Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

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JOURNAL OFVIROLOGY, Oct. 1971, p. 455-468

Vol.8, No. 4 Printed in U.S.A.

Heat

Induction

of Prophage 4105 in

Bacillus subtilis:

Replication of the Bacterial

and

Bacteriophage

Genomes

RICHARD W. ARMENTROUT AND LARS RUTBERG

DepartmentofBacteriology, Karolinska Institutet,S-10401Stockholm60,Sweden

Received for publication 14 June 1971

A temperature-inducible mutant oftemperate Bacillus bacteriophage 4105 was

isolated and used tolysogenize a thymine-requiring strain of Bacillus subtilis 168.

Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by

sucrose gradientcentrifugation and density equilibrium centrifugation of DNA

ex-tracted from induced bacteria. The distribution of DNA in thegradientswas

meas-ured by differential isotope and density labeling of DNA before and after induction

and by measuring thebiological activity of the DNA in genetic transformation, in

rescue ofphage markers, and in infectivityassays. At early times after induction,

but after at least oneround of replication, phage DNA remains associated with

high-molecular-weight DNA, whereas, later in the infection,phage DNA is

associ-ated with material ofdecreasing molecular weight. Genetic linkage between phage

and bacterial markerscanbedemonstrated inreplicated DNA from inducedcells.

Prophage induction is showntoaffectreplication of the bacterial chromosome. The

overallrateofreplication ofprelabeled bacterial DNA is identical in

temperature-induced lysogenics and in "mock-induced" wild-type 4105lysogenics. Therateof

replication of the bacterial marker phe-l (and also of nia-38), located closetothe

prophage in direction of the terminus of the bacterial chromosome, is increased in

induced cells, however, relativeto other bacterial markers tested. In

temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks

phage DNA synthesis, replication of both phage and bacterial DNA stops after

about 50% of the phage DNA has replicated once. The results of these

experi-ments suggestthat theprophage isnot initially excised in inducedcells,butrather

itisspecifically replicatedinsitutogether with adjacentpartsofthebacterial

chro-mosome.

The Campbell model (5) provides an elegant solutionto the

problem

of

integration

and

exci-sionoftemperate phage genomes.

Integration

is

proposed

to occur through a reciprocal recom-binational event between a

specific region

on a

circularized

phage chromosome and a

specific

attachment site on the bacterial chromosome. Prophage

excision

is thought to represent a reversal

of

the

integration

event. There isstrong

genetic

evidence for

specific integration

enzymes coded forby phagesP2(6),P22 (22),and lambda

(8),aswellasanother enzyme, xis, in lambda (11)

which together with the int product promotes excision ofa resident prophage. Excision is

be-lievedto be theprimaryevent inprophage

induc-tion. This is supported by experiments which

show, forexample, that genetic linkage between

bacterial markers bracketing the prophage

in-creases after prophage induction

(13)

or that

prophage

deoxyribonucleic

acid

(DNA)

can be recovered

unreplicated

in mature

particles

after heteroimmune

superinfection

(15).

The primary DNA productafter induction is thoughtto bea circularized DNA molecule

equivalent

in size to

mature

phage

DNA.

Presumably,

excision

of

lambda prophage

requires

protein

synthesis but notDNA

synthesis

(24).

DNA extracted from Bacillus

bacteriophage

4105

particles is a homogenous

collection

of

moleculesof lowinfectivity (3, 4, 18). Thepoor infectivity reflects some structural characteristic of the ends ofthe molecules

(17).

To study the structural

basis

forthe

biological

activityof

4105

DNA, we sought to isolate excised prophage DNA from

lysogenic

bacteria carrying a tem-perature-inducible mutant of

4105

as prophage. 455

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The results of these experiments indicate, how-ever,that prophage replication rather than

pro-phage excision is the primary event associated

with heat induction of 4105 lysogenic bacteria.

The experiments alsosuggest a common

mecha-nism of control of replication of both bacterial

andphage DNA in induced cells.

MATERIALS AND METHODS

Bacteriaandphage.The bacterial strainsemployed

arelisted in Table 1. Someofthesestrainswereused

as recipients in transformation experiments, and the

map order of the markers usedonthebacterial chro-mosomeisorigin-purA16-leu-2, leu-S, -ilvAJ-prophage ¢105-phe-J-nia-38-metB5-terminus (7, 16).

Phage

0105

and the temperature-sensitive (ts) and

suppressor-sensitive (sus) mutants employed have

recentlybeen described (2). As aconvenientmarker

of low background, susl9 in gene C was used in

markerrescueexperiments.MutationtsN31ingeneK

(2) isreferredtohereasKts3l.Ithasbeenpreviously

shown that geneK is essential for phageDNA syn-thesis (2). The temperature-inducible mutant cts23

was isolated after treatment of infected cells with

N-methyl-N-nitroso-N'-nitroguanidine (2). The cts23

mutation mapsclose to susil ofgene J (unpublished

data). Lysogenicderivatives ofT bacteria were

iso-lated from the central growthin plaques formedon

these bacteria at 30 C. The growth rates of T- and

its lysogenic derivatives are identical in the media

employed; in Min-CH (see below) at 30C, the

dou-blingtime ofthese strains is about 60min.

Media and growthofbacteria andphage. Bacterial

strains were maintained on Tryptose Blood Agar

Base (TBAB, Difco) plates. Bacteria were grown in

Spizizen's minimal medium (23) supplemented with

0.05%'o

casein hydrolysate (Difco) and 20 MAg(per ml)

ofany amino acid required and 10 ,ug ofthymidine

per ml when required. We refer to this medium as Min-CH. Phage assays and preparation of phage

stocksweredoneas described(2, 16, 18).

Assaysof the"biological activity"ofDNAsamples.

The relativeamounts of bacterialgenes, phagegenes,

andcomplete phage genomes present inDNA

sam-plesweremeasuredbytransformation, marker rescue,

ancd ttatisk;ction, respectively. For these

measure-metiEs, competentcells were obtainedby the method

ofAnagnostopoulosandSpizizen (1). Transformants

weteassayed byspreading0.1 ml of appropriate

dilu-tions of the competent culture on selective media

atterexposureofthe cellsto a dilutionofthe DNA

sample (2, 18). Inmarkerrescueexperiments,

compe-tentSR135 cellswereexposedtoaDNAsample,and,

20min after additionofDNA, thecells were

super-infected withan excessofmutantphage k105 CsusJ9.

