γ-Ray mutagenesis in bacteriophage T4 is not enhanced by oxygen

121

Mutation Research, 61 (1979) 121--127

© Elsevier/North-Holland Biomedical Press

T-RAY MUTAGENESIS IN B A C T E R I O P H A G E T4 IS N O T E N H A N C E D BY OXYGEN

J.F. BLEICHRODT and W.S.D. ROOS-VERHEIJ Medical-Biological Laboratory TNO, 139, Lange Kleiweg, 2280 AA Rijswijk Z.H. ~ (The NetherlandS)

(~eceived 20 June 1978) (J~evision received 10 January 1979) ( Accepted 16 January 1979)

Summary As in the induction of r mutants in bacteriophage T4 by T-rays, the radiation-induced reversion of T4 a m b e r mutants to wild-type was found to depend on the product of the DNA-repair gene x of the phage. Neither the efficiency of induction Of r mutants nor the efficiency o f reversion of ambers was enhanced by the presence of oxygen during irradiation. T4 differed in this respect from phage T7, for which no indication has been found that T-ray mutagenesis results from error-prone repair of DNA damage. Notwithstanding the substantial contribution of misrepair to mutation induction in T4, the efficiency of induction per base-pair observed for irradiation under oxygen was lower than that found previously for T7.

Reversion o f a m b e r mutants of bacteriophage T7 by 7-rays to particles able to propagate in a suppressorless host is more efficient when the suspension of phage is oxygenated than when irradiation is performed under anoxic conditions [2]. Several a m b e r mutants of the single-stranded DNA phage (PX174 behave similarly [3,4]. No indication has been found that induction of mutants in these phages is due to error-prone repair of their damaged DNA b y bacterial repair processes [2,3]. Conkling, Grunau and Drake [7] studied-/-ray mutagenesis in bacteriophage T4B. The mutants induced comprised deletion, frame-shift and base-pair substitution mutants, and mutation induction was found to depend strongly on the t w o phage-repair genes x and y, i.e. a large fraction of the mutants resulted from misrepair. The present note shows that this kind of misrepair does n o t yield more mutants when the phage has been T-irradiated under oxygen than when irradiated anoxically.

122 Materials and methods Bacterial and bacteriophage strains Bacteriophages T4D, T4x [9], T4amE51 (gene 56) [15] and T4amB256 (gene 5) [8] were provided by Dr. B. de Groot, Leiden University. Escherichia coli BB was used to propagate T4D [7]. A m b e r mutants of T4 were grown and assayed on E. coli BBw/1 [10], a gift of Dr. R. Hausmann, Universit~it Freiburg. E. coli B was used to grow T4x and to assay T4D, T4x, wild-type revertants of T4 ambers and r mutants of T4D. The double mutants T4x araB256 and T4x amE51 were obtained by crossing T4x with the appropriate am ber mutants and screening for increased sensitivity to ultraviolet radiation among the ambers in the progeny [12]. E. coli K12 HfrH thi-8 sustrp-101 suslac-101 supE was used to plate the ambers in the screening procedure. An amber with normal sensitivity isolated from the progeny of a cross was used in the experiments as the corresponding T4x÷arr~. T4x amB256 and its x am* revertants showed larger plaques on E. coli BBw/-~ and B, respectively, than T4amB256 and its revertants. Phage was propagated at 37°C in L-broth [14]. Lysis of the cells was completed by shaking the culture with chloroform. Bacterial debris was removea by low-speed centrifugation. Irradiation and assay Phage stocks were diluted in an equal volume of irradiation broth (8% Difco Nutrient Broth and 1% NaC1 in 0.02 M phosphate buffer, pH 7.1) and irradiated in a Gamma Cell 200 6°Co source (Atomic Energy of Canada Ltd.) at 0°C. The dose rate was measured by ferrous sulphate dosimetry. During the period of the experiments the average value amounted to 200 rad • sec -1 (2.0 G y . s-l). Either 02, or N2 containing less than 2 × 10-3% 02 [cf. 2], was bubbled through the suspension before and during irradiation. If required, a droplet of Dow Coming Antifoam A was added to prevent excessive foaming. Immediately after irradiation, samples were diluted at least 10-fold in tryptone broth (1% Difco Bacto Tryptone, 0.5% NaC1) at 0°C to slow down reactions of long-lived radiation products. As soon as possible, the phage was assayed for survivors and mutants by the agar-layer method [ 1 ], with standard Drake agar [7,14]. In experiments with T4 amber mutants no glucose was added to the b o t t o m agar. Super-soft Drake agar was used for screening of r mutants [7]. Bacteria were grown in nutrient broth (0.8% Difco Nutrient Broth, 0.5% Difco Yeast Extract, 0.5% NaC1). For assay of wild-type revertants of amber mutants at least 3 × 109 E. coli B cells were added per petri dish to keep the multiplicity of infection as low as possible. Plates were incubated at 37°C. Following Conkling et al. [7], r mutants were scored on the basis of aberrant plaque appearance on E. coli B. To obtain an appropriate number of plaques per plate, dilution and plating of the phage for assay of r mutants was performed as soon as the fraction of survivors in the irradiated samples was known, i.e. a few hours after plating of the survivors. If doubtful, aberrant plaque morphology was confirmed by replating of phage from the plaque.

