161

Mutation Research, 45 (1977) 161--167 © Elsevier/North-Holland Biomedical Press

PHAGE YIELD DURING W-REACTIVATION OF BACTERIOPHAGE ?~ P. CAILLET-FAUQUET and M. DEFAIS Laboratoire de Biophysique et Radiobiologie, Universitd Libre de Bruxelles, rue des Chevaux, 67, 1640-Rhode-St-Gen~se (Belgium)

(Received 4 March, 1977) (Revision received 5 May, 1977} (Accepted 8 June, 1977)

Summary Phage production in liquid medium during W-reactivation parallels the extent of W-reactivation of infective centres on plates. The mean burst size is independent of W-reactivation; thus the reactivated phage yields a normal burst. As on plates, the lex- mutant shows no W-reactivation in liquid medium. It is concluded that W-reactivation is a consequence of an induced DNA repair which reactivates the damaged parental phage DNA to its full biological activity.

Introduction Weigle reactivation (W-reactivation [16]), previously called UV reactivation [20], is the increased survival of infective centres of UV-irradiated phage due to certain mutagenic treatments of the host cell before infection. W-reactivation is accompanied by an increase in the frequency of phage mutants [10,20]. These phenomena are dependent on functional recA and lexA genes in the host [5, 13]. W-reactivation and mutagenesis of phage ?~ are host-dependent, inducible phenomena which are, in fact, a measure of some cellular repair and mutagenic activities [4--6,16,17]. Bacterial mutants that conditionally induce W-reactivation and mutagenesis of phage k also induce their own DNA repair and mutagenesis as well as prophage ?t [3,22]. Bacterial mutants that are deficient in W-reactivation are also deficient in host DNA repair, mutagenesis and lysogenic induction (for review see [17]). This is the basis of the "SOS repair hypothesis" [16,17], which states that bacterial mutagenesis, induced by radiation and many chemical mutagens, depends upon induction [4] of a cellular, error-prone, DNA repair [21], called SOS repair. W-reactivation, together with mutagenesis o f phage k, is a consequence of SOS repair, and is still the most reliable measure of this important cellular phenomenon. Therefore, it was of interest to determine u n k n o w n parameters

162 of W-reactivation assay. For example, since all experiments on W-reactivation are based on analyses of phage infective centres on plates, we wanted to determine whether this phenomenon occurs in liquid medium and what is the burst size (i.e. the number of individual phages produced by a single infected bacterium [ 18 ] of W-reactivated, heavily mutagenized phage [ 5 ] ). The former question is important for the attempts to study the biochemistry of W-reactivation where large amounts of bacteria must be grown, and the latter question might be related to the biological activity of the SOS-repaired, hence mutagenized, phage DNA. In the present work, we have tested whether there is also an increase in overall phage production under conditions of W-reactivation. Assuming that W-reactivation occurs as efficiently in liquid medium as on plates, the following possibilities could be expected. (a) Induction of W-reactivation decreases the mean burst size with the following possible consequences: (1) a decrease in W-reactivation; (2) no W-reactivation; or (3) a decrease in overall phage production, i.e. negative W-reactivation. (b) Induction of W-reactivation does not influence the mean burst size; therefore the W-reactivation factor reflected in phage production will be equal to the W-reactivation factor of infective centres on plates. (c) Induction of W-reactivation increases the burst size; therefore the W-reactivation will be more pronounced in liquid medium than on plates. Our results are compatible with option (b). Materials and methods Phage

Wild t y p e k. E. coli s t r a i n s a n d t h e i r r e l e v a n t g e n o t y p e s A B l 1 5 7 rec ÷ u v r ÷ lex÷; AB1886 rec ÷ u v r A 6 lex+; and AB2494 rec ÷ u v r ÷ l e x A 1 . These strains were isolated and described by Howard-Flanders and

Boyle [8]. Media

TBI (tryptone broth), TA12 (plate agar) and TA7 (soft a g a r ) h a v e been described b y Kaiser [9]. TB1-Mg-mal : t r y p t o n e broth supplemented with 10 -2 M MgSO4 and 0.25% maltose. TM was 10 -2 M Tris--HC1 (pH 7) containing 10 -2 M MgSO4. P h a g e assays

Phage assays were standard [1]. U V irradiation

UV irradiation was carried out with a Mineralight lamp having maximal output at 254 nm. Dose rates were measured with a Latarjet dosimeter [11]. Phage were irradiated at concentrations of 108 to 109 particles/ml in TM n o t deeper than 1 ram. Bacteria, suspended in 10 -2 M MgSO4, were irradiated under iden-

163 tical conditions at concentrations between 2 and 5 × 10 a bacteria/ml. Photoreactivation o f irradiated bacteria was avoided by using dim yellow light.