Bacillus subtilis SR 135 carries a suppressor gene

which permits the superinfecting phage to grow and

recombine withphage DNA taken up bycompetent

cellsfromthe DNA sample. Priortolysis, the

super-infected cells were diluted and 0.1-ml samples were

plated with W168asindicator; W168doesnotpermit

growth of the superinfecting mutant phage. Details

ofthe methodhave recentlybeendescribed byus (2,

18). Assayofcomplete phagegenomes wasby

trans-fection as described (2, 18). Competent cultures of

SR135 were exposed to an appropriate dilution of

DNA sample, and prior to lysis the bacteria were

diluted and 0.1-ml samples were plated by using

SR135 as indicator bacteria. The number of plaques

that appearedontheplateswastakenas ameasureof

thenumber of whole "active" phageDNAmolecules

takenupbycompetentcells.

By use of these assays, the distribution of

trans-forming activity, marker rescue, and infectivity was

determinedoveragiven gradient.Thecompetenceof the different strains used in theseassays varied, and the results obtainedrepresentrelativeactivities rather

than specificactivities (e.g.,transformantsper

micro-gram of DNA corrected for constant competence).

The dataare presented onconvenient scalesto show

the distributions of activities overthe gradient, and

thescales neednotbe thesamefor allgraphs.

Inductionofheat-induciblelysogenicbacteria.

Lyso-genicbacteriaweregrownin20mlof Min-CHat30 C

toa density ofabout 5 X 107bacteria perml. For

prelabeling, 10,MCiof3H-thymidine (specific activity,

5Ci/mmole; The RadiochemicalCentre,Amersham)

wasaddedper20 ml inadditionto10,ugofcold

thy-midine per ml. The cells were centrifuged, washed

once with Min-CH, and suspended in0.5 volume of

fresh, thymineless Min-CH at 45 C. After 5 min at

TABLE 1. Bacterial strains

Strain Relevantproperties4 Source

W168 Prototrophic, suI J.

Spizizen

SR135 spoA9, trp-7, su+3 J. A. Hoch

BR26 trpC2, leu-2 J.

Spizizen

BR95 trpC2,phe-1, ilvAI J.

Spizizen

BR95 (l105

AtsJ5)

trpC2,

phe-J,

ilvAl lysogenic for l105

carrying

mu- Ourcollection tation tsNIS

BD25 purA16, leu-5, nia-38,metB5 A. Lindahl-Adams

168T- trpC2, thyAl, thyBI C.

Anagnostopoulos

aProperties:su+,carries thesu3suppressor gene; su-,

nonsuppressing,

spo,asporogenous; trp,

trypto-phan; phe, phenylalanine; ilv, isoleucine-valine; pur, purine

(adenine);

leu,

leucine,

nia, niacin, met,

methionine, thy, thymine.

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PROPHAGE

0105

IN B. SUBTILlS

thistemperature, the culturewasshiftedto42 C and

an equal volume ofprewarmed Min-CH containing

20 ,ug of thymidine per ml was added. For density

labeling at42C,thymidine was omitted and instead

theMin-CHcontained20 ,ugof5-bromodeoxyuridine

(BUdR) perml(Sigma Chemical Co., St.Louis, Mo.)

and '4C-thymidine (specific activity, 58 mCi/mmole;

The Radioactive Centre, Amersham) to give afinal

concentration of 0.25 MCi per ml. Thebacteria were

incubated at 42 C with aeration, and samples were

withdrawn for phage and bacterial assays and for

extraction of DNA. T- bacteria lysogenic for

0105

wild type are not induced by temperature shifts.

WhenT-

(0105)

wastreatedasdescribed above, it is

referredto asmock-induced.

Extraction of DNA. Culture samples containing

2 X 108 to5 X 108 bacteria werepouredoverfrozen

Min-CH andcentrifuged.Thepelletsweresuspended

in2 ml of 0.2 M NaCl-0.001 M

ethylenediaminetetra-aceticacid(EDTA) pH8(saline), lysozymewasadded

to afinal concentration of 200

,g,/ml,

and the

sus-pensionwasincubatedat37 Cfor5 to10min. When

lysis occurred,sodiumdodecylsulfatewasaddedto a

final concentration of about 0.5%. Afterstanding a

few minutes at room temperature, 2 ml of washed

[0.1 M tris(hydroxymethyl)aminomethane

(Tris)-0.01 M EDTA, pH 8] phenol was added. DNA was

extracted bygentle mechanicalrolling of the sample

at roomtemperature for 30 min(3). Thesamplewas

thencentrifugedandthe aqueousphasewas removed

with a "U"-shapedPasteur pipette. The DNA

solu-tionsweredialyzedat4Cfor 16 hragainstonechange

of about1,000 volumesofsaline. 14C-labeledT7 DNA

was prepared asdescribed (3); 32P-labeled P2 DNA

wasagiftfrom Jon Jonasson.

Sucrose gradients. All sucrose gradients were 5 to

20% sucrose in 0.05 MTris(pH 8.0)-0.001 M EDTA,

made up from boiled-stock solutions. Gradients

werecentrifugedat 14CinaSpinco modelL

centri-fuge. The SW50rotorwas run at 35,000rev/min for

150min, whereasthe SW25.1 rotorwas run at24,000

rev/min for 180 min.Fractionswerecollectedthrough

a needle inserted into the bottom of the tubes.

Ten-dropfractions werecollected from the SW50

gradi-ents, and 45-drop fractions were collected from the

SW25.1 gradients. In the case of SW50 gradients,

0.05 ml from eachfractionwasaddeddirectlyto5ml

ofdioxane-based scintillation fluid (3) and counted

forradioactivity. Samples (0.1 ml) from the SW25.1

gradientswereapplied tosquaresof Whatman no. 3

filter paper, placedin cold 10% trichloroaceticacid,

washedoncewithcoldethanol,and thendriedunder

vacuum. The radioactivity on the filter papers was

counted in 5 ml oftoluene-based scintillation fluid

(3).Inall cases, thescintillation vials were precounted

and the background radioactivity for each vial was

subtracted from the sample counts. The biological

activityof thefractionswasassayed.