123 Results

The yield of r mutants as a function of dose is depicted in Fig. 1 for both Yirradiation under 02 and N2. The inset shows the corresponding survival curves. As usual [e.g. 5] the sensitivity of T4 was alittle lower under 02 than under N2. The efficiencies of induction of r mutants, i.e. the slopes of the induction curves, did not differ significantly for irradiation under 02 and N2, and amounted to 6.6 × 10 -9 mutations per phage particle per tad. This is 52% of the value observed by Conkling et al. [7]. When expressed as mutations per lethal hit (in the case of exponential inactivation curves an average of I lethal hit per phage particle corresponds with a survival of 37%}, the efficiencies of induction corresponded with 3.5 X 10 -4 and 3.1 X 10 -4 for irradiation under O2 and N2, respectively. The absence of an enhancement of the induction of r mutants by oxygen is not unique for this type of forward mutation. Fig. 2 shows that, at doses below 300 krad, the reversion of T4amB256 was also hardly affected by oxygen, if at all. At the highest dose the induction under nitrogen exceeded that under oxygen significantly. This non-linearity of the curve for nitrogen is not understood as yet. It is highly unlikely that it is due to cross-reactivation (marker

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rescue) because of multiple infection of cells in the assay of revertants. Indeed, the ratio of the numbers of phage particles (inactivated plus viable particles) and bacteria was of the order of 1--2 at the highest dose, although at the highest dose but one this ratio was the same, and cross-reactivation is expected to be more efficient at lower doses (mutation induction increases linearly with dose, but survival decreases exponentially}. Further, increasing the ratio of phage and bacteria on the plate when assaying revertants in a heavily irradiated phage sample did not result in a significant increase in the yield of wild-types (Table 1). Finally, the recent observation by Priemer and Chan [13] that the

TABLE 1 I N F L U E N C E O F M U L T I P L I C I T Y O F I N F E C T I O N ON T H E N U M B E R OF R E V E R T A N T S D E T E C T E D IN P H A G E I R R A D I A T E D H E A V I L Y U N D E R A N O X I C C O N D I T I O N S a Titre o f E , coU B ( m 1 - 1 )

1.1 X 10 t 0

3,7 × 10 9

1.9 × 10 9

47/46

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n u m b e r of wild-type plaques n u m b e r o f petri d i s h e s a T i t r e o f T 4 a m B 2 5 6 s u s p e n s i o n b e f o r e irradiation: 8.7 X 1010 (m1-1 ): F r a c t i o n of survivors a f t e r irradiat i o n : 4 . 2 X 1 0 - 5 . E q u a l v o l u m e s ( 0 . 5 m l ) o f E. coli B a n d irradiated T 4 a m B 2 5 6 s u s p e n s i o n w e r e a d d e d t o m o l t e n t o p agar (3 m l ) a n d t h e m i x t u r e w a s p o u r e d o n t o t h e p l a t e .

125 gene is involved in multiplicity reactivation of T4, stimulated us to an experim e n t on the induction of mutations under anoxic conditions in which an E. coli K12 r e c A 1 3 and an accompanying r e c ÷ strain served as indicator bacteria in the recA

assay of revertants. The induction curves (not shown) were identical and exhibited an upward curvature, similar to the curve for N2 in Fig. 2. Reversion of an amber codon was much less efficient in a T4x than in a T4x + strain (Figs. 3, 4). This indicates that, as in the induction of r mutants [7], reversion of amber particles is mainly due to misrepair. Analogous results were obtained for irradiation under oxygen and nitrogen. The radiosensitivity of x strains was higher than that of x + strains, although the difference was very small for irradiation under nitrogen (cf. [5] and inset of Figs. 3 and 4). Survival curves for inactivation under oxygen often showed a small shoulder (Fig. 3 and [2,5,7]; but cf. Figs. 1, 2, 4). The m u t a t i o n induction curve for irradiation of T4amE51 under N~ did not show a significant curvature (Fig. 4). However, the efficiency of induction of revertants was much lower in amE51 than in amB256, so that the maximal attainable radiation dose was much lower for the former phage strain.

