A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C II. Reduction of TSP-1-Specific Receptor Sites on Cells Grown at 37 C or 45 C, and the Temperature-Dependent Inactivation of Replicating Phage

(1)

Vol. 9, No. 4 Printed in U.S.A.

A

Bacteriophage of Bacillus subtilis

Which

Forms

Plaques Only

at

Temperatures

Above

50 C

II.

Reduction

of

TSP-i-Specific Receptor

Sites

on

Cells Grown

at

37 C

or

45 C,

and the

Temperature-Dependent

Inactivation

of

Replicating Phage1

J. R. LAMONTAGNE2 AND W. C. McDONALD

Departmenit ofMicrobiologyanidImmunlology, Tulante Uniiversity School of Medicinie, anid Biology Department,

Tullanie

Uniiversity,

New

Orleans,

Louisiania

70118

Received forpublication 26 October 1971

The adsorption ofsubtilisphage TSP-1 to Bacillussubtilis WI 68wastested with

cells grownat 37, 45, and 53 C. Kineticdata, electron micrographs, and

quantita-tive measurement of the per cent phage adsorbed all indicated that irreversible

adsorption occurs only with cells grown at 53 C. IfTSP-1 was allowed to infect

cells at 53 C, subsequent shifts to 37 C inhibited phage replication and resulted in the inactivation ofplaque-forming units.

Thephysical andchemicalcharacteristics of the

subtilisphage,TSP-1, haverecentlybeendescribed (8). It was isolated from soil, contains

double-stranded deoxyribonucleicacid (DNA)withouta

thymineanalogue, and ismorphologically similar

toothersubtilisphages (3,5,10).Itdiffers drama-tically, however, from other B. subtilis

bacterio-phages in that it will formplaques only at

tem-peratures above 50 C. Clear plaques are formed

on any of the B.subtilis strains that grow above 50C, andonB. licheniformis9945A above 50C. This rigorous restriction to temperatures above 50 C (toanupperlimitof55C) maybea reflec-tion of the interaction of one or more factors, actingsinglyorinconcerttoinhibitplaque forma-tion below 50 C. These factors include: (i)

a temperature-sensitive restriction mechanism

which is inactivated above 50 C (6); (ii) the

ab-senceofphagereceptor sites below 50C, dueto

changes in cell wall structure(4);and(iii) TSP-1 may haveintermediatereplicativeenzymeswhich

require the higher temperature for optimal

activity.

As aninitial step inidentifying which of these factors mayaccountfor thisstringenttemperature

requirement, we have asked the following ques-tions: (i) will TSP-1 adsorb to B. subtilis W168 growing attemperatures below 50 C, and (ii) if

1This paper wastaken in partfrom adissertaition submitted by J. R. LaMontagne inpartial fulfillmenit ofthereqluiremiients for thePh.D.degreeatTulanieUniversity,NewOrleans,La.70118.

2Present address: Depairtment of Microbiology, School of Medicine,Universit) ofPittsbturgh,Pittsburgh,Pa.15213.

adsorption is a limiting factor, can TSP-1

replica-tion continue below 50C once phageadsorption and DNA injection have occurred at 53 C? We

approached the first question by analyzing the kinetics of phage adsorption with experiments similar tothe classicexperiment of Hershey and Chase (7), by measuring the amount of phage which would adsorb to B. subtilis cells growing at

various temperatures and by examining negative stains of TSP-1-infected B. subtilis with the elec-tron microscope. To answer the secondquestion,

the initial steps in phage replication, phage

ad-sorption, and DNA injection were allowed to occur at 53 C. The infected cultures were then transferred to37 Cto determine if phage replica-tion could be completed at this temperature.

MATERIALS AND METHODS

Bacterial strains and bacteriophage used. All of theseexperiments were donewith TSP-1 infecting B.

suibtilis

W168, a prototrophic transformant of B.

suibtilis

168, unless otherwise noted.

