Bacteriophage P22 controlled exclusion in Salmonella typhimurium

J. Mol. Biol. (1968) 85, 607-622

Bacteriophage P22 Controlled Exclusion in

Salmonella typhimurium R. NAGARAJA RAO

tDe~artment of Human Genetica The University of Michigan Medica~ School Ann Arbor, M~higan 48104, U.S.A. and Del~artment of Microbiology The Johns Hopkins University School of Medicine Baltimore, Maryland 21205, U.S.A. (Received 27 January 1968, and in reviaedform 29 Al~ril 1968) In Salmonella typhimurizem complementation between the resident prophage P22 and the superinfecting phage" P22, after induction of the prophage by ultraviolet irradiation, is decreased by a factor of 20 to 100 compared to the complementation of the same phages in rniTed infectious of a non-lysogenic cell. Superinfecting phage genomes can function and grow at decreased levels in only about 7% of the induced lysogenic cells. Poor adsorption and injection c~nnot account for the superinfection exclusion. Superinfecting phage DNA is not degraded to produce acid-soluble fragment~. Isolation of phage mutants which as a prophage do not exclude the superinfecting phage suggest that exclusion is prophage controlled. Hetero-lmmune phages which do not plate on P22 lysogeus plate with normal efllciency on non-excludlng lysogeus. The exclusion does not operate when the superinfecting phage enters by bacterial conjugation. This suggests that the route of entry of the superinfecting genome plays a part in the exclusion mechanism. 1. Introduction I f Esc/~rich~ co//lysogenic for phage A is superinfected with a genetically marked A and the immunity is destroyed, thus allowing the growth of the phages, both types of phages multiply and are represented in the burst. The superinfecting phage and the prophage complement, each other just as they would in a rniYed infection. This situation does not exist in SaZmonsZla typhimurium lysogenic for phage P22 (M. Levine, personal communication; Walsh & Meynell, 1967). The superinfecting phage and the prophage do not complement each other even after the immunity is destroyed. In addition, cells lysogenie for phage I)22 do not plate the hetero-lmmune phage L (Bezdek & Amati, 1967), whereas phage L lysogens plate phage P22 normally. This study was initiated to define the problem of exclusion of superinfecting phage and to determine if this is related to the absence of hetero-lmmune phage plating on P22 lysogens. t Reprint request address. Two mutant phages are said to complement each other if on miTed infection the yield is signiilctmtlygreater th~n the yield produced by single infection wish either mut0mS. 607

608

R.N.

RAO

2. M a t e r i a l s a n d M e t h o d s (a) Ma~eriala Carrier-free 32p as inorganic p h o s p h a t e was obtained from E. R. Squibb & Sons, I~.Y. N - m e t h y l - N ' - n i t r o - N - N i t r o s o g u a n i d i n e was o b t a i n e d from Aldrich Chemical Co. Lysozyme (mur,.m~dase, 2 × crystallized) a n d DNase I (pancreatic DNase, $ × crystallized) were o b t a i n e d from W o r t h i n g t o n Biochemical Corp.

(b) M e d ~ Buffered saline (BS), diluent b r o t h (DB), L b r o t h (LB), indicator agar a n d soft agar for top layers have been previously described (Levine, 1957). Minimal agar has been described b y Smith (1968). Minimal soft agar differed from mln~mal agar in having 7 g]l. instead of 20 g/1. of Bacto-agar. K m e d i u m contained 0.022 ~-KH2PO4; 0-042 M-Na2HPO4; 0.018 ~-NH4CI; 2.5 X 10-s ~-MgSO4; 8.5 × 10-3 ~-NaCI; 0.2% glucose; a n d 1"5~o Dffco v i t a m i n free Casamino acids decolorized b y filtration t h r o u g h N o r i t A. N O P m e d i u m contained 0-1 M-Tris ( p i t 7.5); 0-018 ~-iN-H4C1; 2 . 5 × 1 0 -3 ~-MgS04; 8"5×10 - s M-NaCI; 0"2~o glucose, a n d 0.2% v i t a m i n a n d phosphorus-free Casamino acids (Nutritional Biochem. Corp.) The concentration of phosphate was a d j u s t e d b y varying t h e a m o u n t of K a H P O , a d d e d to t h e N O P medium. (c) Strain.9 Bacterial and phage strains are listed in Table 1. (d) Cultivation of bacteria and phage superinfection Bacteria were grown in K medium, a t 37°C w i t h aeration to a concentration of a b o u t 2 × l0 s cells]ml. W h e n cells were to be superinfected, phage were a d d e d a t an m.o.i, t of a b o u t 10. Adsorption was allowed to proceed for 10 rain a n d the unadsorbed phage were i n a c t i v a t e d b y antiphage serum ( K = 3). Cell concentration was assayed either after addition of t h e serum or a t the time of a d d i t i o n of t h e superinfecting phage. Serum t r e a t m e n t was t e r m i n a t e d b y dilution of the cells into BS. These superinfected complexes were then i r r a d i a t e d with ultraviolet light from a 15-w G.E. germicidal l a m p a t a distance of 50 cm for 20 sec to induce the prophage (throughout t h e p a p e r this operation will be referred to as lifting of immunity). After ultraviolet irradiation, t h e cells were allowed to lyse a t either 25°C or 37°C. 25°C a n d 37°C are, respectively, permissive a n d non-permissive for t h e growth of t h e temperature-sensitive (tg) p h a g e m u t a n t s used in this study. Lysates were assayed a t permissive a n d non-permissive t e m p e r a t u r e s using strain 18 as indicator bacteria.

