Hershey 1952 Original 39

8/2/2019 Hershey 1952 Original 39

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I N D E P E N D E N T F U N C T IO N S O F V I R AL P R O T E I N A N D N U C L E I CA C I D I N G R O W T H O F B A C T E R I O P H A G E *

B~ A. D. H ERSH EY AND MARTHA CHASE(From the Department of Genetics, Carnegie Institution of Washington, C old SpringHarbor, Long Island)

(Received for publication, April 9, 1952)The wor k of Doer maml ( 1948) , Doer mann and Dis sosway ( 1949) , and

Ander son and Doer mann ( 1952) has shown tha t bac te r iophages T2 , T3 , andT4 mul t ip ly in the bac te r ia l ce l l in a non- inf ec t ive f or m. The same i s t r ue ofthe phage ca r r ied by ce r ta in lysogenic bac te r ia ( Lwof f and Gutm ann, 1950).L i t t l e e l s e i s known about the vege ta t ive phase of these v i r uses . The exper i -ments r epor ted in th i s paper show th a t one of the f i rs t s teps in the gr owth ofT2 is the release f rom i ts protein coat of the nucleic acid of the virus par t ic le ,a f te r which the bu lk of the su l f ur - conta in ing pr o te in has no f ur the r f unc t ion .

Materials and Methods.--Phage T2 means in this paper the variety called T2H(Hershey, 1945); T2h means one of the host range m utan ts of T2; UV-phage meansphage irradiated with ultraviolet light from a germicidal lamp (General ElectricCo.) to a fractional survival of 10 5.Sensitive bacteria means a strain (H) of Escherichia coli sensitive to T2 and itsh mutant; resistant bacteria B/2 means a strain resistant to T2 but sensitive to itsh mutant; res istant bacter ia B/2h means a strain resistant to both. These bacteriado not adsorb the phages to which they are resistant."Salt-poor ' broth contains per liter 10 gin. bacto-peptone, 1 gm. glucose, and 1gin. NaC1. "Bro th" contains, in addition, 3 gin. bacto-beef extract an d 4 gm. Na Cl.Glycerol-lactate medium contains per liter 70 m~ sodium lactate, 4 gin. glycerol,5 gin. NaC1, 2 gin. KC I, 1 gin. NI-I_aCI, 1 mm MgC12, 0.1 m~ t CaC12, 0.01 g m. gela tin,10 rag. P (as orthop hosph ate), an d 10 mg. S (as MgSO4), at pH 7.0.Adsorp tion me dium contains p er liter 4 gin. NaC1, 5 gm. K~SO4, 1.5 gin. KH~P O,,3.0 gm. Na~ -IP0 ,, 1 mE MgSO,, 0.1 m_~ CaC12, and 0 .01 gm. gelatin, a t p H 7,0.Veronal buffer contains per liter 1 gm. sodium diethylbarbiturate, 3 mE MgS04,and 1 gin. gelatin, at p H 8.0.The HCN referred to in this paper consists of molar sodium cyanide solutionneutralized when needed with phosphoric acid.

* This investigation was supported in part by a research grant from the N ationa lMicrobiological Institu te of the Nationa l Institut es of Health, Public Hea lth Service.Radio active isotopes were supplied b y the O ak Ridge National La bora tory on alloca-tion from the Isotopes DivisiOn, United States Atomic Energy Commission.

39

The Journal of General Physiology


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40 VIRAL PROTEIN AND NUCLEIC ACID Ilq BACTERIOPHAGE GROWTH

Adsorpt ion of i sotope to bacter ia was usual ly measured by mixing the samplein adsorpt ion medium wi th bacter ia f rom 18 hour broth cul tures previously heatedto 70C. for 10 minutes and washed with adsorption medium. The mixtures werewarmed for 5 minutes at 37C., diluted with water, and centrifuged. Assays weremade of both sediment and supernatant f ract ions .

Precipitation of isotope with antiserum was measured by mixing the sample in0.5 per cent saline with abo ut 1011 per m l. of non-radioactive phag e and slightlymore than the least quanti ty of antipha ge serum (final dilution 1:160) th at wouldcause visible precipitation. The mixture was centrifuged after 2 hours at 37C.