Single burst experiments [19] Bacteria were harvested in exponential phase and resuspended in 1 0 - 2 M MgSO4. Unirradiated or irradiated phage were pre-adsorbed at 37°C for 10 min to unirradiated or irradiated bacteria with a multiplicity of infection (m.o.i.) inferior to 0.5. Ten more volumes of 10-2 M MgSO4 were added and bacteria were sedimented twice to remove the unadsorbed phage. The pellet was then resuspended in TB1-Mg-mal broth, diluted to obtain 0.5 infective centre/ml, and 0.5 ml was distributed to each tube. Incubation with aeration proceeded at 37°C for 60 min when unirradiated phage was used and for 150 min with irradiated phage.

phage production in liquid medium Bacteria were grown to a concentration of 5 × 10 a bacteria/ml, pelleted and resuspended in 10 -2 M MgSO4. Infection of intact and irradiated bacteria was performed at 0°C for 20 min with intact or irradiated h phage at a m.o.i. inferior to 0.5. The culture was washed twice in 1 0 - 2 M MgSO4 and then resuspended in an equal volume of warm medium (time = 0). At 10-min intervals, aliquots of infected bacteria were lysed with lysozyme (100 gg/ml) in 10 -2 M Tris--MgSO4 (pH 8) saturated with chloroform. Infective particles were then titrated on plates. Each result given is an average of at least 3 experiments. Results

Phage production in liquid medium The relative yield of phage produced by bacterial cultures carrying mutations in DNA repair was studied. We analysed the production of phage h on a wild-type strain and its two radiosensitive mutants, uvrA and lexA [8]. Fig. l a shows that unirradiated and irradiated bacteria produced the same a m o u n t of unirradiated phage. However, under W-reactivation conditions [4], a 100-fold increase in the production of irradiated phage was observed with wild-type bacteria. A uvrA mutant, deficient in excision repair, exhibited maximal W-reactivation at lower inducing UV doses than the wild type [4,15] and showed an increase in phage production of about 50-fold (Fig. lb). These experiments document an efficient W-reactivation in liquid medium. LexA mutants showed no W-reactivation of }~ phage on plates [4]. The same was true in liquid medium (Fig. lc): a negative W-reactivation on plates was observed when irradiated cells were infected by irradiated phage [5]. Ineffective particles of irradiated phage appeared with an additional delay of 60 to 70 min irrespective of whether or not host cells were irradiated (Fig. la,b,c). This was due both to a prolonged lag period and to an extended phage production period in the one-step growth of irradiated phage, when compared with intact phage {Fig. la,b,c).

Single-burst size experiments The mean single-burst size was studied in intact and irradiated AB1886

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Time (rain) Fig. 1. R e l a t i v e p h a g e y i e l d p r o d u c e d b y b a c t e r i a l c u l t u r e s . E v e r y 1 0 r a i n a l i q u o t s o f i n f e c t e d b a c t e r i a were lysed, and infective phage was titrated on plates. The following combinations were tested for each b a c t e r i a l s t r a i n : (1) u n i r r a d i a t e d p h a g e (e a n d o) l e f t o r d i n a t e a n d (2) i r r a d i a t e d p h a g e (A a n d A) r i g h t ordinate each infected unirradiated (open symbols) and irradiated (closed symbols) bacteria. Phage and b a c t e r i a w e r e i r r a d i a t e d w i t h d o s e s giving m a x i m a l W - r e a c t i v a t i o n [ 4 ] . (a) Wild t y p e b a c t e r i a A B l 1 5 7 a n d A p h a g e w e r e i r r a d i a t e d w i t h 5 0 J / m 2 a n d 1 5 0 J / m 2 r e s p e c t i v e l y . (b) A B 1 8 8 6 uur A- a n d p h a g e r e s p e c tively a t 7 J / m 2 a n d 5 0 J / m 2. (c) A B 2 4 9 4 l e x A - a n d p h a g e r e s p e c t i v e l y a t 1 5 J / m 2 a n d 1 5 0 J / m 2. T h e p h a g e U V d o s e s w e r e c h o s e n t o o b t a i n s i m i l a r survival in e a c h b a c t e r i a l a s s a y .