(e) Preparation of radioactively labeled phage P h a g e labeled with a2p were p r e p a r e d as described b y Botstein (1968). (f) Mutagenization of phage Strain 18 cells were grown in K m e d i u m a t 25°C to a concentration of 108 ceUs/ml. These were infected with a ts phage a t an m.o.i, of one a n d N-methyl:2q'-nltro-N-nitrosoguanidine was a d d e d to a final concentration of 4 ~g/ml. After cell lysis a n d chloroform t r e a t m e n t , t h e mutagenized phage were purified b y differential centrifugation. (g) 18elation of prophage ~nutants unabl~ to exclude superinfevting phage Strain 18 cells were infected with mutagenized ts2.1 ts12.1 phage a t an m.o.i, of a b o u t 20 a n d grown overnight a t 25°C to s t a t i o n a r y phase in K medium. The frequency of lysogenization was 99%. These cells were diluted in L b r o t h a n d t h e n distributed into tubes so t h a t each t u b e contained a b o u t t e n cells. The cells were grown at 37°C to app r o x i m a t e l y l0 s cells/ml, a n d then superinfected w i t h p h a g e tsS.1 cz a t a n m.o.i, of 5. A f t e r a 10-rain period of adsorption, t h e cells from each t u b e were serially diluted in Microt i t e r t r a y s (Cooke Engineering Co., Alexandria, Va.). The t r a y s containing t h e dilutions were i r r a d i a t e d with ultraviolet light for 20 sec. All dilutions were picked, in order, w i t h I" Abbreviations used: m.o.i., multiplicity of infection; ~e, superinfeetion exclusion; t~ phage, a phage mutant the growth of which is temperature sensitive.

EXCLUSION

IN ~ A L M O N E L L A

609

TABLE 1 ~train~ u~ext Strain

Relevant genotype

Sources and/or references

BACTERIA

18 136 198 200 204 205 210 217 219 230 234 235 241 242 243 112 247 258 262 280

LT2 cured for a B type phage J'18(~ c~) 18(t~12.2) 18 (c +) 18 (~5.1) 18 (~2.1 ~12.1) 18 (ts2.1 ~s12.1 a/e 1) 18 (tal3.1) LT2 lysogenie for L phage 18 (as) 18 (taS.1 sic 5) 18 (~13.1 sic 6) 18(MG40) 18(MG178) 18(L) Hfr p u r e 7 stxA Hfrll2(L) H£rll2(ts£.l t~1~.1) rectA22 ~vroAB47 P22 r rectA22 P22 r (ta2.1 ~12.1)

Zinder, 1958 From 18 From 18 From 18 From 18 From 18 Present study From 18 D. Botstein From 18 Present study Present study From 18 From 18 From 18 SU576 of K. E. Sanderson From 112 From 112 H . O . Smith Made from the cross 258 × 262

wild type, also called e + cl c~ ~mnt sa ta ¢2 aI

M. Levine (1957) M. Levine (1957) M. Levine (1957) H . O . Smith Levine & Smith (1964) Young, Fukazawa & Hartman (1964) M. Levine Present study Gough & Levine (1968) M. Levine (in preparation) M. Levine M. Levine Present study Present study M. Levine (in preparation) M. Levine Induction of 219 Grabnar & Hartman (1968) Orabnar (1967)

PHAGES

P22

L MG40 MG178

t,2.1 tal2.1 tag.1 tal2.1 sie 1 tal2.1 e 1 t~13.1 t~5.1 taS.1 cl tsS.1 s~s 5 t~13.1 s ~ 6 ts13.1 cl t~3.1 cl wild type wild type wild type

t Lysogenic strains carry phage P22 and markers of prophage are these of P22 except where noted. rant is a new name given by M. Gough (manuscript in preparation) for the vl locus of Zindcr (1958). I t has been observed by Gough that vl ÷ function is required for the maintenance of lysogeny. a needle onto plates seeded w i t h strain 18 as indicator bacteria. The plates were i m m e d i a t e l y incubated at 43°C for an hour and t h e n transferred to 37°C for overnight incubation. Results were compared w i t h a control consisting of superinfected 18(t~2.1 ts12.1). Six out of sixty tubes showed plaque formation at greater dilutions t h a n t h a t given b y t h e control. These tubes presumably contained a significant proportion of non-excluding lysogens. T h e procedure was repeated on two cell lines o u t of t h e six. Using t h e same criterion, four

610

R.N.

RAO

sublines enriched'for the mutant were picked. These were further purified on plates spread with anti-P$2 serum. Individual lysogenic colonies were tested for their exclusion properties and one non-excluding mutant lysogen was picked for further study. Another method was used to isolate two more mutants after it was observed that hetero~mmune phage neither grew nor plated on regular P22 lysogeus but did plate on the nonexcluding lysogen. Phages ~13.1 and ~6.1 were mutagenized and used to lysogenize strain 18 cells as described before. About 200 mutant lysogens were spread with about 2 × 105 heteroimmune phage MG178 on each of a number of indicator plates and incubated overnight at 37°C. Colonies which could support growth of the hetero-lmmune phage grew as dark green colonies due to phage infection and cell lysis within the colony. Colonies on which the phage could not grow, and occasional colonies which escaped infection, remained lightcolored. Single-colony isolates from the dark green colonies were tested for P22 and MG178 ~mmunity. From the colonies which were P22 immune and MG178 sensitive one colony was isolated and finally tested for its exclusion properties. One mutant lysogen was obtained from each of the two t~ phages.

