Coliphage 186 Replication is delayed when the host cell is UV irradiated before infection.

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JOURNAL OFVIROLOGY, Nov. 1981, p. 341-349 Vol.40, No. 2 0022-538X/81/110341-09$02.00/0

Coliphage 186

Replication

Is

Delayed

When the Host

Cell

Is

UV

Irradiated Before

Infection

IVAN HOOPER,t WALTER H.

WOODS,4

ANDBARRY EGAN* Departmentof Biochemistry, University of Adelaide, Adelaide, South Australia 5001

Received10November 1980/Accepted2July 1981

In contrast to

results

withinjections by A and P2, the latent period for

infection

by coliphage

186 is

extended

when

the host

cell is UV

irradiated before infection.

We

find

that 186

replication is significantly delayed

in such a

cell,

even

though

the

phage

itself has not been irradiated. In contrast,

replication

of the closely

related phage

P2under

the

same

conditions

is not

affected.

We

have previously

shown that the

prophage

186

is UV

inducible (26).

In

further studies we

observed

that the latent

period

was

appreciably

longer for UV induction of the

prophage

when

compared

with the latent

period

seenafter

pro-phage

induction

by

heat. Wereport these studies in

this paper

and show that the 186 life

cycle

is

extended in aUV-irradiated host and that the

delay isatthelevel of

replication.

MATERIALS AND

METHODS

Bacterial and phage strains. Strains C600 supE (1) and a tonA derivative of594

supo

(7), both Esche-richia coli K-12strains, and E. coli C strain C1055 (24)wereused,togetherwith thecoliphages186cItsp (2),186cIam53virl and 186Baml7vir2 (S. M. Hocking and J. B. Egan,manuscriptinpreparation), Xc+ and AindcIts857(20), P2 and P2vir22 (8), and theplasmids

pEC11andpEC13(10).

Media. LG broth (26), H-1 medium (14), and

TPGCAA,a Tns-basedminimal medium (17), have

beendescribedpreviously.

Determination of latent periods. (i)Infection. Log-phasebacteria, C600 inall cases, were grown in LGB at370Cto 2.5 x108colony-forming units (CFU) perml. Ifrequired,the bacteriaweresuspendedin H-1andsubjectedtoUV irradiation for 10 or 30 s (90 and 50%survival, respectively)under standard condi-tions; then0.1volume oflOxLGB added.Phagewere added at a multiplicity of 0.6, and the mixture was incubatedat370Cwithout aeration for5min(for P2, 10 min incubation in the presence of4mM CaC12). Adsorption under these conditionswasonly10 to15%,

givingamultiplicityof infectionof less thanorequal

to 0.1. The mixture was diluted 10-fold into LGB containing antiserumtothephageconcerned (K=1.5

min-1;

antiserumprepared against phage299 wasused against P2) and incubated5minat

370C

(free phage reduced 1,000-fold) before dilution103 and105into

tPresent address:DepartmentofAgricultural Biochemnis-try, Waite Agricultural ResearchInstitute, Urrbrae, South Australia.

tPresent address: School ofPharmacy, South Australia Institute of Technology, Adelaide, SouthAustralia.

LGB. These dilutions were shaken at

370C

and as-sayedperiodicallyforphage activityonC600(P2,on C1055) withoutpreadsorption.

(ii) UV induction. Log-phase C600lysogensof A and186 weregrowninLGBat37°Cto2.5x108CFU/

ml, suspendedinH-1, and UV irradiated for 10, 20,or

30 s.Thesewere immediately diluted

l0-5

and 10-7 intoLGB and shakenat

370C.

Sampleswereassayed

periodicallyforphageactivityonC600 without

pread-sorption. Free phage (that is, chloroform-resistant PFU)wereassayed immediately after the irradiation and foundtobeinsignificant.

(iii)

Heatinduction.Log-phase C600lysogens of

XindcIts857and186cItspweregrown inLGBat

300C

to2.5 x 108 CFU/ml. These were heat induced by

shakingat

450C

forminbefore dilution 10-5 and10-7 into LGB. The dilutions were shaken at

370C

and assayedperiodicallyfor phage activityonC600

with-outpreadsorption. Freephage (assayedasdescribed

above) wereinsignificantimmediatelyafter the heat

treatment.