Tests with DNase (desoxyribonuclease) were performed by warming samplesdiluted in veronal buffer for 15 minutes at 37C. with 0.1 rag. per ml. of crystall ineenzyme (Worthington Biochemical Laboratory) .

Acid-soluble isotope was measured after the chil led sample had been precipitatedwith 5 per cent trichloroacetic acid in the presence of 1 mg./m l, of serum albumin, andcentrifuged.

In all fractionations involving centrifugation, the sediments were not washed, andcontained about 5 per cent of the supernatant . Both f ractions were assayed.Radioact ivi ty was measured by means of an end-window Geiger counter , us ing

dried samples sufficiently small to avoid losses by self-absorption. For absolute m eas-urements, reference solutions of P~ obtained from the National Bureau of Standards,as well as a permanent simulated standard, were used. For absolute measurementsof Ss5 we relied on the assays (4-20 per cent) furnished b y the supplier of the isotope(Oak Ridge Nat ional Laboratory) .

Glycerol - lactate medium was chosen to permi t growth of bacter ia wi thout un-desirable pH changes at low concentrations of phosphorus and sulfur, and proveduseful also for certain experiments described in this paper. 18-hour cultures of sensitivebacteria grow n in this medium contain abo ut 2 109 cells per ml., which grow ex-ponentially without lag or change in l ight-scattering per cell when subcultured inthe same medium from either large or small seedings. The generation t ime is 1.5 hoursat 37C. The cells are smaller than those grown in broth. T2 shows a latent periodof 22 to 25 minutes in this medium. The phage yield obtained by lysis with cyanideand UV-phage (described in context) is one per bacterium at 15 minutes and 16per bacterium at 25 minutes. The final burst size in diluted cultures is 30 to 40 perbacterium, reached at 50 minutes. At 2 l0 s cells per ml., the culture lyses slowly,and yields 140 phage per bacter ium. The growth of both bacter ia and p hage in thismedium is as reproducible as that in broth.

For the preparatio n o f radioactive phage, P= of specific activity 0.5 mc./mg , orS35 of specific a ctivity 8.0 mc./m g, was incorpo rated into glycerol-lactate medium,in which bacteria were allowed to grow at least 4 hours before seeding with phage.After infection with phage, the culture was aerated overnight, and the radioactivephage was isolated by three cycles of alternate slow (2000 6") and fast (12,000 G)centrifugation in adsorption medium. The suspensions were stored at a concentrationnot exceeding 4 i .tc./ml.

Preparations of this kind contain 1.0 to 3.0 10- " gg . S and 2.5 to 3.5 10-1~ttg. P per viable phag e particle. Occasional preparation s con taining excessive a mou ntsof sulfur can be improved by absorption with heat-kil led bacteria that do not adsorb


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A. D. HERS HEY AND MARTHA CHASE 41the phage. Th e radiochemical pu rity of the preparations is somewhat uncertain, ow-ing to the possible presence of inactive phage particles an d e m pty phage membranes.Th e presence in our preparations of sulfur (abo ut 20 per cent) tha t is precipitated byantiphage serum (Table I) and either adsorbed by bacteria resistant to phage, ornot adsorbed by bacteria sensitive to phage (Table VII), indicates contaminationby membrane material. Con tamin ants of bacterial o rigin are prob ably negligible forpresen t purposes as indicated b y the da ta given in Table I. Fo r proof that our prin-cipal findings reflect genuine properties of viable phage particles, we rely on someexperiments with inactivated phage cited a t th e conclusion of this paper.

The Chemical Morphology of Resting Phage Par t ides. - -An ders on (1949)f o u n d t h a t b a c t e r i o p h a g e T 2 c o u l d b e i n a c t i v a t e d b y s u s p e n d i n g t h e p a r t ic l e sin h igh con cen t ra t ions o f s od ium ch lo ride , and rap id ly d i lu t ing the s us pens ionwi th wa te r . T he inac t iva te d phage was v i s ib l e in e l e c t ron mic rog raphs a s t ad -po le - s haped " ghos t s . " S ince no inac t iva t ion occu r red i f t he d i lu t ion was s low

TABLE IComposition of Ghosts and Solu tion of Plasmoly'zedPkage

P e r c e n t o f i s o t o p e]

Acid -so lubl e . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . .Ac id -so lubl e a f t e r t r ea tmen t w i th DNase . . . . . .Adso r bed to sens i t i ve bac t e r i a . . . . . . . . . . . . . . . .P r e c i pi t a t e d b y a n t i p h a g e . . . . . . . . . . . . . . . . . . .