(h) P ~ i n g The ~ phages were plated and incubated at 25°C or 37°C. I n some cases the plates were incubated for 30 to 60 mln at 43°C to accelerate warming. After ultraviolet induction, the plates were kept at 43°C for 30 mln before incubating at 37°C. Strain 18 bacteria were used as indicator and all platings were done without pre-adsorption. (i) Battered matings The method of Sanderson & Demerec (1966) was followed for the matings. At different times mating was interrupted by agitation of a portion on a Vortex Genie at top speed for 1 mln and a sample was immediately plated on selective plates to determine the time of entry of the prototrophie marker. The remaining sample was incubated at 37°C until lysis of the zygotieally induced cells. The lysates were treated with chloroform and assayed for phage. 3. R e s u l t s

(a) Definition of the problem Two complementing ts phage m u t a n t s give a nearly normal burst on mixed infection of a non-lysogen under non-permissive conditions. Complementation might be expected when one is a resident prophage and the other is a superinfecting phage, provided i m m u n i t y is lifted. This was not found to be the case (Table 2). W h e n phage t~3.1 c 1 was used to superinfect lysogens 18(ts5.1), 18(ts12.1) and 18(ts13.1) and imm t m i t y lifted, the burst size was less b y a factor o f a b o u t 100 compared to mixed infections with the same phages. Single infections and non-superinfected induced lysogens gave v e r y low burst sizes, indicating t h a t the m u t a n t s were relatively nonleaky. This experiment shows t h a t there is v e r y little complementation between the superinfecting phage and the prophage, even after the i m m u n i t y h a d been lifted. I t can also be seen from this experiment t h a t the n u m b e r of recombinants produced (last column of Table 2) is similarly decreased. The clear m u t a n t rant c2 was used to superinfect cells lysogenic for ts mutants, to test whether the superinfecting phage is able to grow in an induced lysogen under conditions where the resident prophage is unable to grow. Table 3 shows t h a t v e r y few rant c2 phages were present in the lysates produced a t either 25°C or 37°C, as indicated b y the low yields when plated at 37°C. The low yield of superinfecting phage was found whether or not the resident prophage was permitted to grow. A t 25°C,

E X C L U S I O N I N ,,~ALMONELLA

611

T~BLV. 2

Abseuce of normal complementa2ion i~ ~ e r i n f e c t e d lysogens

No.

Bacteria

Infeoting phages

1

18

ts3.1 cl

5.3

2 3

18 18

2.6 3.1

4 5

18 18

6

18

7 8

18 18(ts5.1)

re5.1 c~ ~12.1 ¢i ts13.1 ci re3.1 cl, tsS.1 cl ~3.1 c~, te12.1 cx ~3.1 c1, ts13.1 cl 0

9

18(ts12.1) 18(ts13.1)

10 11 12 13

18(ta12.1)

0 0 re3.1 c2 ts3.1 cz

18(ts13.1)

ta3.1 cx

18(tsS.1)

Average burst size at 37°C Plated Plated at 25°C at 37°C

m.o.i.

0.0008 0.033 0.066

3.3 5.3, 2.6 5.3, 3.1

5.3, 3.3

0.073 80.0 83.4

6.1

55.7 0.0004 <0.00002 <0.0002 0.73

6.4 6.6

0.56 0.094

0.00025 0.0022 0.005 0.003 3.3 3.0 3.8 <0.00002 <0.00002 <0.00002 0.025 0.018 0.003

The cells were grown in K medium at 37°C to a concentration of about 2 × 10s cells/m]. The phage were adsorbed for 10 mln and the free phage were inactivated by serum (K----20). The superinfected cells were diluted away from the serum. Nos 8 to 13 were irradiated with ultraviolet light for 20 sec to lift the immunity. After cell lysis, the lysates were treated with chloroform and assayed for phage. The burst size was calculated on the basis of total cells.

sizeable yields were o b t a i n e d b u t t h e p h a g e were p r e d o m i n a n t l y of p r o p h a g e t y p e as e v i d e n c e d b y t h e f a c t t h a t t h e y were b o t h t u r b i d a n d ts. T o e s t i m a t e t h e f r a c t i o n o f t h e s u p e r i n f e c t e d lysogenie cells w h i c h could b e comp l e m e n t e d b y t h e s u p e r i n f e c t i n g phage, a s i n g l e - b u r s t a n a l y s i s was carried out. L y s o g e n 18(ts2.1 ts12.1) was s u p e r i n f e c t e d w i t h p h a g e tsS.1 cl, a p h a g e c a p a b l e of c o m p l e m e n t i n g p h a g e ts2.1 t812.1 in m i ~ e d infections, t h e i m m u n i t y was lifted a n d lysis was allowed t o proceed a t 37°C. T h e lysates were a s s a y e d a t 25°C. T a b l e 4 shows t h a t t h e a v e r a g e b u r s t size was o n l y 2. O n l y 7 % of t h e b a c t e r i a y i e l d e d a n y p h a g e a n d t h e s e yielders g a v e a n a v e r a g e b u r s t size of 30.

TABLE 3

Inability of lysogens to support the growth of superinfeeting r a n t ca after induction

Lysogens

18(ts2.1 ta12.1) 18(tsS.1)

m.o.i, of the superinfeeting phage 7.6 9.4

Average burst size at 25°C Average burst size at 37°C Plated Plated Plated Plated at 25°C at 37°C at 25°C at 37°C 72.6 43.6

0-31 7.5

0.41 8.3

0.10 4.7

The lysogens were grown in LB at 37°C to a concentration of about 2 × 10e eells/ml, and were superinfected with mn~ c2 phage. The unadsorbed phage were inactivated by serum. The superinfected lysogens were induced with ultraviolet light and the ceils were allowed to lyse either at 25°C or at 37°C.