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Fig. 3. I n f l u e n c e o f g e n e x o f T 4 on t h e r e v e r s i o n o f t h e a m b e r m u t a t i o n a r a B 2 5 6 b y 9'-rays. I r r a d i a t i o n u n d e r 0 2 . (o) T 4 x + a r a B 2 5 6 ; ( s ) T 4 x a r a B 2 5 6 , V e r t i c a l bars r e p r e s e n t s t a n d a r d d e v i a t i o n s b a s e d o n t h e n u m b e r of p l a q u e s c o u n t e d ; a v e r a g e o f 2 e x p t s . I n s e t : c o r r e s p o n d i n g survival c u r v e s . Fig. 4. R e v e r s i o n o f T 4 a m E 5 1 and T 4 x a m E 5 1 t o p a r t i c l e s able t o p r o p a g a t e in a s u p p r e s s o r i e s s h o s t b a c t e r i u m . ( e ) a m E 5 1 , i r r a d i a t i o n u n d e r 0 2 (2 e x p t s . ) ; (o) a m E 5 1 , N 2 (3 e x p t s . ) ; (z~) x a m E 5 1 , N 2 (3 e x p t s ) . Vertical b a r s r e p r e s e n t s t a n d a r d d e v i a t i o n s b a s e d o n t h e n u m b e r of p l a q u e s c o u n t e d .

126 Discussion

The above results show that in T4 induction of mutations by 7-rays, which results mainly from misrepair of DNA damage, is not enhanced by 02, as it is in T7. For the latter phage no evidence has been found that error-prone repair is involved in 7-ray mutagenesis [2]. Either the various types of pre-mutational damage inflicted in T4 DNA under 02 and N2 (at not too high a dose, cf. Fig. 2) are similar or, more likely, the misrepair process does not discriminate appreciably between different types of DNA damage induced under the two gas conditions. The efficiencies of induction of mutations for the amber mutants investigated are given in Table 2, together with data from the literature. Only efficiencies for irradiation in the presence of 02 are compared, because of nonlinearity of the induction curve for reversion of T4amB256 under anoxic conditions. The efficiency of reversion obtained for T4amB256 compares favourably with the value estimated by Conkling et al. [7] for the induction of point mutations in T4. T4amE51, which is deficient in DNA synthesis [15], is mutated with a much lower efficiency. Its value equals that obtained by Bridges et al. [6] for reversion of this mutant by irradiating suppressorless bacteria infected by this phage. When irradiating complexes of T4amE51 and bacteria which did contain an amber suppressor, Bridges et al. [6] observed a higher efficiency of reversion and they concluded that synthesis of phage DNA is necessary for the production of the majority of the reversions at the amber site. These observations are understandable in the light of the findings of Maynard-Smith and Symonds with ultraviolet radiation [11] that the gene carrying the mutation amE51 is involved in the x-mediated repair pathway.

TABLE 2 E F F I C I E N C Y O F I N D U C T I O N OF P O I N T M U T A T I O N S IN B A C T E R I O P H A G E BY 7 - R A Y S IN T H E PRESENCE OF O X Y G E N Strain

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1, Figs. 2 a n d 3; t a r g e t size in t h i s and f o l l o w i n g d a t a o n r e v e r s i o n of a m b e r s a s s u m e d to be 3 base-pairs. 2. Fig. 4, a v e r a g e of 2 e x p t s . 3. B r i d g e s e t al. [ 6 ] . I r r a d i a t i o n of p h a g e - b a c t e r i u m c o m p l e x e s ; t h e b a c t e r i a d i d n o t c o n t a i n a n a m b e r suppressor. 4. B l e i c h r o d t et al. [ 2 ] . R e v e r t a n t s a n d survivors w e r e a s s a y e d o n t h e s a m e b a c t e r i a l strains as t h o s e used in t h e p r e s e n t w o r k . 5. C o n k l i n g et aL [ 7 ] . In e s t i m a t i n g t h e e f f i c i e n c y of i n d u c t i o n o f p o i n t m u t a t i o n s o n l y , t h e a u t h o r s a s s u m e d a t a r g e t size o f 3 5 0 0 base-pairs. G a s c o n d i t i o n s n o t specified, t h e r e f o r e a s s u m e d t h a t irradiat i o n w a s p e r f o r m e d in air.