Whale phage labeled with ' Plasmolyzedp h a g e l a b e l e d withP~' ~ ~ , ~ P ~ S'S

1 t 1 8 0 18 s I 9 0 I 2 I 9 09 o i ~ I s I 9 7

h e a t t r i b u t e d t h e i n a c t i v a t i o n t o o s m o t i c sh o c k , a n d i n f e r r ed t h a t t h e p a r t ic l e sp o s se s se d a n o s m o t i c m e m b r a n e . H e r r i o t t ( 19 5 1) f o u n d t h a t o s m o t i c sh o c kre lea sed in to s o lu t ion the DN A (des o xyp en tos e nuc le ic a cid ) o f the phagep a r t ic l e , a n d t h a t t h e g h o s t s c o u l d a d s o rb t o b a c t e r i a a n d l y s e th e m . H e p o i n t e dou t t ha t t h i s w as a beg inn ing tow ard the iden t i f i c a t ion o f v i ra l func t ions w i thv i ra l s ubs tances .W e have p la s m olyzed i s o top ica l ly l abe led T 2 b y s us pend ing the phag e(1011 pe r m l . ) i n 3 M s od ium ch lo r ide fo r 5 minu te s a t room te mp e ra tu re , an drap id ly pour ing in to the s us pens ion 40 vo lumes o f d i s t i l l ed wa te r . T he p la s -m o l y z e d p h a g e , c o n t a in i n g n o t m o r e t h a n 2 p e r c e n t s u r v i v or s , w a s t h e n a n -a lyzed fo r phos phoru s a nd s u l fu r in the s eve ra l way s s hown in T ab le I . T h ere s u l t s con f i rm and ex tend p rev ious f ind ings a s fo l lows : - -I . P l a s molys i s s e pa ra te s phage T 2 in to ghos t s con ta in ing nea r ly a l l t hes u l f u r a n d a s o l u ti o n c o n t a in i n g n e a r l y a ll t h e D N A o f t h e i n t a c t p a r t i cl e s.

2 . T he ghos t s con ta in the p r inc ipa l an t igens o f the phage pa r t i c l e de tec t -ab le by ou r an t i s e rum. T he DN A i s r e l e as ed a s the f re e ac id , o r pos s ib ly l inkedto s u l fu r - f ree , appa ren t ly non-an t igen ic s ubs tances .


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4 2 VIRAL P R O T E I N A N D N U C L E I C A CI D I N BACTERI OP HAGE G R O W T H3 . T h e g h o s t s a r e sp e c i f ic a l ly a d s o r b e d t o p h a g e - s u s c e p t i b l e b a c t e r i a ; t h e

D N A is n o t .4 . T h e g h o s t s r e p re s e n t p r o t e i n c o a ts t h a t s u r r o u n d t h e D N A o f t h e i n t a c t

p a r ti c le s , r e a c t w i t h a n t i s er u m , p r o t e c t t h e D N A f r o m D N a s e ( d e s o x y r ib o -n u c l ea s e ), a n d c a r r y t h e o r g a n o f a t t a c h m e n t t o b a c t er i a .

5 . T h e e f fe c ts n o t e d a r e d u e t o o s m o t i c s h o c k , b e c a u se p h a g e s u s p e n d e d i ns a l t a n d d i l u t e d s l o w l y is n o t i n a c t iv a t e d , a n d i ts D N A i s n o t e x p o s e d t oD N a s e .

T A B L E I ISensitization of Phage DNA to DNase by Adsorption to Bacteria

Phage adsorbed to

L i v e b a c t e r i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bac te r ia hea ted be fore infec tion . . . . . . . . . . . . . . .

B a ct er ia he at ed a f te r in fe ct ion . . . . . . . . . . . . . . . .