R, N. R A O

612

TABL~ 4

Single burst analysis of strain 18(ts2,1 tsl2.1) sul~erinfected with ~hage tsS.I c1 Composition of the yield Positive plates 17/60 Turbid plaques Clear plaques (prophage type) (superinfecting phage type) 0 1 2 2 4 3 2 3 8 4 9 2 25 0 74 59 87

1 2 1 3 1 3 6 8 4 8 4 16 26 71 0 25 36

Total plaques 285

215

Average burst size (total no. of plaques/total no. of input cells) = 1.85. Average burst/yielder (total no. of plaques/total no. of plates cont.aln~ng the burst ) = 29.4. Average no. of yielders/plate ~ 0.33. Average no. of bacteria/plate ~ 4.5. Yielders/100 bacteria ---- 7.4. Lysogen 18(ts2.1 t~12.1) was grown in K medium at 37°C to a concentration of about 2 × l0 s eells/mh The cells were Superinfeeted with phage t~5.1 cl. After 10 r,~n of adsorption, free phage were inactivated with serum (K = 20), The cells were induced with ultraviolet light and diluted to a concentration of 20 cells/ml. From this, 0-25 ml. each was distributed into each of 60 tubes and the cells were allowed to lyse at 37oc. The lysate from each of these tubes was assayed for phage at 25°C. The preceding experiments h a v e shown t h a t t h e c o m p l e m e n t a t i o n which occurs n o r m a l l y in m i x e d infections is decreased b y a factor of 100 if one of t h e phages is a prophage, even after the immunity has been Zifted. Only in a b o u t 7 % e f t h e lysogenic cells can t h e p r o p h a g e a n d t h e superinfecting p h a g e c o m p l e m e n t each other. L a c k o f complementation, in a m a j o r i t y o f t h e lysogenic cells, between t h e p r o p h a g e a n d the phage, even after t h e i m m u n i t y h a d been destroyed, will be called "superinfection exclusion". W i l d - t y p e p h a g e (sis +) is able t o exclude t h e superinfeeting phage. The order Of superinfection a n d lifting of i m m u n i t y does n o t seem t o be imp o r t a n t . T h e exclusion m e c h a n i s m c a n n o t be escaped b y superinfeeting t h e lysogen at different times after lifting t h e ~mmunlty. (b) A priori

explanations

Several explanations for superinfection exclusion can be visualized. (1) T h e lysogens do n o t adsorb t h e superinfeeting phage. Zinder (1958) has c o m m e n t e d t h a t P22 lysogens do n o t a d s o r b p h a g e P22 well. ( 2 ) T h e r e is no injection or there is a f a u l t y

EXCLUSION IN BALMONELLA

613

injection o f t h e p h a g e D N A . (3) T h e superinfecting phage D N A is degraded. Degradation has been shown to occur in t h e restriction of phage ~ a n d o f non-gluc0sylated T-even phages (Arber, 1965). (4) The superinfecting phage D N A is excluded w i t h o u t D N A degradation. (c)

Adsorption of the superinfecting 19hage

The adsorption rates of phage P22 t o strains 18 a n d 18(c +) were 3.06 a n d 1-26 × 10 -9 iul. -1 rain -1, respectively. I n other experiments (see Table 8) t h e adsorption r a t e for a l y s o g e n was decreased b y a factor o f 5. These differences c a n n o t a c c o u n t for t h e exclusion effect (see Discussion). (d)

Injection of the sulgerinfecting~ l ~ e DNA

P22 is a short-tailed p h a g e (Anderson, 1961) a n d consequently it has n o t been possible effectively to shear off adsorbed phages using blenders (Takebe & H a r t m a n , 1962; H. O. Smith, personal communication). Freeze-thawing once in t h e presence of l y s o z y m e does n o t b r e a k open t h e phage particles, whereas it does break open bacteria (see Table 5). This suggested the possibility t h a t susceptibility of p h a g e D N A to D N a s e after freeze-thawing could be used to determine if t h e phage D N A has entered t h e bacteria. I t was assumed t h a t adsorbed phage which h a d n o t injected its D N A could w i t h s t a n d t h e t r e a t m e n t like free phage. V a r y i n g a m o u n t s of s2P-labeled p h a g e were adsorbed t o strains 18 a n d 18(c +). The adsorbed complexes were separated f r o m t h e free p h a g e a n d t h e fraction o f D N A t h a t remained acid-insoluble after D N a s e digestion was determined. The values given in Table 5 are average determinations of TABLE 5

Susceptibility of sulgerinfecting phage DNA to DNaze action Treatment

% Acid-insoluble counts

Phage ~- DNase Phage + DNase, freeze-thaw Phage ~- lysozyme, DNase, freeze-thaw Superinfected strain 18 cells ~- DNase lysozyme, DNase, freeze-thaw Superinfected strain 18 (c+) cells

94 86 79

+ DNase + lysozyme, DNase, freeze-thaw

89 46

92 26

Strains 18 and 18(c +) were grown in K medium at 37°C to a concentration of about 2 × 108 cells/ mL Phage ts13.1 labeled with s~p (5 × 10 -4 ets/mln/phage) were adsorbed to the bacteria for 10 min. The unadsorbed phage were removed by centrifuging at 12,000 g for 5 rain. The cells were washed once in Tris buffer (0.01 •-Tris (pH 7.4), 0.005 M-MgSO4) and resuspended in 1 ml. of Tris buffer containing DNase 40 pg/ml, and 10 -s ~-CaCI~. A sample was removed ~mmediately to determine the total counts. Another sample was incubated for 40 rn~n at 37°C to determ~o the amount of DNA that would be susceptible to DNase prior to freeze-thawing. To the rema~nlng volume, 0.03 ml. of 10 mg/ml, lysozyme was added. This was freeze-thawed once and incubated at 37°C for 40 min. Samples were taken to determine the total radioactivity and the acid-insoluble radioactive material. As controls, s~P-labeled phage was treated with DNase with or without freeze-thawing, and with DNase, lysozyme and freeze -thawing. The controls were incubated at 37°C for 40 min and the amount of acid-precipitable radioactive material was determined. The control values are an average of three determinations and the experimental numbers are an average of five determinations.

