of bacteriophage P1 to permit further mapping by Pl-mediated transduction. The other strains grew very poorly for u n k n o w n reasons. The first problem stems from the conditional nature of m u t a t o r effects which are n o t fully expressed on minimal but only on enriched medium. This has been previously observed for m u t D mutants by Degnen and Cox (1974) and in our studies, e.g. m u t a t o r M2-T2, the medium effect produced a 52--164fold difference in m u t a t i o n levels (Table 3). Crosses of Hfr U-2 with strains X4 and X8 suggested that the mutations responsible for the m u t a t o r effect were located close to lacI which is near to the known m u t a t o r mutD. Furthermore, mapping by Pl-mediated transduction, where this was possible, showed 11 of the 12 strains to be carrying m u t a t o r mutations cotransducible with proA. These results are summarised in Table 4.
III. Characterization o f one o f the mutators (X8) The map position of the 11 mutators which could be mapped is very close to that of m u t D (Degnen and Cox, 1974) which is cotransducible with proA and argF. We looked at the properties of one of the m u t a t o r strains which had been designated X8 to determine whether our mutators were identical to mutD. From Table 4 it can be seen that the m u t a t i o n responsible for the mutator effect of X8 is linked to proA (11%) and from Table 2 it can be seen that the m u t a t i o n exerts a considerable m u t a t o r effect, in the range of 3 orders of magnitude for the forward mutations on enriched medium. To determine whether the m u t a t o r of X8 was d o m i n a n t or recessive, sexductions were carried out using strain KV388 carrying F'128proA ÷, proB ÷, lac ÷,
12 TABLE 4 C O T R A N S D U C T I O N F R E Q U E N C I E S OF M U T A T O R M U T A T I O N S WITH proA Parent strain
Mutator
Pro*mut-/pro + ,
Frequency (%)
W1485
M1 M2 M3
6/ 61 121142 lO/174
9.8 8.5 5.7
X478
X2 X3 X4 X5 X6 X7 X8
21150 10/225 51/350 20/183 2/118 1/ 81 19/175
1.3 4.4 15 11 1.7 1.2 I1
X478-1
Y1 Y2
O/ 74 3/ 70
A recA-DEPENDENT MUTATOR OF ESCHERICHIA COLI K12: METHOD OF ISOLATION AND INITIAL CHARACTERIZATION
GERD HOMBRECHER * and WALTER VIELMETTER
Institut fiir Genelik der Universit~'t zu K6ln (West-Deutschland) ( R e c e i v e d 27 N o v e m b e r 1 9 7 8 ) ( R e v i s i o n received 27 M a r c h 1 9 7 9 ) ( A c c e p t e d 5 April 1 9 7 9 )
Summary A number of m u t a t o r strains of E. coli were isolated using histochemical techniques which allow the identification of a single m u t a t o r colony on agar plates with as many as 2000 colonies. Several mutators isolated in this way were found by Pl-mediated transduction to map to the proA--proB region of the E. coli chromosome. The map position of these mutators is very close to that of the conditional mutator, mutD. However, in contrast to mutD, one of these newly isolated mutators was suppressed in a thermosensitive recA strain at 43°C, but not at 30°C. This m u t a t o r m u t a t i o n has been named rout-8. Besides being dependent upon recA, rout-8 is also dependent upon growth in enriched medium for the expression of its m u t a t o r activity. The m u t a t o r activity of mut-8 was f o u n d to be recessive to the wild-type allele.
The appearance of new mutants in a population is an essential part of evolution and there is little d o u b t that the rate of spontaneous mutagenesis is subject to genetic control. A major portion of spontaneous mutagenesis is due to errors in DNA replication, recombination and/or DNA repair. Mutations affecting the spontaneous m u t a t i o n rate have been identified in several organisms and depending on whether they increase or decrease the m u t a t i o n rate they are classed as mutators and antimutators (Drake and Baltz, 1976; Cox, 1976). There are two approaches which can be used to increase our understanding of the processes controlling fidelity of DNA replication and the associated p h e n o m e n a of recombination and DNA repair. One can look at the effect of genes known to be involved in these processes, e.g. polA (Berg, 1971; Kondo, 1973; Vaccaro and Siegel, 1975; Hombrecher, 1978), polC (dnaE) (Hall and * Present address: G. Hombrecher, John Innes I n s t i t u t e , C o l n e y Lane, Norwich (Great Britain).