Heated unadsorbed phage: ac id-soluble t m f 70 . . . . . .8 0 .9 0 0t l O 0 .

Phage labeledwith

S $tp n

835p ~885tmp82p np np n

Non-sedimentable isotope,"per ce~aAfte r DNase No D N a s e

2 18 7

15 1176 1312 1466 235138188

Phag e adsorbed to bacter ia for 5 minutes a t 37 C. in adsorpt ion medium, fol lowed bywashing.Bacteria heated for 10 minutes at 800 C. in adsorption m edium (before infection) o r inveronal buffer (after infection).Unadsorbed phage heated in veronal buffer, treated with DNase, and precipitated withtrichloroacetic acid.All samples fractionated by centrifuging 10 minutes a t 1 300 G.

S e n s it i za t i o n o f P h ag e D N A t o D N a s e b y A d s o rp t i o n t o B a c t e r i a . - - T h e s t r u c -t u r e o f t h e r e s t i n g p h a g e p a r t i c l e d e s c r i b e d a b o v e s u g g e s t s a t o n c e t h e p o s s i b i l-i t y t h a t m u l t i p l ic a t i o n o f v i r u s i s p r e c e d e d b y t h e a l t e r a t i o n o r r e m o v a l o f t h ep r o t e c t i v e c o a t s o f t h e p a r ti c le s . T h i s c h a n g e m i g h t b e e x p e c t e d t o s h o w i t s el fa s a s e n s i ti z a t i o n o f t h e p h a g e D N A t o D N a s e . T h e e x p e r i m e n t s d e s cr i b e di n T a b l e I I s h o w t h a t t h i s h a p p e n s . T h e r e s u l t s m a y b e s u m m a r i z e d a s f o l-l o w s : -

1 . P h a g e D N A b e c o m e s l ar g e l y s e n s it i v e to D N a s e a f t e r a d s o r p t i o n t oh e a t - k i l l e d b a c t e r i a .

2 . T h e s a m e i s t r u e o f t h e D N A o f p h a g e a d s o r b e d t o l iv e b a c t e r ia , a n d t h e n


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A . D. H E R S H E Y A N D M A R T H A C H A S E 43h ea t ed t o 8 0 C . fo r 1 0 min u t es , a t wh ich t emp era tu re u n ad so rb ' ed p h ag e i sn o t s e n s i ti z e d t o D N a s e .

3 . T h e D N A o f p h a g e a d s o r b e d t o u n h e a t e d b a c t e r i a i s r e s i s ta n t t o D N a s e ,p r e s u m a b l y b e c a u s e i t i s p r o t e c t e d b y c e l l s t r u c t u r e s i m p e r v i o u s t o t h e e n -z y m e .

G r a h a m a n d c o l l a b o r a t o r s ( p e r s o n a l c o m m u n i c a t i o n ) w e r e t h e f i r s t t o d i s -c o v e r t h e s e n s i ti z a t io n o f p h a g e D N A t o D N a s e b y a d s o r p t i o n t o h e a t -k i l le db a c t e r i a .

T h e D N A i n i n f e c te d c el ls is a ls o m a d e a c c e s si b le t o D N a s e b y a l t e r n a t ef r eez in g an d t h awin g ( fo l lo wed b y fo rm ald eh y d e f i x a t i o n t o i n ac t i v a t e ce l lu -l a r en zy mes) , an d t o so me ex t en t b y fo rmald eh y d e f i x a t i o n a lo n e , a s i l l u s -t r a t e d b y t h e f o l lo w i n g e x p e r i m e n t .

Bac teria were grown in broth to 5 10~ cells per ml., centrifuged, resuspended inadsorp t ion medium, and in fected wi th ab out tw o P=-labeled phage per bacter ium.After 5 minutes for adsorption, the suspension was diluted with wa ter containing perliter 1.0 mxs MgSO 4, 0.1 m ~ CaCI~, and 10 rag. gelatin, a nd rec entrifuged. Th e cellswere resuspended in the fluid last me ntioned at a conce ntration of 5 108 per ml.This suspension was f rozen at -15 C. and thawed wi th a m in imum of warming ,three t im es in succession. Imm ediate ly afte r the third thawing, the cells were fixed bythe add i t ion of 0 .5 per cen t ( v / v ) of fo rmal in (35 per cen t HC HO ). After 30 minutesat roo m tem perature , the suspension was dialyzed free from forma ldehyde and cen-trifug ed at 2200 G for 15 minutes. Samples of P~-labeled phage, frozen-thawedl fixed,and dialyzed, a nd of infected cells fixed only and dialyzed, were carried along ascontrols.