614

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five different samples~.In both cases a large portion of the DNase-resistant radioactive material became sensitive to DNase action after the freeze-thaw lysozyme treatment, namely 66% for the non-lysogen and 43% for the lysogen. The difference observed is not sutficient to account for the superinfection exclusion effect.

(e ) 18 the mVl~erin/ectingphage DNA degraded to acid-soluble fragment,? A ts ca lysogen which is thermo-inducible at or above 40°C (Levine & Smith, 1964) was used in this experiment to avoid the complication of repair following ultraviolet irradiation, saP-labeled ca phage was adsorbed to strains 18 and 18(ts ca) at 43°C and the acid solubiIization of the input [saP]DNA was followed until the beginning of lysis of the cultures (Fig. 1). Small amounts of acid-soluble fragments (less than 10%) were produced in the phage~infected 18 cells, under which conditions the phage is able to grow. Acid solubilization of superinfecting phage DNA was actually less in the case of the 18(ts ca) lysogen, where the exclusion mechanism operates. Thus, this experiment does not support the idea that the mechanism of exclusion involves degradation of the superinfecting phage genome to acid-soluble fragments.

=8 20/

l

i

i

-

.~

0

I

20

40

60

Time (rain)

FIe. l. Acid solubilization of the superinfeeting phage P$2 DNA. 18, (--O--O--); 18(t~c=), (--O--O--). Strains 18 a n d 18(t~ o2) were grown in K medium a t 25°C to a concentration of a b o u t 10 e cells/ml. The cells were shifted to 43°C a n d infected witJa 32P-labeled e= phage (2× 10 -5 ct~/m~/phage) a t a n m.o.i, o f 20. 1-ml. samples were t a k e n a t different times to determlne acid-soluble radio. active material.

(f) Isolation oJ phage mutants which do not exclude the ,uperinfect~ng phage Since the exclusion depends on the presence of s prophage, it was thought that phage mutants might exist which, as a prophage, did not exclude the superinfecting genomes. Three such mutants were isolated (see Materials and Methods for the isolation procedures). These mutant phages were serologically identical to P22 and homo-lmmune with P22. When the mutant lysogens were superinfected with rant v2 and the immunity was lifted by ~traviolet light, rant c2 was able to multiply (Table a). The fraction of the bursts which plated at 37°C, and thus represented the superinfecting phage, was higher by at least a factor of 30 compared to the corresponding control *ie + lysogens. These ale lysogens could also be complemented b y superinfecting ts phage (Table 6). When the phage obtained from these sic lysogens were used to relysogenize strain 18 cells, the newly made lysogens did not exclude the superinfecting phage, indicating that exclusion is phage-mediated.

EXCLUSION

IN S A L M O I V E L L A

615

TABLE 6

Ability of mutant and wild-type lysogens to Termit au~erinfestirag ~hage to function and/or coml~lement the resident ~rtrphage

Lysogens 18(t,s2.1 ts12.1) 18(t~5.1) 18(t~13.1) 18(ts2.1 t~12.1 sie 1) 18(t~5.1 sie 5) 18(~s13.1 s/e 6) 18(~2.1 ts12.1) 18(~5.1) 18(t~13.1) 18(ts2.1 t~12.1 s~e 1) 18(tsS.1 sie 5) 18(t~13.1 ~¢ 6)

Superinfeeting phage

m.o.i.

rant ¢~ rant c2 mn~ c~ rant ca rant c2 rant c2 t~5.1 c~ t~3.1 c~ t~3.1 cl t~5.1 cl t~3.1 c~ t~3.1 cl

9.5 7.4 14.8 12,4 8.2 10.6 2.8 3.2 6.9 5.4 5.2 5.0

Average b u r s t size a t 37°C P l a t e d a t 25°C P l a t e d a t 87°C 1.32 0-31 2.4 41.6 27.8 18.9 0.34 0.21 0.25 27.2 30.2 9.55

0.31 0.13 0.26 10.8 10.3 8.2 0.014 0.022 0.017 1.28 2.42 0.94

The various lysogens were grown in K m e d i u m a t 37°C to a concentration of a b o u t 2 × l 0 s cells/m], a n d were supcrinfected b y t h e phage for 10 rain. The free phage were i n a c t i v a t e d w i t h serum. The superinfected lysogeus were induced w i t h ultraviolet light a n d allowed to lyse a t 37°C. The values of m.o.i, given h a v e b e e n corrected for t h e unadsorbed phage.