UVirradiation. UV irradiationwascarriedouton 15ml of the bacterialsuspension,whichwasswirled

gentlyinapetridish(14-cm diameter) ina

370C

room,

50 cmfrom a 15-W General Electric germicidal lamp

delivering 1.5

J/m2

pers at254nm. Theculturewas

theninfectedasdescribedbelow, and incubationwas continued.TPGCAAwaseffectivelyUV transparent, andbacterial survival (5%after90J/m2) was no dif-ferent from that obtained with buffersuspensions.

Infection procedure. Bacterial cultures were

growninTPGCAA mediumat

370C

with aerationto

early log phase (1.4x 108CFU/ml)and UV irradiated

ifrequired.Thephage infectionwassynchronizedby

the addition ofphageto10ml of cultureat a multi-plicity of50for 186(20forP2)andbyincubation for 3miinat

370C

before removal of the unadsorbed phage by membrane filtration (Sartorius; 0.45 ,um, 42-mm diameter). The cellsonthefilterwerewashed with10 ml of TPGCAAat

370C

(threetimes)and recovered

byagitatingthe filter2to3min in 10ml of TPGCAA

at

370C,

removingthefilter,andcontinuingthe incu-bation.Unadsorbed phagewerereducedtolessthan

107 PFU/ml bythisprocedure.For186infection,65%

of the cellsregisteredas infectious centers, andthis couldnotbe increasedbytheuse ofhighertitersof 341

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342 HOOPER, WOODS, AND EGAN

infecting although the number ofbacterial survivors decreased from the 9% colony formers found at a multiplicity of50. For P2 infection, only30% ofthe cellsregisteredasinfectious centers,andthis was not increased withhighertitersofphageatinfection.The percentages of infectiouscentersforboth 186andP2 infections of UV-irradiated bacteria were similar to those for infections ofnon-irradiatedbacteria.

DNAsynthesis. Therateof DNAsynthesisin an infected culturewasmeasuredbypulse-labelingwith [3H]thymidine at various times during infection and determining the trichloroacetic acid-precipitable countsincorporated. Attminafterinfection,a0.2-ml sample of the infected culture wasaddedto 50

[1I

of prewarmed TPGCAA containing 2.5ttCiof [3H]thy-midine (21 Ci/mmol), andincubation was continued at37°C for90 s(at maximal bacterial density, incor-poration waslinearfor>4min). A

50-A1

samplewas withdrawn andspottedonto aWhatman GF/A filter, and thefilterwasdroppedintocold10%trichloroacetic acid. The filterswerebulkwashedwiththreechanges of 10% trichloroacetic acid, then with redistilled ethanol, and finally with diethyl ether. After drying, the filters were counted in a Packard scintillation spectrometer, using 2,5-diphenyloxazole-1,4-bis-(5-phenyloxazolyl)benzene (PPO-POPOP) scintillation fluid. Background counts deducted (50 to 100 cpm) weredeterminedby usingchilled cellsand an incuba-tion temperatureof40Cin the aboveprocedure. The representation of phage and bacterial DNA in the DNA synthesized duringapulse wasdeterminedby DNA-DNAhybridization.

DNApreparations.(i) PhageDNA.PhageDNA

waspreparedby suspending1013purified(CsCl density gradient) phage in 10ml of1 Msodium perchlorate followedby three extractions withanequalvolume of 4% isoamyl alcoholin chloroform. The phage DNA was thendialyzed against TE buffer (10 mM Tris, 1 mM EDTA [pH 8.0]). 3H-labeled 186DNAwas pre-pared by nick translation (19).

(ii) Bacterial DNA. A 500-ml culture ofW3350 was grown inLGat37°Cto 3x108CFU/ml,thecells werecollectedby centrifugation, suspended in 20ml of 0.5% sodiumSarkosyl (Geigy)-10mMNaCl-10mM Tris, pH 8.0-10 mM EDTA, 6 mg of pronase was

added, and cellswerelysed by incubatingat37°C for 3h.Nucleic acidwasextracted withredistilledphenol (three times), ethanol precipitated, and resuspended in10mlof TEbuffer,2mgofpancreatic ribonuclease (preheated at 80°Cfor 10 min) was added, and the mixture was incubated at 37°C overnight. After a furtherphenol extraction, the DNA solutionwas di-alyzed againstTEbuffer.