Th e an a ly s i s o f t h ese mate r i a l s , g iv en i n Tab l e I I I , sh o ws t h a t t h e e f f ec to f fr e e z in g a n d t h a w i n g i s t o m a k e t h e i n t r a c e l lu l a r D N A l a b il e t o D N a s e ,wi th o u t , h o wev er , cau s in g mu ch o f i t t o l each o u t o f t h e ce i l s . F reez in g an dth awin g an d fo rm ald eh y d e f ix a t i o n h av e a n eg l ig ib le e f f ec t o n u n ad so rb edp h ag e , an d fo rm ald eh y d e f i x a t i o n a lo n e h as o n ly a m i ld e f f ec t o n i n f ec t ed cel ls .

Bo th se n s i t i za t i o n o f t h e i n t r ace l l u l a r p 3~- t o DN ase , an d i t s f a i l u re t o l eacho u t o f t h e ce ll s, a r e co n s t an t f ea tu res o f ex p er im en t s o f th i s t y p e , i n d e p en d en t l yo f v i s i b l e l y s is . I n t h e ex p er im en t j u s t d esc r i b ed , t h e f ro zen su sp en s io n c l ea redd u r in g t h e p e r i o d o f d i a ly s i s . P h ase -co n t r as t mic ro sco p y sh o wed t h a t t h e ce i l sc o n s is t e d l a r g e ly of e m p t y m e m b r a n e s , m a n y a p p a r e n t l y b r o k e n . I n a n o t h e rex p er imen t , s amp les o f i n f ec t ed b ac t e r i a f ro m a cu l t u r e i n sa l t -p o o r b ro thw e r e r e p e a t e d l y f r o z e n a n d t h a w e d a t v a r i o u s t i m e s d u r i n g t h e l a t e n t p e r i o do f p h a g e g r o w t h , f ix e d w i t h f o r m a l d e h y d e , a n d t h e n w a s h e d i n t h e c e n t ri f u ge .C lea r i n g an d mic ro sco p i c l y s i s o ccu r r ed o n ly i n su sp en s io n s f ro zen d u r in g t h es e c o n d h a l f o f th e l a t e n t p e r i o d , a n d o c c u r r e d d u r i n g t h e f i r s t o r s e c on d t h a w -in g . In t h i s case t h e l y sed ce l l s co n s i s t ed wh o l ly o f i n t ac t ce l l memb ran es ,a p p e a r i n g e m p t y e x c e p t f o r a f e w s m a l l , r a t h e r c h a r a c t e r i st i c r e f r a c ti l e b o di e sap p aren t l y a t t ach ed t o t h e ce l l wa l l s . Th e b eh av io r o f i n t r ace l l u l a r p 3 2 t o wardDN ase , i n e i t h e r t h e l y sed o r u n ly sed ce ll s, was n o t s i g n i f i can t l y d i f f e r en t f ro m


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44 VIRAL PROTEIN AND NUCLE]C ACID IN BACTERI OPHAGE GROWTH

that shown in Table III, and the content of Pa was only slightly less afterlysis. The phage liberated during freezing and thawing was also fitrated inthis experiment. The lysis occurred without appreciable liberation of phagein suspensions frozen up to and including the 16th minute, and the 20 min-ute sample yielded only five per bacterium. Another sample of the cultureformalinized at 30 minutes, and centrifuged without freezing, contained 66per cent of the p a in non-sedimentable form. The yield of extracellular phageat 30 minutes was 108 per bacterium, and the sedimented material consistedlargely of formless debris but contained also many apparently intact cellmembranes.