Equilibrium densitycentrifugation.To 2mlof DNA

samplewasadded1ml ofsaline and 3.8 g of CsClto

givea

56%7

(w/w) solution. The samples were

centri-fugedat14 CinanSW50rotorat 31,000rev/min for

about 64 hr. Fractions ofeight drops were collected

asdescribed above. The refractive index of every fifth

fractionwasread on anZeissAbberefractometer. In

each case, alineardensity gradientwas obtained.To

each fraction was added 1 ml ofboiled saline, and

0.5mlwasadded to 2 ml ofcold 10% trichloroacetic

acid. These samples were filtered through HA filters

(Millipore Corp., Bedford, Mass.). The filters were

dried in air and counted forradioactivity byusinga

toluene-based scintillation fluid (3). The biological

activityof thesamples was assayed.

RESULTS

Heat induction of4105 cts23. When T-

bac-teria lysogenic for the 4105 mutant cts23 are

grown at 30 Candthen shiftedtoinducing

tem-perature, anincreased phage DNA synthesis is

seensome10minafter the shiftand afteran

addi-tional20 to 30 min the bacterialyse andliberate

aburstof phage (Fig. 1).

The 4105 cts23mutant wascrossed with4105

Kts3l and adouble mutant carrying both ofthe

ts mutations was isolated. This double mutant wasthenusedtolysogenizeT- bacteria to give the lysogen

T-(q605

cts23, Kts31). Mutation

Kts3J

is located in gene K which has previ-ously been shown essential for phage DNA synthesis (2). After a shifttoinducing

temper-ature,

T(cts23,

Kts31) does not produce any

phageas longasit iskept at the high

temper-ature. Samples of DNA extractedfrom such a

culture afterthe temperature shift show no or very little increase in specific infectivity when assayed by transfection of competent SR135 cells (Fig. 1). The Kts31 mutation thus effect-ively blocks phage DNA synthesis also after

temperatureinduction of

lysogenic

bacteria.

Irreversible induction of4105 cts23 lysogenic bacteria alsooccursintheabsence ofDNA syn-thesis;

T-(q505

cts23) is rapidly killed in the absence of thymine when shifted to inducing

temperature. The

viability

of T lysogenic for

wild-type

4105

is not affected under the same conditions

(Fig.

2). When cells from tempera-ture-induced and

thymine-starved

cultures are plated with indicator bacteriain the presence of

thymine,

there is no decline in the number of

infectious

centersformed duringa30-min incuba-tion

period

without thymine (Fig. 2). Thus, the lack ofthymine aftertemperatureinduction does not damage the ability of the cells to produce

phage when returned to a thymine-containing

medium. On the other hand, there is evidence

that damageto theprophage does occur during incubation in the absence ofthymine. After 35 minwithoutthymine,thereis abouta 10-fold

de-creasein thespecific infectivity ofDNAextracted

from induced bacteria (Fig. 2). Marker rescue,

however,is not affected (not shown).

This damage to the prophage DNA must be 457

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in

z

0

.10

OA

z iC

VW

:3

10 20 30 40 50 60

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Minutes

FIG. 1. Temperature inductioni of T- (cts23) and

T(cts23, Kts31). The bacteria were grown to about

5 X107cellsperml,centrifuged,andsuspendedinfresh

medium at 42C. Samples were taken for infectious

centers andfor DNA extraction. The purified DNA

samples were assayedfor their infectivity by using

competenit SR135; the infectivity is expressed as

plaquesper microgram ofDNA. Symbols: 0-- -0,

7(cts23) infectivity of DNA; U---0, T-(cts23,

Kts31) infectivity of DNA; O--O, T-(cts23)

inifectious centers; O--EO, T(cts23, Kts3I)

in-fectiouscenters.

reversible because the bacteria willproducephage

when supplied with thymine. In mock-induced

wild-type lysogenics, no or avery slightdecrease

in prophage DNA infectivity is observed when

the cells are starved of thymine. Thus, during

induction in the absence of DNA synthesis,

prophage DNA suffers reversible damage which

decreases itsinfectivity.

Sucrose gradients of DNA from induced or

in-fected bacteria. To investigate whether the

dam-agethatprophage DNAsuffersafterinduction in

the absence of DNA synthesis (thymine

starva-tion) reflects some excision process, DNA from

induced lysogenswasexamined insucrose

gradi-ents. It was expectedthat high-molecular-weight

bacterial DNA could be separated from the

smaller phage-sized piece, and the excision

processmightbe examined.

T-(cts23)wasgrownat 30 C inthepresenceof

3H-thymidine. The prophage was induced by

heat, and the bacteria were incubated at 42C

with or without cold thymidine. Thirty minutes

after induction, DNA was extracted and

sedi-2

c

.n106

0

t

U

c

E4

O105

c

p

0

vc T-(cts

-2 0 5 10 15 20 25 30

M nutes 35

z

0

Qm

:k

Z

nL

z

c

FIG.2. Effects of temperature iniductionz oil

T-lysogenic bacteria in the absenice (f thymine. The

bacteriaweregrownto adenisityof about5 X 107cells

perml, centrifuged, andsuispenidedinfresh medium at

45C. Samples were takeniforviable counts, infectiouis

centers, andfor DNA extraction.Symbols: -- ,

T(p105) viable cells (v.c.); ----, T-(O105)

infectivityofDNA; O--Q ,7(cts23) v.c.;*--*,

T-(cts23)inifectiouscenters (i.c.); *---0, T-(cts23)

infectivity of DNA; A--i\ , T(cts23,KtsN31) v.c.;

A--A, T-(cts23, Kts3J) i.c.; A---A, T-(cts23,

Kts31)infectivity ofDNA.

mented in neutralsucrosegradients together with

"C-labeled T7 DNA as a size marker. After a complete run, fractions were collected and as-sayed forradioactivity, forphageDNA activity, and for phe-l transforming activity. A similar experiment was also performed with DNA

ex-tractedfromT-bacteria infected with 4105cts23

at inducing temperature. The results of these experiments areshowninFig. 3a-c. IntheDNA

sample extracted from lysogenic bacteria

in-duced in the absence ofthymine, tritium

radio-activity and phage and bacterial DNA

sedi-ment together as

high-molecular-weight

species

(>200 x 106), indicatingthatprophageexcision

doesnot occurunder theseconditions (Fig. 3a).