To determine the fraction of the superlnfccted 8/e lysogens allowing complementation, a single-burst analysis was done. Strain 18(t82./t812.1 sle 1) was superinfected with phage t85.1 cl. The superinfccted cells were induced by ultraviolet light. These cells were appropriately diluted and distributed into tubes incubated at 37°C. The lysates were assayed at 25°C (Table 7). The average burst size was 83, a 40-fold increase over that obtained with a sic + lysogen (see Table 4). The frequency of yielders was 88~, at least 10 times greater than for sic + superinfection, and the average burst. size per yielder was 120 compared to 30 when the superinfected lysogen was sic + (sea Table 4). (g) Rate of phaqe adsorption in Iysoeens carrying sie ltrrolohage Phage 1>22 controls the production of a new and distinct somatic antigen called antigen 1 (Robbins & Uchida, 1962). A phage mutant, ax, unable to convert lysogenio cells, has been isolated by Young, Fukazawa & Hartman (1964). Lysogons carrying this mutant prophage adsorb superinfected P22 at a more normal rate than do lysogens carrying wild-type prophago (Hartman, personal communication). In view of this, the adsorption rates of sic lysogens were compared with that of sic + and a 1 lysogens (Table 8). This experiment shows that az lysogens adsorb phage more rapidly than a + lysogens. The adsorption rates of sic lysogens vary from non-lysogenio to lysogenic rates. (h) Do a1 lysogens exclude suTerinfectlng phage ? Superinfection of an a 1 lysogen with phage rant ca was carried out in an experiment similar to that presented in Table 6. The a x lysogen did not support the growth of the superinfecting phage. Experiments of Walsh & Meynell (1967), showing that superinfection of an ax lysogen with a + phage did not lead to the production of antigen 1, supports the idea that ax lysogens are phenotypically sic +. 40

R.N.

616

RAO

TaBT.~ 7

~ingle.burst analyai~ of atrai~ I 8 ( t s 2 . I tsl2.1 sie 1) ~erinfected with phage tsS.1 e 1 Composition of the yield Positive plates 22/60 Turbid plaques Clear plaques (prophage type) (superinfeeting phage type) 0 7 9 5 24 25 41 73 83 86 97 64 91 100 85 135

2 3 4 17 18 22 16 32 30 34 26 59 34 29 44 27

94

81

120 103 149 276

66 85 54 67 45

1859

795

192

Total plaques

Average burst size (total no. of plaques/total no. of input cells) = 83-4. Average burst/yielder (total no. of plaques/total no. of plates containing the bursts) ---- 120.0. Average no. of yielders/plate -----0.47. Average no. of bacteria/plate ---- 0-53. Yielders/100 bacteria ---- 88-6. Lysogen 18(t~2.1 ~sI2.1s/e 1) was grown in Kmedium at 37°C to a concentration of about 2 × 10a cells/ml. The experimental details were slrnl]ar to that described for the experiment in Table 4, except that the induced superinfected lysogenic cells were diluted to about 2 cells/ml, and 0.25 ml. each was distributed into each of 60 tubes.

(i) Are hereto-immune phages excluded? P h a g e s L (Bezdek & Amati, 1967), MG40 (Grabnar & H a r t m a n , 1968) a n d 1KG178 (Grabnar, 1967) are h e t e r o - i m m u n e t o p h a g e P22. I t is therefore interesting t h a t these phages do n o t plate on 18(c +) lysogens (Table 9). I n t h e ease o f p h a g e L, t h e efficiency o f plating behaves a s y m m e t r i c a l l y (Bezdek & A m a t i , 1967), i.e. P22 plates on LT2(L) b u t L does n o t plate o n LT2(P22). All these phages h a v e near n o r m a l efficiency o f p l a t i n g w h e n p l a t e d o n a sie lysogen (Table 9) indicating t h a t thes6 phages are norl n a l l y excluded b y t h e presence o f sie + function in P22 lysogens. P h a g e P22 does n o t p l a t e on 18(MG40) a n d 18(MG178) even t h o u g h MG40 a n d MG178 plate on t h e sie lysogen. The lysogen 18(az) does n o t plate a n y of these h e t e r o A m m u n e phages, t h u s e o n ~ r m ; n g t h a t it behaves like sie*.

EXCLUSION

617

IN ,SALMONELLA TABLE 8

Rate of ~8orption of phage to ~on-lysoge~ = ~ to r~rio~ ~ysoge~

Bacteria

18 18(az) 18(ta2.1 t~12.1) 18(t~2.1 ~12.1 8is 1) 18(teS.1 ~ s 5) 18(~13.1 ~e 6)

m.o.i.

Adsorption rate X (10 ~ ml. mln)

7-1 4.6 4.5 4.8 3-9 4-2

2.04 0.78 0.46 1.43 0.67 1-68

The cells were grown in K medium at 37°C to a concentration of about 2 X 1Os cells/ml. At t = 0, phage c2 was added and at various times, up to 10 rnln~ samples were diluted into DB containing chloroform. The free phage were assayed and the K values calculated from the slope as described by Adams (1959). (j) Does excl~io~ depend on the route of entry of the phage D N A ? P h a g e L n e i t h e r p l a t e s o n a sic + l y s o g e n n o r p r o d u c e s s i g n i f i c a n t p r o g e n y i n a o n e s t e p g r o w t h cycle. T h i s a l l o w s o n e t o t e s t w h e t h e r p h a g e L c a n g r o w i n a 8is ÷ l y s o g e n w h e n i t is i n t r o d u c e d b y b a c t e r i a l m a t i n g . A n H f r s t r a i n l y s o g e n i c f o r p h a g e L w a s

I

I

I

]

I

600

12,000

-r

/y

"

200-

000

!

0

,,~ 15

I

30 Tim (mi,)

I

45

60

FzG. 2. Escape from exclusion in zygotic induction, met ,4, ( - ' - A - ' - A - ' - ) ;

262, ( - - © - - © ~ ) ;

28o, (--O--O--). Strains 247, 262 and 280 were grown in nutrient broth at 37°0 to a concentration of 3 X l0 s cells/ml. Mating was carried out on a filter paper and the cells were resuspended in nutrient broth. 1-ml. samples were taken at different times and the mating pairs separated. These were appropriately diluted and plated on selective plates for the time of entry of m6~A + marker. The rem-.;u~ng sample was incubated at 37°C to allow the zygoticaUy induced cells to lyse. The lysates were assayed for infective phages.