T A B L E I I ISensitization o f Intracdlular Phage to DN ase by Freezing, Thawing, and Fixationwith Formaldehyde

J Unsdsorbed Infec ted cells Iphage frcaen , frozen, hawed, Infected cellsthawed, fixed fixed fixed onlyL o w s p e e d s e d i m e n t f r a c t i o n

T o t a l P ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - 7 1 8 6A c id - so l ub l e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - 0 0 . 5A c id - so . lu b le a f t e r D N a s e . . . . . . . . . . . . . . . . . . . . . - - 5 9 2 8

L o w s p e e d s u p e m a t a n t f r a c t i o nT o t a l P = . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - 2 9 1 4A c id - so l ub l e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 . 8 0 . 4A c i d -s o l u bl e a f t e r D N a s e . . . . . . . . . . . . . . . . . . . . . 1 1 2 1 5 . 5

T h e f igu res exp res s pe r cen t o f to ta l P ' ~ in the o r ig ina l phage , o r i t s ads o rb ed f r ac t ion .We draw the following conclusions from the experiments in which cells

infected with PS!labeled phage are subjected to freezing and thawing.1. Phage DNA becomes sensitive to DNAse after adsorption to bacteria in

buffer under conditions in which to known growth process occurs (Benzer,1952; Dulbecco, 1952).2. The cell membrane can be made permeable to DNase under conditionsthat do not permit the escape of either the intracellular P= or the bulk of thecell contents.3. Even if the cells lyse as a result of freezing and thawing, permitting escape

of other cell constituents, most of the p3~ derived from phage remains insidethe cell membranes, as do the mature phage progeny.4. The intracellular p3~ derived from phage is largely freed during spon-taneous lysis accompanied by phage liberation.


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A . D . H E R S H E Y A N D M A R T H A C H A S E 45W e i n t e r p r e t t h e se f a c t s to m e a n t h a t i n t ra c e ll u la r D N A d e r i ve d f r o m p h a g e

i s n o t m e r e l y D N A i n s ol u t i o n , b u t i s p a r t o f a n o r g a n i z e d s t r u c t u r e a t a l l t i m e sd u r i n g t h e l a t e n t p e r i o d .Liberation of D NA from Phage Particles by Adsorption to Bacterial Frag-

ments.--The s e n s i ti z a t io n o f p h a g e D N A t o s p e c if i c d e p o l y m e r a s e b y a d s o r p -t i o n t o b a c t e r i a m i g h t m e a n t h a t a d s o r p t i o n i s f o l lo w e d b y t h e e j e c t i o n o ft h e p h a g e D N A f r o m i t s p r o t e c t i v e c o a t . T h e f o l l o w i n g e x p e r i m e n t s h o w s t h a tt h i s i s i n f a c t w h a t h a p p e n s w h e n p h a g e a t t a c h e s t o f r a g m e n t e d b a c t e r i a lcells .

TABLE IVRdease of DNA from Phage Adsorbed to Bacterial Debris

P h a g e labeled withSs s ] pal

Sediment fractionS ur viv in g p hag e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 22T o ta l iso to pe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 55A cid -so lu bl e i so to pe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . 0 2A cid -so lu ble a f te r D N as e . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 29

Supematant fractionS u r v i v i n g ph age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5T o ta l iso to pe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4 5A cid -so lu ble i so to pe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 .8 0 . 5A cid -so lu ble a f te r D N as e . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 .8 3 9

S 35- and P~-labeled T2 were mixed with identical samples of bacterial debris in adsorptionmedium and warm ed for 30 minutes at 37C . The mixtures wer e then centrifuged for 15minutes at 2200 G, and the sediment and supernatant fractions we re analyzed separately.The results axe expressed as per cent of input phage or isotope,B a c t e r i a l d e b r i s w a s p r e p a r e d b y i n f e c t i n g c e l l s i n a d s o r p t i o n m e d i u m w i t h