Growth conditions. All bacterial cultures were

growninDifco BrainHeartInfusion (BHI) broth

me-dium.Brothcultureswereincubatedat53,45,or37C

in reciprocating shaker-water baths (New Brunswick Scientific Co.).Plaquecounts weredoneonBHI agar

(1.5',) by the agaroverlay technique (1) with0.1 ml ofa B.

suibtilis

W168sporesuspension (108spores per

ml)astheinoculum forthelawn. Serialdilutionswere

made in BHIbroth,and theoverlaywasBHIsoftagar

(0.65,).

Preparationof TSP-1 antisera.Anti-TSP-1 antisera

wasprepared in rabbits withTSP-1 suspensions

puri-652

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fied as previously described (8). Primary inoculations consisted of 0.25 ml of a TSP-1 suspension (1012 plaque-forming units) emulsified in an equal volume of complete Freund's adjuvant (Difco) and injected into the footpad. This was followed by two 0.5-ml subcutaneous injections ofTSP-1 at 10-day intervals. Rabbits were bled by cardiac puncture 6 to 8 weeks after the primary inoculation. The sera were collected andheated for 30 min at 36 C to inactivate comple-ment. Antisera obtained by this procedure had K values of at least 400.

Temperature shift experiments. Bacteria were incu-bated in 300-ml Nephelo flasks (side arm = 14 by 130mm;Bellco, Vineland, N.J.) containing 40 ml of BHI in areciprocating shaker-water bath at 53 C. The bacterial growthwasmonitored witha Klett-Summer-soncolorimeter with a red filter (640 to 700 nm) and incubated until the culture reached a turbidity of 75 Klettunits (equivalent to 5 X 107 bacteria per ml). At thistime, TSP-1 wasaddedtothedesired cultures at a multiplicity of infection (MOI) of 1. The shift to a lower temperature was made by transferring the desired flask to a second reciprocating shaker-water bath at the appropriate temperature Under these

conditions,aculturetransferred from53 to 37C,for

example, reached 37 C within 1.5 to 2.0 min after

transfer. In those experiments which involved the transferfrom53 to37C,followed byareturn to53C, 0.1 mlof undiluted phage antiserawas addedshortly after the shift to 37 C.

Radioactive labeling of TSP-1 DNA. TSP-1 was

labeled with 32P-orthophosphate (285 Ci/mg; Inter-national Chemical and Nuclear Corp.). The radio-active phosphatewasadded to aculture ofB.subtilis W168 growing in BHI to afinal concentration of 3

,gCi/ml

2 hr before addition of TSP-1. The labeled phage were purified by the polyethylene glycol-sodiumchloride procedurepreviouslydescribed (8).

Kinetics of TSP-1 adsorption and DNA injection. Thekinetics of TSP-1 adsorption and DNAinjection

werestudied byaproceduresimilartothat ofHershey

andChase (7). Phagelabeled with 32pwere addedto

late log-phase culture of B. subtilis W168

(approxi-mately 108 bacteria perml) growingat53 C at aMOI of 10.Samples (3ml)weretakenat5-minintervals and divided into twofractions as follows: (i) 0.5 ml was

filteredimmediatelyontonitrocellulose filters(25mm

diameter; 0.45 ,um pore size; Millipore Corp.) and washed with 15 ml ofminimal salts (7); (ii) the

re-maining 2.5 ml washomogenized in aSorvall

Omni-mixer (IvanSorvall, Inc., Norwalk, Conn.),equipped

witha5.0-mlmicroattachment, for 60 secat asetting

of5. A0.5-ml sample of this homogenate was then

filtered and washed. This homogenization procedure

wassufficientto removeany adsorbed phageparticles

asjudged by removal ofTSP-1 particles labeledwith

'4C-isoleucine. Thefilterswere placed in scintillation vials, dried, and counted with Spectrafluor (Amer-sham/Searle)-toluenein aBeckman Instruments 200B

liquid scintillation counter.