Brammar, 1973; Siegel, 1973; Sevastopolous and Glaser, 1977), uvrE (mutU) (Siegel, 1973; Mattern and Houtman, 1974). In the case of bacteriophage T4, several of the D N A polymerase mutants have been thoroughly studied and the mechanism by which they affect D N A replication fidelity is understood (Speyer, 1965; Drake and Greening, 1970; Muzyczka et al., 1972). Alternatively, one can directly isolate mutants affecting spontaneous mutation rates and examine these mutants with respect to those processes involved in D N A metabolism. In this study a histochemical staining technique was developed and employed to allow the isolation of mutator mutants of E. coli by the direct examination of colonies derived from mutagenised cultures. Materials and methods Bac teria and phages The bacterial strains used in this study are derivatives of E. coli K12 and are listed in Table 1. Phage P l k c was used for transduction which was performed as described by Lennox (1955). TABLE 1 BACTERIAL STRAINS Strain
Sex
Relevant genotype
S o u r c e , ref.
W1485
F-
p r o t o t r o p h , str r
W. V i e l m e t t e r
m u t a t o r d e r i v a t i v e s o f W 1 4 8 5 ( r n u t a g e n i z e d with M N N G )
this p a p e r
M2 M3 X47S
F-
ara, leu, p r o C , tsx. p u r E , gal, t r p , lys, str r, m e t E , thi, xyl, l a c y
W. V i e l m e t t e r
X2 X3 X5 X6 X7 X8
FFFFFFF-
m u t a t o r d e r i v a t i v e s o f X 4 7 S ( m u t a g e n i z e d with M N N G )
this p a p e r
mut-8
X478-1
F-
derivative o f X 4 7 8 ; proC +, lacY *
this p a p e r
Y1 Y2
F- } F-
m u t a t o r derivatives o f X 4 7 8 - 1 ( m u t a g e n i z e d w i t h M N N G )
this p a p e r
AB1 157 U-2
FHfr
thr, leu, a r g E , his, p r o A , str r l a c I , ara r e g u l a t o r y g e n e s
Broda (1974) K.P. R e i n e r s
KV221 KV388
FF'
A (lac, p r o A , B ) F ' 1 2 8 proA+,B +. lac+/mal, spc, s u p E , A (lac p r o )
W. V i e l m e t t e r W. V i e l m e t t e r
NH4921
F
leu, str r, r e c A 4 4 , tel, thi, a r g E , his, p r o A , thr, ara, galK, lacY, m t l , xyl, tsx, s u p
Hall a n d H o w a r d Flanders (1975)
GHI0 M2-T2
FF-
mut-S, by Pl-transduction from X8 into NH4921 rout, b y P l - t r a n s d u c t i o n f r o m M 2 i n t o A B l 1 5 7
this p a p e r this p a p e r
4358
F-
4359
F-
thi, leu, proC, m e t E , hisF, t h y A , lacZ, ara, m t l , xyl, m a l A , str r, s p e 4358, recA200
L l o y d et al. (1974) L l o y d et al. (1974)
X4
F o r g e n e s y m b o l s s e e B a c h m a n n et al. ( 1 9 7 6 ) .
Chemicals Sources of chemicals were: Nalidixic acid (Nal) and naphthol-AS-MX-phosphate (NAP) (Sigma Chemical Company, St. Louis), fast blue RR salt (FBRR) and 6-bromo-2-naphthyl-fi-D-galactopyranoside (NAG) (Serva, Heidelberg), N-methyl-N-nitroso-N'-nitroguanidine (MNNG) (Koch-Light, Colnbrook), strept o m y c i n (Str) (Bayer, Leverkusen). Antipain was a gift of Bayer (Wuppertal), Giemsa of Merck (Darmstadt).
Media Saline used for dilution was 0.9% NaC1. PB medium contained 1.75% antibiotic medium 3 (Difco) and NB medium 0.8% nutrient broth (Difco) and 0.5% NaC1. TY agar contained 0.8% t r y p t o n e (Difco), 0.5% yeast extract (Difco), 0.5% NaC1 and 1.5% agar (Difco): after autoclaving, MgCl~ was added to a final concentration of 1 mM. Minimal agar (MM) contained 1.5% agar, salt solution was added so that the final concentrations were 0.15% KH2PO4, 0.35% K2HPO4, 0.07% Na-citrate-5~ H20, 0.1% (NH4)~SO4 and in addition 0.1% MgSO4 • 7 H20, 0.2% glucose and 20 pg/ml of the required amino acids and bases. P1 agar contained 1% bacto t r y p t o n e (Difco), 0.5% yeast extract (Difco), 0.75% NaC1 and 1% agar, after autoclaving 0.1% glucose and 2.5 mM CaC12 were added. P1 soft agar had the same ingredients except 0.6% agar only.