f o u r p a r t i c l e s of T 2 p e r b a c t e r i u m , a n d t r a n s f e r r i n g t h e c e ll s t o s a l t - p o o rb r o t h a t 3 7 C . T h e c u l t u r e w a s a e r a t e d f o r 60 m i n u t e s , ~ r/ 5 0 H C N w a s a d d e d ,a n d i n c u b a t i o n c o n t i n u e d f o r 30 m i n u t e s l o n g e r . A t t h i s t i m e t h e y i e l d o f e x -t r a c e l l u l a r p h a g e w a s 4 0 0 p a r t i c l e s p e r b a c t e r i u m , w h i c h r e m a i n e d u n a d s o r b e db e c a u s e o f t h e l o w c o n c e n t r a t i o n o f e le c t r o l y te s . T h e d e b r i s f r o m t h e l y s e dc e l l s w a s w a s h e d b y c e n t r i f u g a t i o n a t 1 7 0 0 G , a n d r e s u s p e n d e d i n a d s o r p t i o nm e d i u m a t a c o n c e n t r a t i o n e q u i v a l e n t t o 3 X 1 0 ~ l y s e d c el ls p e r m l . I t c o n s i st e dl a r g e l y o f c o l l a p s e d a n d f r a g m e n t e d c e l l m e m b r a n e s . T h e a d s o r p t i o n o f r a d i o -a c t i v e p h a g e t o t h i s m a t e r i a l i s d e s c r i b e d i n T a b l e I V . T h e f o l l o w i n g f a c t ss h o u l d b e n o t e d .


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4 6 VIRAL PROTEIN" AND NUCLEIC ACID llg BACTERIOPHAGE GROWTH1 . T h e u n a d s o r b e d f r a c t i o n c o n t a i n e d o n l y 5 p e r c e n t o f t h e o r i g in a l p h a g e

p ar t i c l e s i n i n f ec t i v e fo rm, an d o n ly 1 3 p e r c en t o f th e t o t a l su l fu r . (M u cho f th i s s u l fu r m u s t b e t h e m a t e r i a l t h a t i s n o t a d s o r b a b l e t o w h o l e b a c t e r ia . )

2 . A b o u t 8 0 p e r c e n t o f t h e p h a g e w a s i n a c t i v a te d . M o s t o f th e s u l f u r o ft h i s p h ag e , a s we l l a s mo s t o f t h e su rv iv in g p h ag e , was fo u n d i n t h e sed imen tf r ac t i o n .

3 . T h e s u p e r n a t a n t f r a c t i o n c o n t a i n e d 4 0 p e r c e n t o f t h e t o t a l p h a g e D ~ q A( i n a f o r m l a b il e t o D N a s e ) i n a d d i ti o n t o t h e D N A o f t h e u n a d s o r b e d s u r v i v i n gp h a g e . T h e l a b il e D N A a m o u n t e d t o a b o u t h a l f o f , h e D N A o f t h e i n a c t i v a t e dp h ag e p a r t i c l e s , wh o se su l fu r sed imen ted wi th t h e b ac t e r i a l d eb r i s .

4 . M o s t o f t h e s e d i m e n t a b le D N A c o u l d b e a c c o u n t e d f o r e i th e r a s s u r v iv i n gp h a g e , o r a s D N A l a bi le t o D N a s e , t h e l a t t e r a m o u n t i n g t o a b o u t h a l f t h e D N Ao f t h e i n ac t i v a t ed p a r t i c l e s .

E x p e r i m e n t s o f t h i s k i n d a r e u n s a t i s f a c t o r y in o n e r e s p e c t : o n e c a n n o t t e l lw h e t h e r t h e l i b e r a t e d D N A r e p r e s e n t s a l l t h e D N A o f s o m e o f t h e i n a c t i v a t e dp ar t i c l e s , o r o n ly p a r t o f it .

S i m i l a r re s u l ts w e r e o b t a i n e d w h e n b a c t e r i a ( s t r a in B ) w e r e l y s ed b y l a r gea m o u n t s o f U V - k i l l e d p h a g e T 2 o r T 4 a n d t h e n t e s t e d w i t h P ~ - l a b e l e d T 2a n d T 4 . T h e c h i ef p o i n t o f in t e r e s t i n t h i s e x p e r i m e n t i s t h a t b a c t e r i a l d e b r iss a t u r a t e d w i t h U V - k i l l e d T 2 a d s o r b s T 4 b e t t e r t h a n T 2 , a n d d e b r i s s a t u r a t e dw i t h T 4 a d s o r b s T 2 b e t t e r t h a n T 4 . A s i n t h e p r e c e d in g e x p e ri m e n t , s o m e o ft h e a d s o r b e d p h a g e w a s n o t i n a c t i v a t e d a n d s o m e o f t h e D N A o f th e i n a c t i -v a t e d p h a g e w a s n o t r e l e a s e d f r o m t h e d e b r is .