Electron microscopy. TSP-1 was addedto cultures ofB.subtilis W168(MOI = 50) growingat53or37C inBHI. After 10 minof furtherincubation, 10ml of

the culture was centrifuged in a clinical centrifuge

(International Equipment Co., Needham, Mass.) at maximum speed (ca. 3,000 X g) for 10 min. The pelletwas suspended in 5ml of 10-2 M tris(hydroxy-methyl)aminomethane (Tris)-10-3 M MgSO4 buffer,

pH7.4. Adropof the suspendedpelletwasmixed with

an equal amount of

2%/o

phosphotungstic acid-2%0 normalrabbit serum, pH 7.0,andexaminedon

Form-var-coated grids in aPhillips 300E electronmicroscope operating at 80 kv.

RESULTS

Adsorption of TSP-1 to B. subtilis W168.

TSP-1waslabeledwith

32p,

purified, and added to

B.subtilis W168 growing under various tempera-ture conditions. If adsorption was not

accom-panied with DNA injection, the cell-associated radioactivitywould be removable by homogeniza-tion. In contrast, ifadsorption was followed by DNA injection, the radioactivity would be

re-tainedafterhomogenization.

It is apparentfromtheresults of these experi-ments that TSP-1 will adsorb efficiently to 53 C growncells and thatDNAinjectionaccompanies

this adsorption (Fig. IA). Adsorption at 53 C occurred rapidly, and a plateau wasreached by 5

min after infection. A comparable situation does

not prevail at 37 C, however (Fig. 1B). In this case,TSP-1 adsorbed to 37 C cells, but not to the degree observed with 53 C grown cells, andonly

20%of the labelremained afterhomogenization. These results suggest two alternative explana-tions: (i)B. subtilisgrown at 37 C does nothave TSP-1 receptors on itssurface, and (ii) B. subtilis

grown at37 Chas the proper receptors,butDNA injection will not occur because it is a thermophilic

process.Toanalyze thispossibility,B.sutbtiliswas grown at 53 C, shifted to 37 C 10 min before addition ofTSP-1 (MOI = 10),and the kinetics of phage adsorption and DNA injection were

determined. Theresultsshowthatadsorptionand DNAinjection take place under these conditions, indicating that TSP-1 DNA injection is not a

thermophilic process (Fig. IC). Collectively, these results can be interpreted to indicate that

TSP-1adsorption to 53 C cells isspecific,whereas

adsorption to 37 C cells is not. In this context,

specific adsorption is defined as that adsorption

whichleadstoDNAinjection.Theseexperiments suggest that specific phage receptors are not present on cells grown at 37 C and that any

ad-sorptionto37Ccells isprobably nonspecific.Itis alsoclearthatphageDNAinjectioncan occurat

37Ciftheproperphage receptorsarepresent.

To confirm this suggestion, B. subtilis W168

grown at 37 and 53 C were infected withTSP-1 (MOIca.50),negatively stained,andexaminedby electron microscopy. These micrographs clearly

indicated thatTSP-1 does not irreversibly attach

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LAMONTAGNE AND McDONALD

5 10 5 20 25 5 tO 15 20 25 5 10 15 20 i5

Time After Addition of TSP-1(min)

FIG. 1. KitneticsofTSP-IadsorptioniandDNAinjection. Bacillussubtilis W168 wasgrowti tolate logphase

(108 cells/ml) antd 32P-labeled TSP-I wasadded(MOI = 10) tocellsgrownz at53 C (A), 37 C (B),and 53 C

cells shiftedto37 C 10mitibeforeTSP-Iaddition (C).Cell-associated radioactivity after (0) antdbefore (0) ho-mogenization.

toB.subtilisW168cellsgrownat37 C, but does

attachto53 Cgrowncells.