When

T-(cts23)

lysogens are induced in the

presenceofthymidine, mostof the tritium

radio-activity is still associated with high-molecular-weightmaterial atthe time ofsampling,

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PROPHAGE c105 IN B. SUBTILlS

3Ht

1500H

10ooo

[image:5.489.45.237.70.506.2]

5 10 15 20 25 30 35 *0

FIG. 3. Sucrosegradientcentrifugation ofDNAfrom

induced cts23 lysogens or cts23-infected cells. The

bacteria were grown at 30C with prelabeling of the

bacterial chromosome with 3H-thymidine to about

5 X107cellsperml. Thebacteriawerewashedonceand

suspentdedin Min-CH (without any thymine) at45C for 5 min and then shifted to 42 C. After30 min at

inducing temperature, a sample was takenfor DNA

extraction. The purified DNA together with

14C-labeledT7DNAwascentrifugedinthe S W25.1rotor as

describedinMaterials and Methods. Thefractionswere

tested for 3H (0 ), 14C (X----X). phe-1+

transforming activity

(----

-0), rescue of phage

marker susl9 (0---0), and *for infectivity

(0--O). (a) T(cts23) with no thymine added

during the experiment. (b) T-(cts23) withz thymidine

ing no breakdown of preexisting DNA in the induced bacteria (Fig. 3b). Phage DNA active in the transfection or marker rescue assays is found

in three main peaks inthis sample. One peak of

infectivity and marker rescue cosediments with

the tritium peak. A second, more slowly

sedi-menting,

peak (fraction 29) corresponds to the infectious vegetative DNA previously

demon-strated in pulse-labeled DNA from mitomycin

C-induced c105 wild-type lysogenics (19). A

third peak which exhibits marker rescue only is

mature DNA (18, 19). Most of the phe-J

trans-forming activity cosediments with the tritium radioactivity, but a distinct peak is found at fraction 29. This peak of phe-J activity is con-sistently reproducible and must represent newly synthesized bacterial DNA. Inthe DNAsample from infected bacteria, finally, tritium radioac-tivity andphe-1 transforming activity cosediment as high-molecular-weight material, whereasmost

of thephageDNAactivitysediments moreslowly

and corresponds tovegetativeand mature DNA

(Fig.

3c).

Thus, 15 min after infection of T- bacteria

with phage cts23, there is little phage DNA

present in thehigh-molecular-weight DNAfrom

the bacterial chromosome. Therefore, the

rap-idly sedimenting phage DNA observed after

prophage induction is not a very large form of

vegetativephage DNA. Thepersistence ofphage DNAactivityamonghigh-molecular-weight bac-terialDNA even 30 minafterprophage induction

suggests that excisionof prophageDNAhasnot

occurred under these circumstances.

Also,

the experiments give no evidence for the existence of a uniquely phage-sized DNA early after pro-phageinduction

(see

alsoFig. 10a-c). Rather,the experiments suggest that excision may not be a

necessary early eventin induction and thatafter

induction and subsequent replication, phage

DNA may stillbe associated with bacterialDNA.

Replication of phage and bacterial DNA after

prophage induction. Experiments were next

per-formedto

study

DNA

replication

in temperature-induced

T-(cts23).

The bacteria were grown at

30 C in the presence of

3H-thymidine.

At a

den-sity of about 5 x 10' bacteria per ml, the cells were induced at 45C in theabsence of thymine. After 5 min atthis temperature, theculture was

shifted to42Candanequalvolumeofprewarmed

medium containing BUdR and '4C-thymidine

was added.Samples were then taken at intervals

from the culture, and DNA was extracted. The

addedwheen cellsshiftedto42C. (c) T-infectedwith

cts23ataneffectivemultiplicity of about 2.2; burstsize

was48.Infectivitynotshown inthefigure. Thissample

wastakenat15 minafterinfection.

VOL.8, 1971 459

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DNA was

centrifuged

to equilibrium in CsCl, and the fractions were assayed for radioactivity and for biological activity. In the presence of excessBUdR, phage DNA synthesis will occur, but no burst of infectious particles is produced. InFig. 4 is shown an experiment where DNA was extracted from T(cts23) at 0 and 20 min afterinduction, respectively.Inthe 0-min sample, radioactivity and

biological

activity band at

similar

densities,

about 1.703. After 20 min, about 11%of the prelabeled bacterial DNA has

replicated

ascalculated from the shift of tritium toa

position of

increased

density.

Although

only

a

fraction

of the

bacterial

DNA has

replicated

at 20 min in the induced cells, about 80%

of

the marker rescueactivity is found atahigher

density

thanthe

major

tritium peak

of

unreplicated DNA (the arrows in the figure

de-3H

2000- 200E

a.

E

(0 20 30 4

b)

2000 -200 200

!-i

100010MO100

notethe peaks of marker rescue in the gradient). In other words, phage DNA replicates more rapidly than the bulk of the bacterial DNAafter induction. The gradient fractions were assayed for their contents of the bacterial markersphe-J and leu-2 by transformation. Of these markers, about 45% of the phe-l transforming activity is associated with replicated material of increased

density,

whereas only about 10% of leu-2 trans-forming activity is found outside the main trit-ium peak of unreplicated DNA. The phe-l marker is linked to prophage 4405 by transforma-tion and transduction (14), whereas leu-2 is lo-catedfurther away from the prophage. The phe-l andthe leu-2 markers are replicated at the same rate in mock-induced wild-type lysogenic T or in temperature-induced T(cts23, Kts3l) (Fig. 5a-b). Thus, if the prophage is not induced by thetemperature shift employed or if the prophage

z

c a

Fia.4. EffectoftemperatureinductionofT(cts23)

on the replication of the bacterial chromosome. The FIG. 5. Effectoftemperature-inducingtreatment on

cells were grown and induced as described in Materials replication of the bacterial chromosome in

mock-and Methods and in text. DNA was extracted as induced Th(105)andinT(cts23, Kts3). Cells were

described andcentrifugedtoequilibrium in CsCl. The grown and treated as in Fig. 4. The fractions

ob-fractionsobtained were testedfor 3H(0 O), 14C tainedfrom the CsCl gradient were assayed for 3H

(0---0), phe-l+ transformingactivity (0 0), (0 O), 14C (0---0), phe-l+ transforming

leu-2+transforming activity (a---0),andrescueof activity (--), and leu-2+ transforming activity

phage marker susl9; thepeaks ofmarker rescue are (---0). The DNA samples were taken 20 min

indicatedbyarrows.(a)DNA taken 0minafter shifting aftershiftingofthecells toinducingtemperature. (a),

the cells to inducing temperature. (b) DNA taken 20 T (j4105).(b), T(cts23, Kts3J).Data areexpressedas

minafter shiftingthecellstoinducingtemperature. per centof total recoveredfrom the gradients.