TABLE 1 SINGLE-BURST ANALYSIS FOR NON-IRRADIATED NON-IRRADIATED AND IRRADIATED HOST CELLS.

AND IRRADIATED

PHAGES

INFECTING

T h e l y s a t e s o f m o r e t h a n o n e h u n d r e d t u b e s w e r e p l a t e d o u t f o r e a c h r e s p e c t i v e AlE. coil c o m p l e x in e a c h e x p e r i m e n t (see M a t e r i a l s a n d M e t h o d s ) . T h e n u m b e r s in t h e c o l u m n s s h o w t h e n u m b e r s o f p l a t e s c o n r a i n i n g p h a g e A in e a c h b u r s t size r a n g e . H o s t cell

Phage

Experiment

Burst 1--5

5--20

20--150

> 150

Total number of plates having A phages

NI

NI

I II

0 0

0 1

9 19

0 1

9 21

I

NI

I II V

0 3 0

2 3 0

7 4 6

0 1 0

9 11 6

NI

I

III IV VI

1 0 1

2 1 4

14 7 7

2 1 0

19 9 12

I

I

III IV

0 0

2 3

12 4

6 0

20 7

166

uvrA- bacteria as hosts, each infected by either intact or irradiated k phage. Four possible k/E. coli combinations were washed and distributed to at least 100 tubes (see Materials and methods). Fewer than 25 plates contained infective phage. Under these conditions, only about 2% of the tubes contained two or more infected bacteria. When separated into different classes according to the number of phages produced,, the principal class produced between 10 and 150 phages per single burst (Table 1). There was no significant difference amongst these four combinations. Discussion A simple way to increase the mutation frequency of irradiated phage in infective centres of irradiated bacteria is to increase the number of generations, i.e. of DNA replication rounds before lysis [12]. We show here that this is n o t unlikely, since there was a long delay in lysis when both k phage and host cells were irradiated (Fig. la,b,c). Other experiments (Table 1) permitted us to estimate the burst size and to study the influence of induced repair on its mean value. Production of k phage showed a long lag when phage were irradiated. Moreover, the duration of the lytic cycle was twice as long as it was in unirradiated phage. These two phenomena were independent of SOS repair. The delay in phage production, when the phage was UV irradiated, may have been caused by the presence of pyrimidine dimers in k DNA, which are known to inhibit DNA synthesis. The mean burst size was similar in each of the four cases studied. The average burst size was between 20 and 150 phages in all cases. Bursts giving one phage (Table 1) probably arose either from residual unadsorbed phage or from unlysed infective centres. Therefore, the increased mutagenesis during W-reactivation did not seem to be caused by extra rounds of phage DNA replication. W-reactivation occurred as efficiently in liquid medium as on plates. Since the mean burst size was similar whether or not phage and bacteria were irradiated (Table I), the increase in yield of irradiated phage in irradiated hosts was due only to the increase of infective centres. The fact that irradiation of the host cells that was optimal for induction of SOS repair did not cause any delay in phage production (Fig. la,b,c) suggests that cellular metabolism was not significantly impaired by this irradiation dose. Acknowledgements We t h a n k Dr. M. Radman for helpful advice and Drs. C.H. Evans, M. Radman and E. Witkin for critical reading of the manuscript. This work was carried out under Contract Euratom U.L.B. 0099-72-1BIAB with an agreement between the Belgian Government and Universit~ Libre de Bruxelles on priority action for collective basic research and with the help of the " F o n d s de la Recherche Fondamentale Collective".