An additional experiment was done to verify the absenceofphagereceptorsitesat37C. In this instance, TSP-1 was added to B. subtilis W168

growingat37 C(MOI = 1.0)andincubatedat37

Cforanadditional 5or10min before theculture wastransferredto53 Candmonitored turbidime-tricallyfor thedevelopmentoflysis. IfTSP-1 did

notadsorb due to theabsence ofphage receptor

sites, it should be rapidly neutralized by TSP-1 antiserum, and the culture would be protected frominfection. Theseresultsclearly showthatthis

is thecase, forin allculturestowhich antiserum

was added the cultures did not manifest any

evidenceofphage infection (Fig. 2). All cultures

towhichnonspecificserum wasaddedlysed about

60to70minafter transferto53C.

Estimation of per cent phage adsorbed to B. subtilis W168 growing at53 and 45 C. A simple

method to quantitate the number of bacterio-phage particleswhichadsorbtoa suitablehost is toaddafewdropsofCHC1sto the

bacteria-bac-teriophage suspension andthentoassaythe resul-tant aqueous supernatant fluid for surviving

in-fectiouscenters(2).Thisprocedure actually meas-uresthe number of particles thatareirreversibly

adsorbed to bacteria, since it kills uninfected as

wellasinfected bacteriabut has noeffect on

un-adsorbed particles. The number of adsorbed

phagecanthen be determined by subtractingthe

titer obtained with CHCI3 treatment (i.e., free

phage), from the titer obtained without CHCI3

treatment (i.e.,freephage plusinfectedbacteria). However, when this experiment was done with

TSP-1 andB. subtilis W168 (MOI = 1.0)at53C,

the numberof infectiouscentersobtained withor

without CHC13 was identical. Moreover, if one compares these titers with the titer calculated

from the initial phage inoculum, it is apparent

thatabout 95% of the input phageisinactivated

very soonafteraddition of TSP-1 (Fig. 3B).This

coincidence inphagetiters indicates that infected cellsexposedtotemperaturesbelow 50 C willnot

formplaques. This loss inphage may be due to

the temperaturechangewhichaccompanies

dilu-tionandsample platingbytheagaroverlay

tech-nique. To test the possibility that extracellular

phage particles may be inactivated by a rapid

temperature shift, a TSP-1 suspension was

incu-B

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654 J. VIROL.

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TIME AFTER INFECTION (min)

FIG. 2. Susceptibility ofTSP-I toneutralizationiby phage antiserum ifTSP-I isaddedto B. subtilis W168

growingat 37C. TSP-Iaddedattime(a) (MOI= 1.0), transferto53Cattime(b), andanitiserumornonspecific

serumaddedat lime (c). Key: uninfectedculturetransferredto 53 Cat T = 5mill (A); intfected culture

trans-ferredto53 Cat T = 5min, antiserum addedimmediately before transfer to 53 C(B); infectedculture

trans-ferredto53 Cat T 10 min, antiserum addedimmediately before transferto 53C(C); inifectedculture

trans-ferredto53Cat T =5miii (D); infectedculture transferred to 53 Cat T = 5 miii,nonzspecific serum added

immediately before tranisfer(E); infected culture transferredto53 Cat T = 10 miii, nionispecific serum added

imnmediately before transferto53 C(F).

bated at 53 C, rapidly transferred to 37 C, and

assayedatvarious times before and after transfer. The results of these experiments indicated that

TSP-1 particles per se are not inactivated by a

rapidtemperaturechangesincetherewas noloss ofinput after transfer to 37 C. It wouldappear

then thatreplicating phagearesensitivetoadrop

in temperature, andas aconsequenceplaqueswill

not be formed from infected bacteria. One can

then conveniently measureTSP-1 adsorption by assayinganinfectedculturewithoutCHCl3

treat-ment and comparingthis titer with the one

esti-mated from the phage inoculum. Repeated

ex-periments indicate that TSP-1 adsorption to B.