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PROPHAGE

0105

TN B. SUBTILIS

carries

amutation

(Kts31),

whichprevents

phage

DNA

replication, in additionto thects23

muta-tion,

there is no

preferential

replication of the phe-l

region

over the leu-2region on the

bacterial

chromosome. Heat induction ofT(cts23) thus seems to be

associated

with

preferential

replica-tion of phage genes and withanalteration inthe pattern of replication of the bacterial chromo-some. At least one marker, phe-1, which is linked to the

prophage,

is

replicated

at an increased rate

compared

to another

marker,

leu-2, after prophage

induction.

3H 10000

80001

6000)

1C

X00

20

The effects of prophage induction on

replica-tion

of phage and bacterial DNA were next in-vestigated in the followingexperiments.

Tc(cts23)

and

T-(cts23,

Kts3W)

were induced in the

pres-ence

of BUdR, and DNA was extracted from each culture at various times after induction. Each DNA sample was

centrifuged

in a CsCl densitygradient.InFig. 6a-careshown the CsCl gradients for DNA from

Tr(cts23),

and in Fig. 7a-c, the gradients for DNA from

T-(cts23,

Kts31).

The DNAsamples from

T-(cts23)

were

10 15 20 25 30 35 40

FIG. 6. DNA wasextracted fromtemperature-induced Th(cts23) incubated with5-bromodeoxyuridine, as

de-scribed in Materials andMethods and centrifugedin CsCl. DNA wasextractedat 10min (a), 20 min (b),and

30min(c) after shiftingthecellstoinducingtemperature.Symbols:3H( );14C(0---0);unreplicated

DNA(LL);hybridDNA,replicatedonce(HL);heavyDNA,replicatedtwiceor more(HH).

|(a)

HL LL

I

_o--Cl-10 15 20 25 30 35 40

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3H 1000

800

600 400 200

3H 1000

500

3H

1000

500

0o

20 30 40

FIG. 7. Same experiment as shown in Fig. 6, but

performedwith 7(cts23, Kts31). Symbolsidenticalto

thoseof Fig.6.Sample (c) wasextractedat40min.

also centrifuged in linear sucrose gradients (Fig.

lOa-c).

The fractions from the CsCl gradients were

pooled into fully light (LL), heavy-light (HL),

and heavy-heavy (HH) densitymaterial as

indi-catedinFig.6a-c and 7a-c. Thepooledfractions

were then assayed for radioactivity, for

trans-forming activity, for several bacterial markers,

and forphage markerrescueactivity.

Itshould be noted that the DNAwasextracted

so astominimizefragmentation ofthe material.

Asaresult of thelarge size of much oftheDNA,

e.g., Fig. lOa-c, some '4C-radioactivity appears

in the density gradients between LL and HL

density.

InFig. 8a-carepresented thedatafor the DNA

from induced T-(cts23). The curves represent

the relative ratesof replication of the particular

markersaswellastherateof replication of DNA

labeled with tritium before induction. For

in-stance, in Fig. 8a (per cent activity in LL

frac-tion), it is seen that phage marker rescue (the

lowest curve) leaves the unreplicated, LL, DNA

fraction ata muchfasterrate than, for example,

the terminal metBS transforming activity. By

scanning a fixed time such as the 10-min DNA

sample, it is seen that only 40% of phage DNA

activity (measured as marker rescue) remains

unreplicated (LL) at10 min after induction (Fig.

8a), and 60% of this activityappearsin the

once-replicated (HL) material (Fig. 8b), whereas no

activity is foundinthe HHmaterial(Fig. 8c). In

Fig. 8d-farepresented the results obtained with

DNA extracted from the induced Tr(cts23,

Kts31),toallowcomparison between thepatterns

of DNA replication observed after induction of

thetwo lysogens.

The results of theseexperiments,assummarized

inFig. 8a-f, show that prophage induction hasa

profound effect on DNA synthesis. Essentially

allphage DNA has replicatedatleastonce at30

min after induction of

T-(cts23),

and, of the

bacterial markers, at least 80% of the phe-l

transforming activity has replicated atthis time.

About25% of the phe-J activityisfound in

ma-terialreplicated twiceormoreat30min (Fig. 8c).

However, about 50% of purA16, leu-2, and ilvAl

transforming activity is still in the LL material

at30min after induction (Fig. 8a), and only 10%

of the totalactivityof these markers is foundin

the HH material (Fig. 8c). The terminal metBS

marker replicates verylittle (about 10%) during

theexperiment, and noactivity ofthis marker is

recovered in the HH material. Thus, preferential

replication of phage DNA andthephe-1 marker

isconfirmed.

Itshould be noted that only 25% of the

pre-labeled DNA has replicatedat30min (Fig. 8b),

whereas somephage DNAandphe-l

transform-ing activity is found in the fraction replicated

twiceormore. Thiscould notoccuriftherewere

notpreferential local replication of the bacterial

chromosome. We havealso observedpreferential

replication of the bacterial marker nia-38,

lo-cated closeto phe-1 indirection of the

chromo-somal terminus (Table 2). Thus, preferential

replication of bacterial markers is not peculiar

tothephe-1 marker.

Induction ofTr(cts23, Kts3l) gives a totally

different pictureofDNAreplication (Fig. 8d-f).

About 50% of the phagemarker rescue activity

is found in the once-replicated (HL) fraction at

10to 20minafter inductioncompared toabout

20

Ib, Ic)

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PROPHAGE

0105

IN B. SUBTILIS

T-(cts 23) T-(cts 23, Kts31)

I .iI, I a. . *I I

10 20 30 L0 10 20 30 40

Minutes

FIG. 8. Rateof replication ofbacterialmarkers, phagemarkers, andtotal DNAaftertemperatureinduction of

T(cts23) and T(cts23, Kts3J). ThebracketedfractionsinFig.6 and 7werepooled, dialyzed overnight against

saline,andassayedforbiological activity.The dataaregivenaspercentoftotalactivity recoveredfromthegradient

inagiven timesample. Ontheleftisshown T(cts23) unreplicated (a),oncereplicated(b),andDNAreplicated

twiceor more(c).On therightisshown T(cts23, Kts31) unreplicated (d),oncereplicated(e),andDNAreplicated

twiceormore(f). Symbols:3H(O O),purA16+ transforming activity (O---0), leu-5+ activity (o D),

ilvA1+activity (X X),phe-l activity ( 0),metB5activity (0---0),rescueof phagemarkersusl9

(

--

-*)

15% for the bacterial markers purA16, leu-5,

ilvAl, phe-1. Thus,in the presence of the Kts3J

mutation, preferential replication of phage

DNA still occurs in the induced cells, although

at areduced rate. However, preferential

replica-tionof thephe-l marker isabolished, andphe-J

is replicated at the same rate as other bacterial

markers (Fig. 8d). Twenty minutes after

induc-tion, replication of both bacterial and phage

DNA virtually comes to a stop in T-(cts23,

Kts3J) and is not resumedduring theremaining

20minof theexperiment (Fig. 8e-f).