(iii) 3H-labeled bacterial DNA. W3350 (10 ml) wasgrown inTPGCAAmedium at37°C to 1.2x 108 CFU/ml,

80,uCi

of[3H]thymidine (50Ci/mmol) was added, and incubation at 37°C wascontinued for 5 min. A10-mlsampleof 2% sodiumdodecyl

sulphate-0.1MEDTA(pH 8.5) containing50yg of NaCN and 1 mg lysozyme was added, the cells were lysed by incubatingfor 10 min at 65°C, 22 mg of pronase was added, and the mixture was incubated ovemight at 37°C.Afterisoamylalcohol-chloroformextraction, the nucleic acidwasprecipitatedwithethanol,suspended in 0.1x SSC (standard saline citrate: 0.15 M NaCl,

0.015Mtrisodium citrate[pH 7.4]), treated with0.2N NaOH for20minto removeRNA,and then neutral-ized. TheDNA wasdialyzed against1mM Tris-1 mM EDTA (pH 8.0) and then concentrated 10-fold by rotaryevaporation.DNAwith aspecific activityof105 cpm/yg wasobtained.

(iv) Plasmid DNA. PlasmidDNA waspreparedas described byFinnegan and Egan (10).

Preparation of DNA filters. Nitrocellulose filters (Sartorius; 9-mm diameter)weresoaked inwaterfor 20min. DNA inTEbufferwasdenaturedinboiling waterfor5min and rapidlycooled to4°C, and SSC was added to give afinal concentration of 6x SSC. The denaturedDNA wasloadedontofilters by placing thefiltersonWhatman3MMchromatographypaper, adding5ygofDNA(in less than50

pi),

andallowing the 3MMpaper todraw thesolutionthrough the filter. The filterswerewashedbriefly with2xSSC, driedat 37°C for30min, and thenbakedat80°C undervacuum for2h. Beforehybridization, the filterswere preincu-batedat68°C for6h inDenhardtsolution in2xto 3x SSC (9).

DNA-DNA hybridization. Hybridization of la-beled DNA to DNA immobilized on nitrocellulose filterswasusedto measurespecificDNAspecies syn-thesized afterphageinfection ofbacteria. Hybridiza-tionswerecarriedoutwith DNAisolatedfrominfected bacteria (method A) and with alkali extracts of in-fectedcells(method B).

(i) Method A: hybridizations with purified

DNA. A 10-ytCi amount of [3H]thymidine (21 Ci/

mmol) wasaddedto 10ml oftheinfected cultureat the required time, and 4 min later, the DNA was extracted asdescribed abovefor the preparation of 3H-labeled bacterialDNA.After ethanolprecipitation, the DNAwasdissolvedinTEbufferat 50,ug/ml,0.2 volof5NNaOHwasadded,thesolutionwas heated at94°C for 8min and quickly cooled, and anequal volume of5N HCIwasadded, followed by SSC and sodiumdodecylsulfatetofinally giveDNA(0.5

/ig/75

pl)

in 2x SSC and 0.5% sodiumdodecyl sulfate. The pH wasadjusted, ifnecessary, totherange of 6.5 to 7.5, and0.01volume of1MTris(pH8.0) wasadded. A

75-p1

of thissample solutionwasaddedto avial (10-mm diameter) containing a filter loadedwith DNA, totally immersing the filter, and0.2mlofparaffin oil wasaddedtopreventevaporation.After 24 hof incu-bationat68°C, the filterwastransferredto5ml of2x SSC-0.5% sodium dodecyl sulfate. The filters were

finally dried in vacuo for 12 h and then counted. Hybridizationswerecarried out intriplicate.

The efficiency of hybridization was assessed by determiningthe extentofhybridization after incubat-ing0.5yg ofpurifiedlabeled DNA with a filterholding

5 ,ug of eitherpurified bacterial orphage DNA. 186 DNA showed 39.3 and 42.0% hybridizations to its homologous DNA, and bacterial DNA showed 21.3 and 25.6% to its homologous DNA. The two DNAs showed no cross-hybridization (<0.5%). In an un-knownsampleof DNAisolated fromaninfectedcell, therefore,weassumedthat the amount ofphageDNA was 2.5times the countshybridizedto thephage DNA filter (less background) andthat the amount of bac-terial DNAwas 4.3times thecountshybridizedtothe bacterial DNA filter.