subtilis and B. licheniformis 9945Aoccursrapidly,

with 90to95%input phageadsorbed after 2min

at53C. Thislossofinput phage,however,isnot

evidentifoneaddsTSP-1 to B.subtilis incubated

at45 C(Fig. 3A) or37 C. Inthiscase,thephage

titers obtained withorwithoutCHC13arereduced

by about 40% oftheestimatedphage titer. There

isalsonoevidenceofphage replication (Fig. 3A)

comparable to the burst of new phage particles

observedat53C (Fig. 3B).Addition ofTSP-1to

cells incubated at 37 Cgives thesame results as

thecellsat45C (datanotshown), indicatingthat

irreversible phage adsorption and injection are reduced under these circumstances.

Temperature-dependent inactivation of replicat-ing TSP-1. The results presented above

demon-stratethatTSP-1 specificreceptorsitesareeither

not present or are not accessible on B. subtilis

W168cellsgrownat37 C. Inaddition,itappears

adsorbedphage canbe inactivatedbydecreasing

thetemperatureofincubation. These observations

imply that, although adsorption is a barrier to

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20 40 0 20 40 60

TIME AFTER INFECTION

[image:5.500.69.260.66.297.2]

min

FIG. 3. One-step growtAl ctirvesofTSP-1 doneoiiB.

subtilis W168growiiigat45 C (A) and 53 C (B). Tlte

claslhed linierepreseitts thlephage titercalcuilatedfroni

thephiage inoculione.P/lagetiters obtaiiied with CHCI3

(-) actcd withloit CHCI3 (O).PFU = plaquie-formniflg

units.

TSP-1

replication

below 50

C,

it is not the

only

one. To define more

clearly

this apparent

tem-perature-dependent

inactivation andits effect on

phage

replication,

we undertook several

experi-mentsinwhich TSP-1 wasaddedtoB.subtilisat

53 C

(to

allow

adsorption), transferring

the in-fected cultureto37 Catvarious times after infec-tion and

determining

ifthe

lytic cycle

could be

com-pleted

at this temperature. We

employed

two

indexesof

replication: (i)

theturbidimetric

meas-urementof host cell

lysis

and

(ii)

themeasurement

ofnew

phage synthesis by

infectiouscenterassays.

In the first

experiment,

TSP-1 was added to

B.subtilis W168

(MOI

=

1.0)

at53C.Theculture

wastransferred to 37 C

immediately

after

infec-tion,

and the number of infectious centers was

determined with or without

CHC13

treatment at

various times after infection. These titers were

compared

with the calculated

input phage

titer and

plotted

against time. Over 95 'S- of the

phage

inoculumwas

destroyed,

and the

surviving

phage

titerwasmaintained for the duration ofthe

experi-ment. These results indicate that

replicating

phage

were

destroyed

and that further

phage

adsorption, injection,

and

replication

did not

occur at 37 C. In contrast, infected cultures at

53 Cshowedanidentical loss of

input phage

but

wascharacterized

by

a burst ofnew

phage

parti-cles about 30min after infection

(Fig. 3B).

To determine if it was possible to inactivate

replicatingTSP-1 atvarious times after

infection,

theeffectsofatemperatureshift from53 to37C

on the turbidity of infected and uninfected B.

subtilis W168 were observed. Ten flasks ofBHI

broth were inoculated withB. subtilis W168 and incubated at 53 C until they reached Klett 75

(5 X 107 bacteriaperml). TSP-1 wasthen added (MOI = 1.0) to nine of these flasks. At 5-min intervals through 35 min, cultures were

trans-ferredto 37C alongwith the uninfectedcontrol.

Inthis case, itwas possibleto determine atwhat timethetemperature requirementwas overcome.