127

However, the present observation, that the x mutation affects the reversion of a m E 5 1 too (Fig. 4), does not fit the simple picture that x and a m E 5 1 belong to

a single repair pathway. The efficiencies of reversion of T7 a m b e r s are higher than those observed for T4. (In preliminary experiments it was found that, among several T4 a m b e r mutants, araB256 was not reverted less efficiently by irradiation than were the others.) This is remarkable because v-ray mutagenesis in T7 is probably not due to error-prone repair [2], whereas in T4 misrepair contributes substantially to mutation induction. Possibly, any error-free repair of v-ray damage is more efficient in T4 than in T7, or (and) errors during replication occur more frequently in T7. It should be remembered, however, that part of the difference in efficiency of reversion between different am ber mutants may be due to differences in sensitivity of the protein functions involved to amino acid substitutions at the site corresponding with the a m b e r codon in the DNA of the am ber mutant.

Acknowledgement The authors are indebted to Dr. B. de Groot and Dr. R. Hausmann for providing strains of bacteria and bacteriophage.

References 1 Adams, M.H., Bacteriophages, Wiley Interscience, New York, 1959. 2 Bleichrodt, J.F., A.L.M. Roos and W.S.D. Roos-Verheij, I n d u c t i o n of m u t a t i o n s in bacteriophage T7 by V-rays: Independence of host-repair mechanisms, Mutation Res., 43 (1977) 313--326. 3 Bleichrodt, J.F., and W.S.D. Verheij, Reversion of cistron A a m b e r m u t a n t s of bacteriophage dPX174 by ionizing radiation, Mutation Res., 18 (1973) 363--365. 4 Bleichrodt0 J.F., and W.S.D. Verheij, Influence of oxygen on the i n d u c t i o n of m u t a t i o n s in bacteriophage dPX174 by ionizing radiation, Int. J. Radiat. Biol., 25 (1974) 505--512. 5 Bleichrodt, J.F., W.S.D. Verheij and J. de Jong, Involvement of the excision repair system in recovery of bacteriophage from v-ray damage sustained under oxic and anoxic conditions, Int. J. Radiat. Biol., 22 (1972) 325--335. 6 Bridges, B.A., R.E. Dennis and R.J. Munson, Mtitagenesis in Escherichia coli , n I . R e q u i r e m e n t for DNA synthesis in m u t a t i o n by gamma rays of T4-phage c o m p l e x e d with Escherichia coli, Genet. Res., Camb., 15 (1970) 147--156. 7 Conkling, M.A., J.A. Grunau and J.W. Drake, Gamma-ray mutagenesis in bacteriophage T4, Genetics, 82 (1976) 565--575. 8 Epstein, R.H., A. Bolle, C.M. Steinberg, E. Kellenberger, E. Boy de la Tour, R. Chevailey, R.S. Edgar, M. Susman, G.H. Denhardt and A. Lielausis, Physiological studies of conditional lethal mutants of bacteriophage T4D, Cold Spring Harbor Symp. Quant. Biol.,28 (1963) 375--394. 9 Harm, W., Mutants of phage T 4 with increased sensitivityto ultraviolet,Virology, 19 (1963) 66--71. 10 Hausmann, R., and B. G o m e z , A m b e r mutants of bacteriophage T 3 and T 7 defective in phage-directed deoxyribonucleic acid synthesis,J. Virol.,1 (1967) 779--792. 11 Maynard-Smith, S., and N. Symonds, Involvement of bacteriophage T 4 genes in radiation repair, J. Mol. Biol.,74 (1973) 33--44. 12 Minderhout, L. van, J. Grimbergen and B. de Groot, Nonsense mutants in the bacteriophage T 4 D v gene, Mutation Res., 29 (1974) 333--348. 13 Priemer, M.M., and V.L. Chan, The effects of virus and host genes on recombination a m o n g ultraviolet-irradiatedbacteriophage T4, Virology, 88 (1978) 338--347. 14 Ripley, L.S., Transversion mutagenesis in bacteriophage T4, Mol. Gen. Genet., 141 (1975) 23--40. 15 Wiberg, J.S., Mutants of bacteriophage T 4 unable to cause breakdown of host D N A , Proc. Natl. Acad. Sci. (U.S.A.), 55 (1966) 614---621,