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186 REPLICATION DELAY IN UV-IRRADIATED CELI 343

(ii) Method B: hybridizations with alkali ex-tracts.Thefollowing procedure,describedby Kuem-pel(16), allowed theavoidingof DNA extraction. A

4-mlsample of culturewaspulse-labeled (10 jiCi of [3H] thymidine) at different times after infection. Thepulse wasterminatedbythe addition of 1 volume ofchilled TE buffercontaining 10 mM NaCN. The cells were

collectedbycentrifugation,washed twice with TEplus NaCN buffer, andfinally resuspendedin 0.6volume of TEplus NaCN buffercontaining 3%sucroseand 100

,.g

oflysozyme perml.After40minof incubationat

4°C, 0.1 volume of5 N NaOH was added, and the solutionwasheatedat94°C for8minandneutralized by the addition of0.1volume of5 NHCland Tris(pH 7.0)to mM. Thiswasdiluted 10-foldwithDenhardt solution in 3x SSC-10 mM Tris (pH 7.0).A

500-pd

sample of this DNA solution wasadded to a DNA filter in a vial (10-mm diameter) and overlaid with paraffin oil. After24hofincubationat68°C,the filter waswashed with threechanges of3mMTris(pH 9.4) at room temperature, dried,and counted. Hybridiza-tionswerecarriedoutintriplicate.

The efficiency ofhybridizationwithalkaliextracts wasdeterminedby the addition ofaknownamountof labeledDNA to an infected cultureimmediately be-fore alkali extraction. Theextract wasthen incubated with5ugofDNAon afilter,andthecountshybridized wereassayedasoutlined above.186DNA showed45.0 and 48.9%hybridization ofcountsadded,andbacterial DNAshowed 7.7and 12.4% hybridization. Inathird series,abacterialculturewaspulse-labeledand alkali extracted, and hybridization efficiency to bacterial DNAfilterswasdeterminedtobe9.2and 14.0%. Cross-hybridizationswere again negligible. Inan unknown alkaliextractfromapulse-labeled infected cell, there-fore,weassumed that theamountofphageDNAwas 2.1 times the countshybridized to the phage DNA filter (lessbackground) and that the amountof bac-terialDNAwas9.3times thecountshybridizedtothe bacterialDNAfilter.

RNA synthesis. RatesoftotalRNA synthesisat

various timesthroughout infectionwere measured by pulse-labeling and determining trichloroacetic acid-precipitable counts.Attmin afterinfection,0.2ml of the culturewasaddedto50

pi

ofprewarmed TPGCAA containing 1,uCiof[3H]uridineand incubatedat37°C for 2 min. Trichloroacetic acid-precipitable counts werethenmeasuredasdescribed for DNA synthesis.

RNAextraction. Attminafter infection,4.5mlof the infected culture waspulsedwith [3H]uridine (30 uCi for non-UV-irradiated culture; 180,uCi for UV-irradiated culture) for 2 min, and incorporation was

stopped bythe addition of5mlofice cold TPGCAA

containing20mMsodium azideandchilling.The cells werecollected by centrifugationandsuspended in 1.5 ml of 0.15 M NaCl-10 mM EDTA-0.5% sodium do-decyl sulfate-1mM Tris(pH7.7). Thecellswere lysed by heating at 97°C for 3 min, and the lysate was cooled.The RNA was extractedbyshaking for 3 min with2mlof water-saturated phenol at 60°C. The hot phenolextractionwasrepeatedtwice.The RNA was precipitatedwith ethanol andsuspendedin 0.2 ml of 1.4Mpotassiumphosphate buffer, pH6.8.