Those infected flasks which were transferred to

37 Catless than 30minafterinfection continued

toincreased in turbidityataboutthe same rateas

theuninfected control culture. Flaskstransferred

attimesafter 30 min eventuallylysed,

suggesting

thattherequirement for 53 Cwas overcomeafter 30 min at 53 C. However, this experiment

only

measured changes in turbidityand did not

indi-cate if phage replication was actually taking

place, evenin those cultures transferred to 37 C after 30 min at 53 C.We tested this directlyby

transferring samples ofaninfected cultureto37C

atvarious times after infection, incubating them

at 37 C for 1 hr and thenassaying for infectious

centers. Thetiters of the transferred cultureswere

compared to the titers ofthe mother culture at

the time of transfer. If the transferred culture showed ahigher titer than the mother culture at

thetimeof transfer, thenonecouldconclude that in that sample replication had continued after transfer to 37 C. The results of one of these

ex-periments are shownin Fig. 4 and confirm that infectedcultures must bemaintained at 53 C for

atleast40minafter infectiontoobtainreplication

at 37 C in this experiment.

To determine how long anincubation at37 C wasrequiredtoobtain theirreversibleinactivation ofreplicating phage, infected culturesat53 Cwere

transferredto 37 Cfor2-minintervals atvarious times after infection, phageantiserum was

added,

and the culture was returned to 53 C. This time interval was used because it is just sufficient to

bring thetemperature of the medium from 53 to

37C. Theresultsdemonstrate that 2 min at37 C issufficient to prevent the lysis of infected cultures provided thattheexposure at 37 C occurs during

the first35minafterinfection.

DISCUSSION

The data presented above demonstrate that

there are at leat twobarrierstoTSP-1 replication

attemperatures below 50 C. One obstacle is the

absence of phage receptor sites on B. subtilis grown at 37 C or 45 C. This observation is

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./.

1 16

L~~~~~~~~~~

0 0

20 30 40 50 60

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TIME AFTER INFECTION (min)

FIG. 4. Effect of trantsfer from53to37 ConiTSP-I

replicationinB. subtilis W168. At T = 0, TSP-1 was

added(MOI = 5); after20miii ofincubationiat53C,

3.0-misampleswereremoved andin2cubatedat37 Cfor

1 hr. At theenid ofthle 37 C inicubationiperiod, these

sampleswereassayed forTSP-I. Thephagetitersafter

1hrat37 C(a) werecomparedtothe phage titers in

theoriginalculture at thetimeof tranlsfer(0) to37 C.

strongly supported byevidence obtained through

four different experiments. Kinetic data indicate thatphage adsorptionand phageDNAinjection

occurredonly with cells grown and incubated at

53C,ortoalesser extent, with cells that hadbeen

grownat53 C untilshortly (10min) beforephage

addition and then transferred to 37 C. Phage

DNA injection was shown not to be a

thermo-philic eventsinceit couldoccurwith 53 C grown

cells incubated at37 C. Electron micrographsof

infected cellsgrownat53 and 37Cconfirmedthe

absence of irreversible TSP-1 adsorption. Addi-tional evidence was provided by quantitative

measurementsof the percentphageadsorbedto

cellsgrowingat53, 37,or45 C. Thedisappearance

ofTSP-1receptor sitesmaybe another

manifesta-tion of the cell wallchanges observed by Dul and

McDonald (4) in B. subtilis growing at 53 and 37 C. They observed a thicker cell wall and a greaterthermal resistance in B. subtilis incubated at 53 C. Additionally, the thermal resistance in 37 C grown cells can be induced by antibiotics which inhibit the final cross-linking of the peptido-glycan. It is possible that the phage receptor site for TSP-1 isassociatedwith this non-cross-linked peptidoglycan. Young et al. have shown that the specificity for phage receptor sites resides in the glucosylation of the polyglycerol teichoic acid (11). Perhaps the absence of a cross-linked pepti-doglycan is required for the suitable exposure of phage receptors on the teichoic acid. Another

possibilityis that theteichoic acidpresentin cells grown at 53Cisqualitatively different from that at 37C.