1 ~I0 -s ~I0 -a <10 -e < 10 - e ~ I0- e 1.03

o+ 1 ~ I 0 -8
al l ~ I 0 -B <10 -8 <10 -e < I0 -e ~ I0 - e 0.82

t~2.1 ~12.1 1 ~ i 0 -8 <10 -e <10 -6 < 10 - e ~ I0- o 1.02

$s2.1 t~12.1 eis 1

Phages

Salmonella

I 6.6 X 10 -5 5 × 10 - a 0.43 < 10 - e <~ I 0 - e 0-09

MG 40

1 ~ I 0 -5 <=10 - 6 0.35 < 10 - 5 ~ I0~- 5 0.16

MGI78

phages on various lysogens

I ~ I 0 -6 ~10 -e 0-4 < 10 - e ~ I0- e < 10-°

L

T h e cells w e r e g r o w n i n L B a t 37°C t o a c o n c e n t r a t i o n o f a b o u t 5 × 108 c e l l s / m l , a n d 0.1 m l . o f t h e cells w a s u s e d a s i n d i c a t o r . T h e p l a t e s w e r e i n c u b a t e d a t 25°C.

18 18(a~) 18(~2.1 t,812.1) 18(ta2.1 ta12.1 ei,s 1) 18(MG40) 18(MG178) 18(I,)

Plating bacteria

Bj~ciency of plating of phage P22 and several hereto-immune

T~BT.V, 9

EXCLUSION IN ~ALMO_,~ELLA

619

m a t e d with a aie + lysogen. The mating was interrupted at various times and the lysates obtained from zygotic induction were assayed. As a control, a non-lysogenic female recipient was used. The time of e n t r y of the m e t A + m a r k e r and the prophage were similar in b o t h cases (Fig. 2). The burst sizes a t various times from b o t h matings were comparable within a factor of two. This experiment shows t h a t exclusion does not operate ff the phage D N A enters as a prophage through a conjugation tube. (k) Can a lysogen be induced by h o m o - i m m u n e 8uperinfe~ion? I t was shown earlier t h a t the superinfecting phage genome can neither grow b y itself nor complement the prophage in a n immunity-lifted sie ÷ lysogen. The superinfecting genomes can also manifest their presence b y induction of the prophage. Experiments of this t y p e h a v e been performed with phage A (Weismeyer, 1965) and it has been assumed ~hat prophage induction represents the titration of i m m u n i t y 100

I

I

! 8

¢)

10 ._¢

o

o

0"

0 50 100 150 m.o.i, of superinfecting phage

Fro. 3. Prophage induction by homo-lmmune superinfection. 18(tsS.1 t~lg.1), (--O--O--); 18(t~5.1 t~12.1 eie 1), ( - - 0 - - 0 - - ) . Strains 18(t~2.1 t~12.1) and 18(t~2.1 t~12.1 Re 1) were grown in K medium at 37°0 to a concentration of about 2 X 10a cells/ml, and infected with phage c2 at the indicated multiplicities. After 5 rain of adsorption anti-P22 serum was added (K ----30). After 10 more ruin, the superinfected lysogens were sufficiently diluted and plated for infective centers. 0nly turbid plaques and plaques which wereheterozygous were scored.

repressor b y superinfecting phage DNA. I f this is the mechanism operative in prophage induction, intracellular localization of the superinfeeting phage D N A is a prerequisite. As seen in Figure 3, superinfection of either a sis ÷ or a sis lysogen resulted in induction of the m a j o r i t y of the cells. The difference in the m.o.i, required to achieve comparable levels of induction in the two cases is less t h a n is apparent, because t h e 8/e lysogen adsorbs phage more rapidly t h a n the 8is ÷ lysogen and the m.o.i, has not been corrected for this. 4. D i s c u s s i o n The present

s t u d y has

characterized

an exclusion mechanism operating in

S. t y p h i m u ~ u m lysogenic for phage P22. Superinfecting phage is represented in the

620

R.N.

RAO

yield from the induced lysogen at greatly reduced levels. Complementation of ts prophage mutants by superinfecting ts phage is also substantially decreased, indicating that the exclusion mechanism operates to block gene expression as well as replication. The superinfecting genome is not degraded to acid-soluble fragments. Isolation of phage mutants which produce non-excluding lysogens points to phage control of the exclusion mechanism, although cellular factors might also have a role. The exclusion operates not only on the superinfecting homo-immune phage genome but also on several hetero-immune phages. At what level does exclusion operate ? Since the exclusion results in decreased complementation, it becomes important to show that the superinfecting phage genome enters the cytoplasm of the superinfected lysogen. I t can be shown in the following manner that poor adsorption cannot accout for exclusion. When phage is adsorbed at a cell concentration of 2 × 10a cells/ml, with an input m.o.i, of five for ten minutes, and using the adsorption rates given in Table 8, it can be calculated that m.o.i, after correcting for unadsorbed phage is 4.4 for a non-lysogen and 2.5 for a lysogen. Under these conditions, the fraction o£ cells not superin£ected is, respectively, 0.01 and 0-08. These calculations show that 90% of the superinfected cells do adsorb at least one superinfecting phage. Also, the data presented in Table 6 show that exclusion can be seen even after the m.o.i, has been corrected for unadsorbed phage. Then, do these adsorbed phages inject their DNA ? Experiments presented in Table 5 suggest that they do inject. A rough estimate shows that 65 to 70% of the DNA is injected into a lysogen compared to a non-lysogen. If one phage per lysogenic cell is sufficient for complementation with the prophage, then one would expect that the magnitude of the exclusion to be no more than a factor of 2. In fact the magnitude of exclusion was never less than a factor of 20. This can be interpreted in at least two ways: there is a faulty injection, or the exclusion operates after the phage genome is injected into the lysogenic cell. I f the injected DNA were released between the cell wall and the cell membrane without penetrating into the interior, many of the findings could be accounted for. Two of the results, however, do not support this possibility. A lysogen can be induced by superinfection at high multiplicity, suggesting that the superinfecting genomes are available for interacting with phage immunity repressor. Second, superinfection with the hetero-immune phage L leads to curing of the lysogen (Amati, personal communication; Rao, unpublished experiments) suggesting that the in~ + locus of the entering genome which controls detachment (Gottesman & Yarmolinsky, 1968) is capable of functioning. However, it should be remembered that we have no direct evidence to prove that the superinfecting phage genomes enter the cell and that they do so faultlessly. I f the superinfecting phage genomes enter the cell, then why are most of its genes not expressed ? Simple considerations rule out DNA degradation as having a primary role in the exclusion. Information transfer from the genome involves a series of steps at any one of which the exclusion mechanism might operate. In this regard, it is interesting to note that translational blocks have been proposed by Hattman & Hofschneider (1967) for the interference of RNA phage function by phage T4 in E. coll. W~at is the nature of the recognition in exclusion ? Exclusion operates on a variety of DNA molecules, including genomes of homo-lmmune P22 and heter0-'immune L, MG40 and 1VIG178phages. It seems nnHkely that all these DNA molecules share some common recognition site(s) which is acted on by the exclusion mechanism, since the prophage would also possess the site. A likely possibility is that at least some factors