167

References 1 A d a m s , M . H . , B a c t e r i o p h a g e s , Wiley ( I n t e r s c i e n c e ) N e w Y o r k , 1 9 4 9 , p p . 4 4 3 - - 5 2 2 . 2 C a i U e t - F a u q u e t , P. a n d M. Defais, I n d u c e d r e p a i r o f b a c t e r i o p h a g e k b y t e m p e r a t u r e s h i f t o f a n E. c o l i dnaB m u t a n t , s u b m i t t e d t o Molec. G e n . G e n e t . 3 C a s t e l t a z z i , M., J. G e o r g e a n d G. B u t t i n , P r o p h a g e i n d u c t i o n a n d cell d i v i s i o n in E. c o i l I. F u r t h e r c h a r a c t e r i z a t i o n o f t h e r m o s e n s i t i v e m u t a t i o n tif-1 w h o s e e x p r e s s i o n m i m i c s t h e e f f e c t o f U V i r r a d i a t i o n , Molee. G e n . G e n e t . , 1 1 9 ( 1 9 7 2 ) 1 3 9 - - 1 7 4 . 4 Defais, M., P. F a u q u e t , M. R a d m a n a n d M. E r r e r a , U l t r a v i o l e t r e a c t i v a t i o n a n d u l t r a v i o l e t m u t a g e n e s i s o f k in d i f f e r e n t g e n e t i c s y s t e m s , V i r o l o g y , 4 3 ( 1 9 7 1 ) 4 9 5 - - 5 0 3 . 5 D e t a i s , M., P. C a i L l e t - F a u q u e t , M. F o x a n d M. R a d m a n , I n d u c t i o n k i n e t i c s o f m u t a g e n i c D N A r e p a i r a c t i v i t y in E. coli f o l l o w i n g u l t r a v i o l e t i r r a d i a t i o n , Molec. G e n . G e n e t . , 1 4 8 ( 1 9 7 6 ) 1 2 5 - - 1 3 0 . 6 D e v o r e t , R., M. B l a n c o , J. G e o r g e a n d M. R a d m a n , M e c h a n i s m f o r t h e r e c o v e r y o f p h a g e k f r o m u l t r a v i o l e t d a m a g e , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.) M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f D N A , P l e n u m Press, N e w Y o r k , 1 9 7 5 , p p . 1 5 5 - - 1 7 1 . 7 G e o r g e , J., M. Castellazzi a n d G. B u t t i n , P r o p h a g e i n d u c t i o n o n cell d i v i s i o n in E. coli, III. M u t a t i o n s s f i A and s fiB r e s t o r e d i v i s i o n in t i f a n d Ion s t r a i n s a n d p e r m i t t h e e x p r e s s i o n o f m u t a t o r p r o p e r t i e s o f tif, Molec. G e n . G e n e t . , 1 4 0 ( 1 9 7 5 ) 3 0 9 - - 3 3 2 . 8 H o w a r d - F l a n d e r s , P. a n d R.P. B o y c e , D N A r e p a i r a n d g e n e t i c r e c o m b i n a t i o n s t u d i e s o n m u t a n t s o f E. coli d e f e c t i v e in t h e s e p r o c e s s e s , R a d i a t . Res., ( 1 9 6 6 ) s u p p l . 6 , 1 5 6 - - 1 8 4 . 9 Kaiser, A.D., A genetic study of the temperate coliphage, Virology, 1 (1955) 424--443. 1 0 L a t a r j e t , R., M u t a t i o n i n d u i t e c h e z u n virus p a r i r r a d i a t i o n u l t r a v i o l e t t e d e cellules i n f e c t ~ e s , C . R . A c a d . Sc. Paris, 2 2 8 ( 1 9 4 9 ) 1 3 5 4 . 11 L a t a r j e t , R . , P. M o r e n n e a n d R. B e r g e r , U n a p p a r e f l s i m p l e p o u r le d o s a g e d e s r a y o n n e m e n t s u l t r a v i o l e t s ~mis p a r les l a m p e s g e r m i c i d e s , A n n . Inst. P a s t e u r , 8 5 ( 1 9 5 3 ) 1 7 4 . 1 2 L u r i a , S. a n d M. D e l b r i i c k , M u t a t i o n o f b a c t e r i a f r o m virus s e n s i t i v i t y t o v i r u s r e s i s t a n c e , G e n e t i c s , 2 8 (1943) 491. 