Apparently the absence of TSP-1 receptor sites

on37 C grown cells is nottheonlyencumbrance tophagereplicationat37C. Our results reveal a temperature-dependentinactivation of replicating TSP-1. This becomes evident when one allows TSP-1 toadsorb to cells growing at 53 C, shifts these to 37 C, and observes the culture for evi-dence of TSP-1 replication. These resultsclearly

indicate that if infected cells are transferred to 37Cduringthe initial 30 to 35 minofinfection, theinhibition ofmaturephageparticle production occurs.Thistimeperiodissimilartothe observed latent period of TSP-1 inB.subtilis (8). One can offeratleasttwoexplanations for this effect. The

inhibition could be due to the presence of a

tem-perature-sensitive restriction mechanism.

Evi-dence hasbeenpresentedfor suchasystem in B. subtilis 168 by Gwinn and Lawton (6). They

observed thattwo serologically identical phages,

SP-10and SP-20, formed plaques onB. subtilis W23Srbutnot onB.subtilis 168.These

bacterio-phages could form plaques on B. subtilis 168,

however, if the bacteria were incubated at 53 C

for 10minbeforebeing infectedat37C. Further-more, their evidence suggested a temperature-sensitive repressor, as they were able to rescue

phagemarkersfrom infectedB.subtilis 168aslate

as 3 hr afterinfection. Ifthisrestriction mecha-nismplaysarole in theinhibitionof TSP-1 repli-cationat37C,thenpreincubationat53 Cshould be sufficient to allow phage

replication

at 37 C. This does not occur in TSP-1infections. The alter-nativeexplanationfor thistemperature-dependent

inactivation may be that TSP-1 istrulya

thermo-philic bacteriophage. That is, its phage-specific

enzymesareinactive below50C.If this is thecase

theneitherphage-specific DNA,ribonucleic acid,

or protein synthesis might be

temperature-sensi-tive.

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ACKNOWLEDGMENTS

This investigation was supported by Public Health Service

training grant STOl GM-00079 from the National Institute of General MedicalSciences.

We thank J. Oaks for assistance with electron microscopy. LITERATURE CITED

1. Adams, M. H. 1959. Bacteriophages. Wiley-Interscience Publishers,Inc.,New York.

2. Clowes, R. C., and W. Hayes, (ed.). 1968. Experiments in microbialgenetics.JohnWiley and Sons, Inc., New York. 3.Davison, P. F. 1963. The structure of bacteriophage SP-8.

Virology21:146-151.

4. Dul, M. J., and W. C. McDonald. 1971. Morphological changesandantibiotic-inducedthermalresistance in

vege-tativecells of Bacillus subtilis. J. Bacteriol. 106:672-678. 5.Eiserling,F.A.,and E.BoydelaTour. 1965.Capsomersand

otherstructuresobserved insomebacteriophages. Pathol. Microbiol. 28:175-180.

6. Gwinn, D. D., and W. D. Lawton. 1968. Alteration of host specificityinBacillus subtilis. Bacteriol.Rev.32:297-301. 7. Hershey, A.D.,and M. Chase. 1952. Independent functions

of viralprotein and nucleic acid in growth of bacteriophage. J. Gen.Physiol.36:39-56.

8. LaMontagne, J. R., and W. C. McDonald. 1972. A bacterio-phage ofBacillus subtilis which forms plaques only at

temperaturesabove 50 C.I.Physical and chemical

charac-teristics ofTSP-1.J. Virol. 9:646-651.

9. Spizizen, J. 1968.Transformation of biochemically deficient strains of Bacillussubtilis bydeoxyribonucleate.Proc. Nat. Acad.Sci. U.S.A.44:1072-1078.

10. Tikhonenko,A.S. 1970. Ultrastructure of bacterial viruses. Plenum Press, New York.

11. Young, F.E., C. Smith, and B. E. Reilly. 1969. Chromosomal location ofgenesregulating resistancetobacteriophage in Bacillussubtilis. J.Bacteriol. 98:1087-1097.

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