EXCLUSION

IN. ~ A L M O N E L L A

621

required for exclusion are localized at the cell surface, the act of e n t r y itself constltuting the recognition mechanism. This idea gains support from the fmding t h a t prophage introduced b y conjugation is not apparently subject to exclusion, suggesting t h a t route of entry is of significance. Recently, Walsh & Meynell (1967) have reported independent isolation of nonexcluding phage t)22 mutants b y another method. These mutants are apparently similar in general properties to those reported here. The exclusion phenomenon described here is different from the restriction phenomena seen for T2 and for the ;~ E. co//system in at least two respects. Restricted T2 and ;~ I)NA's are degraded (Hattman, 1964; I)ussoix & Arber, 1962), whereas no degradation is apparent in P22 exclusion. I)espite degradation of the restricted T2 genome, some early genes are expressed (Hattman, Revel & Luria, 1966), and in the case of ;~ infecting a restricting host (Terzi, personal communication) it has been observed t h a t the restricted genomes can complement m a n y different sus mutants in different genes, whereas the excluded P22 genome will not complement the prophage. The exclusion phenomenon shares some features with the restriction o f T 2 b y Shlgella strain Sh lysogenic for phage P2, where T2 is restricted but no I)NA degradation is observed. More important is the observation t h a t there is v e r y little early enzyme synthesis under these conditions (Smith & Pizer, personal communication). I t should be emphasized t h a t the exclusion described here ~iffers from all those cases where the restricted genome is able to function, whether or not the restricted genome is degraded to acid-soluble fragments. I am particularly grateful to Dr M. Levine, in whose laboratory most of this work was carried out. My thanks are due to Dr It. O. Smith for suggesting the problem, for many discussions and for his patient criticisms of the manuscript. I am also indebted to I)rs M. Levine, M. Gough, P. E. Hartman and I). Botstein for their criticisms and eommen~s. I tbA.n~ Dr P. E. Hartman, who provided me with the hetero-~mmune phages, and Drs H. S. Smith, L. I. Pizer, M. Terzi and P. Amati for allowing me to quote their unpublished results. This work was supported by U.S. Public Health Service gran~s GM-09252-05 and AI-07875-01. REFERENCES AdAm~, M. H. (1959). Bacter/ophage~. New York: Interscience Publisher, Inc. Anderson, T. F. (1961). Prec. European Re,g~onal Gon]. ElecSron Microscopy, Delft, 1960. Uppsala: Almqvist & Wiksell. Arber, W. (1965). Ann. Rsv. Microbiot. 19, 365. Bezdek, M. & Amati, P. (1967). Virology, 31, 272. Botstein, D. (1968). J. MoL Biol. 34, 621. Dussoix, D. & Arber, W. (1962). J. Mol. Biol. 5, 37. Gottesman, M. E. & Yarmoliusky, M. B. (1968). J. Mol. Biol. 31, 487. Gough, M. & Levine, M. (1968). Genetics, 58, 161. Grabnar, l~I. G. (1967). Ph.D. Thesis, The Johns Hopkins University. Grabnar, M. G. & Hartman, P. E. (1968). Virology, 34, 521. Hattman, S. (1964). Virology, 24, 333. Hattman, S. & Hofschneider, P. It. (1967). J. MoL Biol. 29, 173. Hattman, S., Revel, H. R. & Luria, S. E. (1966). Virology, 30, 427. Levlne, M. (1957). Virology, 3, 22. Levine, M. & Smith, H. O. (1964). ScOnce, 146, 1581. Robbins, P. W. & Uchida, T. (1962). Eed. Prec. 21, 702. Sanderson, K. E. & Demerec, M. (1965). Genet~s, 51, 897.

622

R . lq'. P~AO

Smith, H. O. (1968). Virology, 34, 203. Takebe, H. & Hartman, P. E. (1962). V&ogo~/, 17, 295. Walsh, ft. & MeyneU, G. G. (1967). J. Gen. ViroL 1, 581. Weismeyer, H. (1965). J . Bact. 91, 89. Young, B. G., Fukazawa, Y. & Hartman, P. E. (1964). V&oZogy, 23, 279. Zinder, lq. D. (1958). Virology, 5, 291.