Prophage inductionhaslittleeffect ontherate

ofsynthesisof totalDNA, althoughit hasa

pro-100

80

60

40

20

4'0

4'

0-80

60

40

20

40

20

0

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TABLE2.Replicationofbacterial andbacteriophage

DNA in heat-inducedT(cts23)G

Percentmarkeractivityin DNAreplicatedonce

Time of or more

DNA

extraction

(min) purA16 leu-5 nia-38 metB5 s_SUS19+105

10 20 23 42 7 59

20 37 30 74 14 81

30 41 43 71 14 97

aDNA extracted from induced

T(cts23)

and

described inFig.6,8,and 10wasusedtotransform

BD25.

found effect on the rate of

replication

of indi-vidual

portions

of the bacterial chromosome

(Fig.

9). T-(105), T(cts23), and

T(cts23,

Kts31) were

induced,

or mock

induced,

in the presence of BUdR.

Samples

were taken at intervals for DNA

extraction,

and the DNA was then

cen-trifuged

in CsCl. Pooled

density

fractions from

the

gradients

were

assayed

as

described above.

InFig. 9a is shown therateof

replication

ofDNA

continuously

labeled with

3H-thymidine

for several

generations

before

induction.

In

Fig.

9b is shown the rate of

replication

(as

measured

by

shift to

higher

density)

of the bacterial

markers

leu-2and

phe-J.

Theresultsofthese

experiments

are in full agreement with those

presented

in

Fig.

8a-f.

During

the first 20minafter

induction,

the rate of

replication

of the

prelabeled

DNAis identical in the three

lysogens.

After this

time,

however,

DNA

replication

stops in

T-(cts23,

Kts3J). Only

in

T(cts23)

is there

preferential

replication of

the

phe-J

marker. The leu-2

marker

is

replicated

at a

higher

rate in

induced

T-(cts23)

compared

tothe

wild-type

lysogen,

although

their

rates

of

replication

of

prelabeled

DNA are

identi-cal,

suggesting

that

reinitiation

ofDNA

synthe-sis may occur after

induction

not

only

in the

prophage and the

phe-J

region

but

also,

less

fre-quently,

inother

places

onthebacterial chromo-some.

The DNA

samples

takenfromtheinducedT

(cts23) were

assayed

in

transformation

for

link-age betweenthe bacterial

phe-1

marker and the phage AtslS marker. A low

degree

of

linkage

could be detected evenin DNAreplicated twice or more

(Table 3).

No

linkage

was

observed

in DNA extractedfromT-cellsinfected withcts23 phage underconditions similartothose usedfor induction. SomephageDNAthusremains cova-lently bound to bacterial DNA after induction and

replication.

The DNA

samples

extracted from

T-(cts23)

and used in the

experiments

shown in Fig. 8a-c

100

10so

a

u i0

c30

11I20

0 10 20 30 40 0 10 20 30 40

Minutes

FIG. 9. Rateofreplicationof the bacterial

chromo-some after temperature induction of li(cts23),

T-(cts23, Kts31), and mock induction of T(.105).

BacterialDNA wasprelabeled with 3H-thymidineawd

induced in the presence of BUdR as described in

Materials and Methods. DNA was extracted 10, 20,

and 30 min after induction and centrifuged in CsCl.

The gradientfractions were pooled as described for

Fig. 6 and 7. Eachfractionwasassayed for its content

of3H-prelabel (a) andforphe-J+andleu-2+

transform-ing activity (b). Results are presented as per cent

activityoftotal, whichisfoundinreplicated DNA. (a)

Per cent 3H-prelabel in replicated DNA: T(4d105),

O--O; T(cts23), * *;

T(cts23, Kts3J),

X--X. (b) Percent transformingactivity in

repli-catedDNA,phe-1+:

T-(105),

0 O; T(cts23),

* *; T(cts23, Kts3l), X---X; leu-2+:

T(0105),

Ai A; 7(cts23), A A; 7T(cts23,

Kts31), +---+.

were also

sedimented

in neutral sucrose with

32P-labeled

P2 DNA asasize marker(Fig.

lOa-c).

With

increasing

time after

induction,

there is a gradual decrease in the

sedimentation

rate of phage DNA, with thefirstappearance ofmature phageDNA at 30 minafter induction.Inno ex-perimenthave wefoundanabrupt, early change inthe

sedimentation

characteristics of prophage DNAafterheat

induction,

such as

might

be ex-pected from an excision event. The relative

amounts of rescue and

infectivity

(Fig. 10c) differ

from those shown in Fig.

3b, probably

due to

theeffects of BUdR onthe

biological

activity of

the DNA (9).

DISCUSSION

Twoconclusionsemergefromtheexperiments presented. First, we do not observe excision of

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PROPHAGE

0105

IN B. SUBTILlS

TABLE 3. Linkagebetweenbacteriophage and bacterial markers in DNAfrom 4105lysogenicbacteriaa

Strainand treatment Fraction ts+/pkc-I+Transformnts

T-(cts23) heat induced (fractions shown in Fig.6) 10 minLL 9/104

HL 3/52

Unfractionated 9/156

Tr(cts23) heatinduced 20 min LL 0/70

HL

5/182

HH

0/80

Unfractionated

4/94

T-(cts23) heatinduced 30 min LL 0/52

HL 2/104

HH 2/80

Unfractionated 7/156

T-(40105)

mock induced 10 min 21/115

Unfractionated

T(r0105)

mock induced 40 min 17/104

Unfractionated

T infectedwith cts23 15min 0/298

Unfractionated

aBR95

(4105

AtslS)

wasgrown forcompetence as described.DNA extractedfrominduced

T(cts23)

anddescribed inFig.6, 8, and10 wasused totransform the cellstophe-l+.The distributionof thets

and ts+alleles of the AtslS markerwasthendeterminedamongthetransformants (14).TenminutesLL,

HL, andHHreferstothefractions described inFig. 6. Unfractionated means thatthe DNAis used

priortoany centrifugation.

prophage DNA in heat-induced

4105

lysogenic

bacteria. That

is,

the

experiments give

no

indica-tion

that both strands

of

the

prophage

are

pre-cisely

cut outof the bacterial

chromosome

prior

to autonomous

replications

of

phage

DNA.