Histochemical staining The staining procedures were modifications of methods used by Messer and Vielmetter (1965) and Vielmetter et al. (1968). Giemsa solution mixture contained 40 mg basic fuchsin, 30 mg methylene blue and 17.5 ml Giemsa solution in 1 1 water. This solution was filtered and 5-ml aliquots were poured out per plate for colony-staining. After sufficient staining (10--20 min) the plates were rinsed with water and were observed with a stereomicroscope. NAG solution as an indicator for ~-galactosidase contained 0.2 g NAG in 1 1 0.08 M Tris buffer pH 8. This solution was added to 1.6 mg FBRR per ml NAG solution (which was predissolved in some drops of dimethylsulphoxide). 5 ml of this dye was used immediately after mixing in the way described above for Giemsa staining. NAP solution for alkaline phosphatase contained 1 g NAP in 1 1 Tris 1 M buffer pH 8. The mixture with the azo dye FBRR was prepared as described for NAG.
Isolation procedure For mutagenesis an exponentially growing culture (5 × 107 cells/ml) in PB was incubated with N-methyl-N-nitroso-N'-nitroguanidine (MNNG) (concentration 40 pg/ml) for 4 min. After a 500-fold dilution cells were grown to allow segregation and expression of the mutations for about 5--6 generations and then plated in a special way to screen for m u t a t o r mutants. The cells were embedded in a double top layer of soft agar. The first layer, containing 1.5 ml of 0.45% soft agar plus 2 × 103 cells, was poured onto a TY agar plate. After solidifying, 1.5 ml of soft agar was added w i t h o u t bacteria as a top layer to prevent colonies from being washed away during the staining procedure. Plates were kept open for 1 h to dry. After about 16 h of incubation at 37°C lens-like microcolonies had formed. For the first screening of mutators we used Giemsa
10
staining because at least 3 different phenotypes could be seen. These genetically uncharacterized mutants absorbed alkaline dyes out of the solution mixture to a greater extent than wild-type cells. Mutant colonies were red, blue or violet and by subculturing such coloured colonies one can demonstrate that the staining reaction is determined genetically. This staining m e t h o d is highly sensitive so that different coloured m u t a n t sectors can be detected in a single colony of the prospective m u t a t o r strain. In order to show that the isolated strains had general m u t a t o r activity we used indicators for known gene functions (NAG, for ~-galactosidase constitutive and NAP for alkaline phosphatase constitutive mutants). For illustration see pictures of stained m u t a t o r colonies (Hombrecher et al., 1972). Results
I. The isolation o f mutator strains In order to isolate m u t a t o r mutants of E. coli we developed a technique based upon a combination of histochemical stains as has been described in Materials and Methods. Techniques were developed by which a single m u t a t o r colony on an agar plate could be quickly and reliably identified among a background of 2000 wild-type colonies. The scanning of more than 2.7 million colonies derived from MNNG-treated bacterial cultures following Giemsa staining resulted in the isolation of some 120 suspected m u t a t o r colonies which were subjected to restreaking and purification followed by staining with Giemsa, NAG for lacI, and NAP for p h o R and p h o S to test forward mutagenesis. This second screening left 24 strains which were classed as having m u t a t o r phenotypes based upon the appearance of predominantly sectored colonies following the staining procedures. The remaining 96 either did n o t show strong m u t a t o r activity or were not general mutators. Mutants were called strong mutators if more than 10% of sectored colonies could be found after staining with NAP corresponds to a m u t a t i o n frequency higher than 10 -4 mutants/plated cell. The m u t a t o r isolates were further examined for their spontaneous mutation frequency for a variety of mutational sites. The results for two of the isolated mutators (Table 2) are typical of those obtained for the mutators tested. From TABLE 2 MUTATION Strain
FREQUENCIES
FOR WILD-TYPE AND MUTATOR
STRAINS
Characteristics examined pho
+ ~
l a c ÷ --* l a c l
hal +-~ naIA
leu-6
~
Leu +
__b _ b 7.6 X 10 -3 ( 5 8 5 0 ) 1.3 X 10 -6
2 1 1.4 1.0
6.7 × 10 -7 (51) 1 . 3 × 1 0 -8 _c _c
his-4
~
His +
phoR,S
XS rout a 10-3 (1000) X 4 7 8 rout + 1 X 10 -6 M 2 - T 2 rout 2.7 × 10 -3 A B l 1 5 7 rout ÷ _ c
× × X X
1 0 -5 ( 2 0 0 0 ) 1 0 -8 10 -6 ( 1 4 0 ) 1 0 -8
M u t a t i o n f r e q u e n c i e s were m e a s u r e d in PB m e d i u m a f t e r o v e r - n i g h t g r o w t h . a Estimate made from densely populated agar plates. b S t r a i n s e x a m i n e d are l a c Y . c Not determined. T h e n u m b e r s i n b r a c k e t s a r e t h e relative m u t a t i o n f r e q u e n c i e s c o m p a r e d t o t h e w i l d - t y p e s t r a i n .