1 3 M i u r a , A. a n d J. T o m i z a w a , S t u d i e s o n r a d i a t i o n sensitive m u t a n t s o f E. coli. III. P a r t i c i p a t i o n o f t h e rec s y s t e m in i n d u c t i o n o f m u t a t i o n b y u l t r a v i o l e t i r r a d i a t i o n , M o l e c . G e n . G e n e t . , 1 0 3 ( 1 9 6 8 ) 1 - - 1 0 . 1 4 M o r s e , L.S. a n d C.C. P a u l i n g , I n d u c t i o n o f e r r o r - p r o n e r e p a i r as a c o n s e q u e n c e o f D N A ligase defic i e n c y in Escherichia coli, P r o c . N a t l . A e a d . Sci. US 7 2 , ( 1 9 7 5 ) 4 6 4 5 - - 4 6 4 9 . 1 5 R a d m a n , M. a n d R. D e v o r e t , U V r e a c t i v a t i o n o f b a c t e r i o p h a g e k e x c i s i o n d e f i c i e n t h o s t : i n d e p e n d e n c e o f rec f u n c t i o n s a n d a t t a c h m e n t r e g i o n s , V i r o l o g y , 4 3 ( 1 9 7 1 ) 5 0 4 - - 5 0 6 . 16 R a d m a n , M., in M o l e c u l a r a n d e n v i r o n m e n t a l a s p e c t s o f m u t a g e n e s i s , P r a k a s h et al. (eds.), C.C. T h o m a s P u b l . , S p r i n g f i e l d , Illinois, 1 9 7 4 , p p . 1 2 8 - - 1 4 2 . 17 R a d m a n , M., S O S r e p a i r h y p o t h e s i s : p h e n o m e n o l o g y o f a n i n d u c i b l e D N A r e p a i r w h i c h is a c c o m p a n i e d b y m u t a g e n e s i s , in P.C. H a n a w a l t a n d R . B . S e t l o w (eds.), M o l e c u l a r M e c h a n i s m s f o r R e p a i r o f D N A , P l e n u m Press, Basic Life S c i e n c e s Series, 1 9 7 5 , p p . 3 5 5 - - 3 5 6 . 1 8 S t e n t , G.S., M o l e c u l a r B i o l o g y o f B a c t e r i a l V i r u s e s , W.H. F r e e m a n a n d C o . , S a n F r a n c i s c o , 1 9 6 3 . 1 9 T h o m a s , R . , C o n t r o l o f d e v e l o p m e n t in t e m p e r a t e b a c t e r i o p h a g e . I. I n d u c t i o n o f p r o p h a g e g e n e foll o w i n g h e t e r o - i m m u n e s u p e r i n f e c t i o n , J. Mol. Biol., 2 2 ( 1 9 6 6 ) 7 9 - - 9 5 . 2 0 Weigie, J . J . , I n d u c t i o n o f m u t a t i o n in a b a c t e r i a l virus, P r o c . N a t l . A c a d . Sci. US, 3 9 ( 1 9 5 3 ) 6 2 8 - - 6 3 6 . 21 W i t k i n , E., M u t a t i o n p r o o f a n d m u t a t i o n p r o n e m o d e s o f survival in d e r i v a t i v e s o f E. coli B d i f f e r i n g i n s e n s i t i v i t y t o u l t r a v i o l e t light, B r o o k h a v e n S y r u p . Biol. 2 0 ( 1 9 6 7 ) 1 7 - - 5 5 . 2 2 W i t k i n , E., T h e r m a l e n h a n c e m e n t o f u l t r a v i o l e t m u t a b i l i t y in a t i f l u v r A d e r i v a t i v e o f Escherichia coli B/r: e v i d e n c e t h a t u l t r a v i o l e t m u t a g e n e s i s d e p e n d s u p o n a n i n d u c i b l e f u n c t i o n , P r o c . N a t l . A c a d . Sci. US, 71 ( 1 9 7 4 ) 1 9 3 0 - - 1 9 3 4 . 2 3 W i t k i n , E.M., T h e r m a l e n h a n c e m e n t o f u l t r a v i o l e t m u t a b i l i t y in a dnaB u v r A d e r i v a t i v e o f Escherichia coil B / r : e v i d e n c e f o r i n d u c i b l e e r r o r - p r o n e r e p a i r , in H a n a w a l t P.C. a n d S e t l o w , R . B . (eds.) M o l e c u l a r mechanisms for repair of DNA, Plenum Publ., New York, 1975, pp. 369--378.

Phage yield during W-reactivation of bacteriophage.

161 Mutation Research, 45 (1977) 161--167 © Elsevier/North-Holland Biomedical Press PHAGE YIELD DURING W-REACTIVATION OF BACTERIOPHAGE ?~ P. CAILLET...
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