Second

prophage

induction has a

profound,

but

covert,effectonthepattern of

replication

of the

bacterial chromosome.

The first

conclusion,

that

prophage excision

need not be an early eventin

induction,

is

sup-ported

by

several

experimental

findings.

In

su-crose

gradients

of DNA

from induced

bacteria,

the bulkofphagegenesand wholephagegenomes cosediment with

high-molecular-weight

bacterial DNA, even when most of the phage DNA has

replicated

at least once

(see Fig. 8b, lOa).

With

increasing

time after

induction,

phage DNA is found in more

slowly

sedimenting material,

with a concomitant decline in the amount of

phage DNA associated withprelabeled bacterial

DNA. Ininduced E. coli (lambda), mostofthe

early

synthesized DNA sediments in neutral

sucrose at a ratecharacteristic formaturephage

DNA (12, 20). Later in infection, this DNA is

converted to more rapidly

sedimenting

material,

which most

likely

represents covalently closed, circularphageDNA (12, 25). ThelambdaDNA

sedimentation pattern isthus the reverse ofthat

found for 4105. With thisphage,wehave never

observed DNA withsedimentation characteristics

of covalently closed circles either in induction, in infection, or in superinfected, immune, lyso-geniccells.

In DNAfrom4105lysogenics, genetic linkage

between phage and bacterial markers can be

demonstrated by transformation (14). The

de-gree oflinkagedecreases afterinduction of

lyso-genic bacteria, butsome

linkage

persistseven in DNAreplicatedtwo or more.

When DNA from T bacteria infected with

cts23 phage, under conditions similar to those

used for induction, is sedimented in sucrose, phage DNA sediments more slowly than, and well separated

from,

bacterialDNA. Nogenetic linkage between phage and bacterial markers is found in such DNA.

Phage genes do not leave the region of

bac-terial DNA inthe sucrosegradients when DNA

synthesis is blocked after induction (Fig. 3a). However, the lysogenic bacteria are fully

com-mitted to

induction;

whenthe cells are returned

to conditions permissive for DNA

synthesis,

morethan

90%

of them

produce

phage.

Although the prophage state is maintained in

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1s

11

3H

125

100

75

50

25

2000 1600*' 1200 c

X

Boo-800

I100

Jn

[image:12.489.61.249.67.541.2]

5 10 15 20 25 30

FIG. 10. Sucrose gradientcentrifugation ofthe DNA

samplesfrom 7(cts23), showninFig. 6,intheSW50

rotor. To the gradients wereadded0.15 ml of DNA

solution and 0.05 ml of appropriately diluted

32P-labeled P2 DNA. Symbols: 3H ( 0), 28p

(X---X),rescueofphagemarker susl9(O---0),

infectivity ofDNA (0 O). DNA samples were

takenat10min(a),20 min(b), and30 min(c) afterthe cellswereshiftedtoinducing conditions.

induced cells in the absence of DNA synthesis,

the infectivityofprophage DNA decreases. The

damage suffered by the prophageinthese

experi-ments is repairable, because in practice all

in-duced cells produce infectious phage when al-lowed to resume DNA synthesis. The thymine starvationdamage might be the result of abortive DNAsynthesis intheprophage region.

When the lysogen

T-(cts23,

Kts3J)

is induced

under conditions nonpermissive for gene K ac-tivity, phage DNAis preferentially copied once (Fig. 8d, e), whereas the bulk of the bacterial DNA remains unreplicated even 40 min after induction. The preferential replication of phage DNAindicatesthatsomemechanism operates in the induced cells, which can specifically initiate replicationoftheprophage.

Noextensivebreakdown of bacterialDNA oc-curseither upon induction of lysogenic bacteria orafter infection of sensitive bacteria. After in-fection, replication ofthe bacterial chromosome

seems to proceed in a normal fashion at least

during thefirst 30min.

Preliminary

experiments indicate no preferential effect on replication of phe-J ininfection.

Itshould bepointedoutthat therateof replica-tion of the bacterial

chromosome,

as measured by the transfer ofprelabeledDNAfrom lightto hybrid

material,

is identical in induced and in mock-induced T

lysogenic bacteria,

whereas the rate of

replication

of

phage

DNA, and at least the bacterial phe-J marker, ismore rapid in in-duced bacteria

(Fig.

9).

After induction of prophage cts23, bacterial

markers closer to the

origin

of the bacterial chromosomethan the

prophage,

replicate

at

simi-larrates. Marker

phe-J,

situated

just

distaltothe

prophage,exhibitsadifferentpattern. Itis

initially

replicated at a faster rate than other bacterial

markers,

and it appears earlier in

fully

heavy

DNA. Induction of the

prophage

thus leads to preferential

replication

not

only

of

phage

DNA but also of an

adjacent

part of the bacterial chromosome. A new initiation site for

replica-tion mustthenbe

exposed

ininduced cellsat or closeto the

prophage,

with

replication

proceed-ing

from this site in direction of the

terminus,

but stopping short of the terminal metB.

Frag-mentsofasize

exceeding

thatof

phage

DNAare

produced, as shown

by

sucrose

gradient

sedi-mentationaswellas

by

preserved linkage

between phage and bacterial markers in

replicated

DNA from induced

T-(cts23).

The

integrated

state of the

prophage,

in

itself,

generates an

important

element of the mechanism

controlling

replica-tion,

for induction alters the pattern of host DNA

synthesis,

whereas infection appears to

have no sucheffect.

Preferential

synthesis

of

adjacent

regions

of the bacterial chromosome after

prophage

induction

iH 32p

Xa)

50 300 i a

II It 200

II I

'K~~~~~~~

u

5 10 is 20 25 30

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PROPHAGE 4105 IN B. SUBTILIS

is not unique to the 4105 system. In lambda

lysogenic E. coli, there is evidence from DNA-DNA hybridization studies that replication of

the prophage may occur in situ after induction

andproceed into the gal operon (10). Such

repli-cation in induced lambda lysogenics has been

implied also in other types of experiments (13).