T h e s e e x p e r i m e n t s s h ow t h a t s o m e o f t h e c e l l r e c e p t o r s f o r T 2 a r e d i f fe r e n tf r o m s o m e o f t h e c e ll re c e p t o r s f o r T 4 , a n d t h a t p h a g e a t t a c h i n g t o t h e s e s p e -c i f i c r e c e p t o r s i s i n a c t i v a t e d b y t h e s a m e m e c h a n i s m a s p h a g e a t t a c h i n g t ou n s e l e c t e d r e c e p t o r s . T h i s m e c h a n i s m i s e v i d e n t l y a n a c t i v e o n e , a n d n o tm e r e l y t h e b l o c k i n g of s it e s o f a t t a c h m e n t t o b a c t e r i a .Removal of Phage Coats from Infected Bavteria.--Anderson (1951) has ob-t a i n e d e l e c t r o n m i c r o g r a p h s i n d i c a t i n g t h a t p h a g e T 2 a t t a c h e s t o b a c t e r i ab y i t s t a il . I f t h i s p r e c a r io u s a t t a c h m e n t i s p r e s e r v e d d u r i n g t h e p r o g r e s s o ft h e i n f ec t i o n , an d i f t h e co n c lu s io n s r each ed ab o v e a r e co r r ec t , i t o u g h t t ob e a s i m p l e m a t t e r t o b r e a k t h e e m p t y p h a g e m e m b r a n e s o f f t h e i n f e c t e db ac t e r i a , l eav in g t h e p h ag e DNA in s id e t h e ce l l s .

T h e f o ll o w in g e x p e r i m e n t s s h o w t h a t t h i s i s r e a d i ly a c c o m p l i s h e d b y s t r o n gsh ear in g fo rces ap p l i ed t o su sp en s io n s o f i n f ec t ed ce l l s , an d fu r t h e r t h a t i n -f e c t e d c e ll s f r o m w h i c h 8 0 p e r c e n t o f t h e s u l f u r o f th e p a r e n t v i r u s h a s b e e nr e m o v e d r e m a i n c a p a b l e o f y i e ld i n g p h a g e p r o g e n y .

B r o t h - g r o w n b a c t e r i a w e r e i n f e c t e d w i t h S3s- or P3*-labeled phage in ad-s o r p t io n m e d i u m , t h e u n a d s o r b e d m a t e r i a l w a s r e m o v e d b y c e n t r i f u g a t io n ,a n d t h e c e l ls w e r e r es u s p e n d e d i n w a t e r c o n t a i n i n g p e r l i te r 1 m ~ M g S O , ,0 .1 rn ~ CaCl~ , an d 0 .1 g in . g e l a t i n . Th i s su sp en s io n was sp u n i n a War in g


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A . D. H E R S H E Y A N D M A R T H A C H A S E 47blendor (semimicro s ize) a t 10,000 g .P.~. The suspens ion was cooled brief lyin i ce wa te r a t t he end o f e ach 60 s econd runn ing pe r iod . Sample s we re removeda t i n t e r v a l s , t i t r a t e d ( t h r o u g h a n t i p h a g e s e r u m ) t o m e a s u r e t h e n u m b e r o fbac te r i a c apa b le o f y i e ld ing phage , an d cen t r i fuged to meas u re the p ro por t ionof i s o tope re l e a s ed f rom the ce ll s.

T he re s u l t s o f one expe r imen t w i th e ach i s o tope a re s hown in F ig . 1 . T hed a t a f o r Sa s and s u rv iva l o f in fec ted bac te r i a com e f rom the s ame expe r im en t ,in wh ich the ra t io o f added phage to bac te r i a was 0 .28 , and the co ncen t ra t ions

JO0

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Hershey 1952 Original 39