__ c _ c 2 × 10 -7 (40) 5 X 10 -9
11
TABLE EFFECT Culture
1 2 3 4 5
3 OF MEDIUM
ON THE MUTATION
Initial frequency
Frequency in minimal medium
1.8 1.2 1.7 7.1 --
2.1 2.8 1.1 1.3 1.4
× × X X
10 -8 10 -8 10 -8 10 -8
The results given are for mutation
X X X X X
FREQUENCIES
10 -8 10 -8 10 -8 10 -8 10 -8
OF STRAIN
M2-T2
Frequency in
Frequency
minimal medium + Thd
in NB
2.7 6.8 5.5 4.8 --
1.1 1.5 1.8 1.1 1.5
X X X X
10 -8 10 -8 10 -8 10 -8
X X X X X
10 -6 10 -6 10 -6 10 -6 10 -6
Factor MM/NB
52 54 164 85 107
t o nal r e s i s t a n c e .
these results and those reported above, it can be concluded that these strains are indeed generalized mutators. This technique is also useful for identifying weak mutators as for example a forward m u t a t i o n frequency of 10 -s mutants/cell gives approximately 1% of sectored colonies.
II. Mapping o f the mutator strains Mapping of the m u t a t o r genes proved to be difficult for at least two reasons: first an effect of growth medium was observed so t h a t the staining techniques could n o t be used on minimal medium plates following conjugation and secondly only 12 of the 24 m u t a n t s were able to produce sufficiently high titres of bacteriophage P1 to permit further mapping by Pl-mediated transduction. The other strains grew very poorly for u n k n o w n reasons. The first problem stems from the conditional nature of m u t a t o r effects which are n o t fully expressed on minimal but only on enriched medium. This has been previously observed for m u t D mutants by Degnen and Cox (1974) and in our studies, e.g. m u t a t o r M2-T2, the medium effect produced a 52--164fold difference in m u t a t i o n levels (Table 3). Crosses of Hfr U-2 with strains X4 and X8 suggested that the mutations responsible for the m u t a t o r effect were located close to lacI which is near to the known m u t a t o r mutD. Furthermore, mapping by Pl-mediated transduction, where this was possible, showed 11 of the 12 strains to be carrying m u t a t o r mutations cotransducible with proA. These results are summarised in Table 4.
III. Characterization o f one o f the mutators (X8) The map position of the 11 mutators which could be mapped is very close to that of m u t D (Degnen and Cox, 1974) which is cotransducible with proA and argF. We looked at the properties of one of the m u t a t o r strains which had been designated X8 to determine whether our mutators were identical to mutD. From Table 4 it can be seen that the m u t a t i o n responsible for the mutator effect of X8 is linked to proA (11%) and from Table 2 it can be seen that the m u t a t i o n exerts a considerable m u t a t o r effect, in the range of 3 orders of magnitude for the forward mutations on enriched medium. To determine whether the m u t a t o r of X8 was d o m i n a n t or recessive, sexductions were carried out using strain KV388 carrying F'128proA ÷, proB ÷, lac ÷,
12 TABLE 4 C O T R A N S D U C T I O N F R E Q U E N C I E S OF M U T A T O R M U T A T I O N S WITH proA Parent strain
Mutator
Pro*mut-/pro + ,
Frequency (%)
W1485
M1 M2 M3
6/ 61 121142 lO/174
9.8 8.5 5.7
X478
X2 X3 X4 X5 X6 X7 X8
21150 10/225 51/350 20/183 2/118 1/ 81 19/175
1.3 4.4 15 11 1.7 1.2 I1
X478-1
Y1 Y2
O/ 74 3/ 70