This regional replication appears to be under control of the 0andPcistrons.

Induction of integration-deficient (L) mutants

ofphageP22gives few infectiousparticles but a

substantial increase in proC andpurE transduc-ing particles (21). These markers are located

close to oneend of the prophage.Thedistribution

of phagegenes inthe defectiveparticlesisstrongly polar with genes distal to proC appearing with diminishing frequency. After induction of L

mu-tants of P22, replication thus seems to be

in-itiated at or close to the prophage and to

pro-ceed in the direction of proC. The mechanism

which ultimately produces phage-size DNA is unknown.

Ofparticular interest in the present context is

the defective phage PBSH carried by B. subtilis

168(9).PBSHisinducibleby mitomycin C. After induction, the relative amount of purA16 trans-forming activity increasesmanyfold due to pref-erentialreplication of this marker (9). However,

itcannot bedecided whetherpreferential

replica-tion of

purA16

isdueto

repeated

reinitiations at thebacterialinitiation site atthe originorto the

appearance of a new initiation site associated

with the phage. Other bacterial markers also replicate after PBSH induction albeit at a slow rate

compared

to purA16. Breakdown of the bacterial chromosome does not occur in the in-duced cells; even late after induction, purA16

containing

DNA exists in

pieces

atleasttwicethe size of the DNA found in the mature

phage

particles (9).

Our

experiments

indicate that there is more

than one step involved in separating bacterial

and phage genes after heat induction of 4105

cts23 lysogens. After

induction, phage

genes

replicatealthough theyare still

genetically

linked to bacterialgenes and appear in

large-size

DNA pieces.

Apparently,

thereisa

gradual

decreasein

the size of the DNA molecules which harbor

phage genes

during

thecourseof induction

(Fig.

10a-c). Rather than an early excision event, the

processof

separating

phageandbacterialgenes in

induced bacteriaseems to be

gradual,

withfinal definition

occurring

with the mature

phage

DNA.

Insummary, excision doesnot seemto be the

only mechanism

by

which a prophage can enter the phase of

vegetative growth

after induction of

lysogenic

bacteria. Whether induction

in-volves nucleases, recombinases, or polymerases, it requires recognition of the prophage region on the

bacterial

chromosome. Our data

indicate

thataDNAreplicationsystem mayrecognize the

4105

prophage after induction. Itwould appear,

then, that in our system the initial events in

lysogenic induction arefundamentally similar to

the processof normal replication ofthebacterial

chromosome, in that some control apparatus directs a DNA replication system to a specific chromosomal site for initiation of

replication.

On thebasisofthedatapresented, this control

apparatus cannot be defined. Itcouldinvolve the

unveiling of an initiation site at the prophage, which is blockedin theuninduced lysogenic bac-teria, followed by binding ofthe

DNA-polymeriz-ingsystem alreadypresent in the

cell

tothissite.

Or, the primaryevent ininduction may involve

some interaction between this

DNA-polymeriz-ing system and some (phage-coded) product

such thatthe specificity of the polymerizing

sys-tem is altered. Further

experiments

are now in

progress trying to resolve these

possibilities.

ACKNOWLEDGMENTS

Kerstin Nilsson provided excellent assistancethroughoutthis

work.

Theworkwassupported by grants from TheSwedishMedical

ResearchCouncil, Karolinska InstitutetsForskningsfonder, and

Emil ochWeraCornellsstiftelse. One of us (R.W.A.) holds a

postdoctoral fellowship from the American Cancer Society. LITERATURECITED

1. Anagnostopoulos, C., and J. Spizizen. 1962. Requirements

fortransformationinBacillus subtilis. J. Bacteriol.

81:741-746.

2. Armentrout, R. W., and L. Rutberg. 1970. Mapping of prophage and maturedeoxyribonucleicacid from temperate

Bacillus bacteriophage )105 by marker rescue. J. Virol. 6:760-767.

3.Armentrout, R. W., L. Skoog, and L. Rutberg.1971. Structure

andbiologicalactivity ofDNAfrom Bacillus bacteriophage

4105; effects of Escherichia coli exonucleases. J. Virol.

7:359-371.

4. Birdsell, D. C., G. M. Hathaway, and L. Rutberg. 1969.

Characterization oftemperate Bacillusbacteriophage4105. J.Virol. 4:264-270.

5. Campbell,A.1962.Episomes. Advan.Genet.11:101-145. 6. Choe,B. K.1969.Integration-defectivemutantsof

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7. Dubnau,D., C.Goldthwaite,I.Smith, and J. Marmur. 1967. GeneticmappinginBacillus subtillis. J.Mol. Biol. 27:163-185.

8.Gingery,R., and H.Echols. 1967. Mutantsof bacteriophageX unabletointegrate into the host chromosome.Proc. Nat. Acad.Sci.U.S.A.58:1507-1514.

9. Haas,M., andH.Yoshikawa. 1969. Defective bacteriophage

PBSH in Bacillus subtilis. II. Intracellular development of theinducedprophage.J.Virol.3:248-260.

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11. Kaiser,A.D., and T.Masuda. 1970.Evidencefor a prophage

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lar DNAafter lysogenic induction in Escherichia coli CR34 (X). J.Mol. Biol. 21:517-525.

13.Nishimune, Y. 1970. Prophage excision:onthestatusof host chromosome afterinduction.Virology41:541-548.

14.Peterson, A. M., and L. Rutberg. 1969. Linked transformation of bacterialandprophagemarkers inBacillus subtills 168

lysogenic for bacteriophage0105.J.Bacteriol. 98:874-877. 15. Ptashne,M. 1965. The detachmentandmaturationof

con-servedlambdaprophageDNA. J.Mol.Biol. 11:90-96.

16.Rutberg, L. 1969. Mapping ofa temperate bacteriophage activeonBacillus subtilis. J. Virol. 3:38-44.

17. Rutberg, L., and R. W. Armentrout. 1970. Low-frequency rescue ofa geneticmarker from Bacillus bacteriophage 0105bysuperinfecting bacteriophage. J. Virol. 6:768-771. 18.Rutberg, L., J.A.Hoch,andJ. Spizizen. 1969.Mechanism

oftransfection withdeoxyribonucleicacid fromthe tem-perateBacillusbacteriophage0105.J.Virol. 4:50-57.

19. Rutberg, L., and B. Rutberg. 1970. Characterization of

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