RNA-DNAhybridization. (i) Liquid. Hybridiza-tion in 70%formamidepermitsRNA-DNAassociation

without DNA-DNA reassociation (21). T-- 'Llvbridi-zationmixture contained 20yilof RNA evrract, 7,5,lI offormamide, and 2.5,ugof 186 DNA ini11Vlf 0.7 M potassium phosphate, pH6.8.This was heated at 90°C for5 min and then incubated at 45°C for 5 h. Two milliliters of3xSSCwasadded,and 100tdwas with-drawn to determine trichloroacetic acid-precipitable counts.Pancreatic RNase A (40

[Lg)

wasadded to the remainder, and incubation at 37°C was continued for 8 min before determination of trichloroacetic acid-precipitable counts. Nonspecific background RiNase-resistantRNA wasdetermined for each RNA prepa-ration by carrying outthe procedure with no added DNA.

(ii) Filter. Inliquid RNA-DNA hybridization, the nonspecific backgroundwas, on the?verage, 0.65% of thecountsadded. In anassay ofR, 'iybridizing to afragment of186present as a cloned nseettn pBR322, this level was severalfold higher than the level of countsexpectedtohybridize.Wetherefore usedDNA immobilizedon anitrocellulose filterfor RNA hybrid-ization (4), where the backgroundwassmaller. DNA filterscarrying (a) 0.5 pgofpECII DNA, (b) 0.4 ,ug of pEC13 DNA,or (c)noDN ^ were preparedessentially asdescribed above (howlver, without preincubation inDenhardtsolution). The amount of186 DNA car-riedonfiltersa and b was equivalent to at least 1.5

jig

ofcomplete 186chromosomalDNA perfilter. Fil-tersa, b, andc were added to 2 ml ofhybridization buffer (8% phenol in 2x SSC) containing 100,ul of RNA extract and incubated at 65°C for 20 h. The filters were then washed at room temperature by incubation in 2x SSC for 1 h, treatedwith 40 of pancreatic RNase in2 ml of 2x SSC,

wihtcd

in 2X SSC for 1 h, and finally washed twice with

-h..

.l. The filtersweredriedand counted.

RESULTS

Latent

period

of

phage

product

-.

Whereas in thecaseof Ait wasknown that the

latent

period after UV induction was a little

longer than that after heat

induction,

we had

noticed that the difference in thecaseof 182was

most marked. This is seen in Fig. 1,where the

latent

periods

for

phage

production

after

infec-tion and heat

induction, compared

with that

after

UV

induction,

areshown. For

X,

the latent

period

after UV induction (65

min)

was 30%

longer

than that after either infection or heat

induction (50 min).

However,

for186, the latent

period

after UV induction

(87 min)

was 135%

longer

than that after either infection or heat

induction(37 min). We therefore

suspected

that

the UV-irradiatedhost mustbe less

hospitable

to 186infection thantoX infection.

Latent

period

of

phage

infecting

UV-ir-radiated bacteria. The latent

period

forX

in-fection of host bacteria irradiated

(45

J/m2)

be-fore infection remained

unchanged

(50 min)

compared with infection of unirradiated host

bacteria(Fig. 2).Incontrast,thelatent

period

of

186 infection increased from 37minfor

unirra-VOL. 40,1981

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CL~

(L10

30 60 90 30 60 90 120

MINUTES AFTER INFECTION OR INDUCTION

FIG. 1. Latentperiod of 186 and A after infection (INF), heat induction (HI),orUV induction (UVI;dose;

308= 45J/m2).

186 P2

10~~~~~~~~~~~~~~~~

_.E

uJ

i

10-30 60 30 60 90 30 60

MINUTES AFTER INFECTION

FIG. 2. Latentperiod after infection by either A, 186, or P2 of C600 bacteria either unirradiated (0), or irradiatedfor20s(30J/m2;El)or30 s(45J/m2;X) before infection.

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[image:4.497.120.402.64.355.2] [image:4.497.117.405.394.650.2]

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186 REPLICATION DELAY IN UV-IRRADIATED CELL 345

diated host

bacteria

to 72

min

for irradiated

bacteria (45

J/m2).

The latent periods for

inter-mediate

doses showed

corresponding

increases,

namely,

44min for 15

J/m2

and 54 min

for

30J/

mi2.

The

latent

period for infection by

the

coli-phage

P2,

which is

closely related

to 186, was

unaltered

inthe

irradiated

host from that in the unirradiated host.

Effect of the

UV-irradiated

host on 186

DNA

synthesis.

We first

determined the extent

of DNAsynthesis in the

non-irradiated

cell after

186

infection

by

pulse-labeling

atvarious times

throughout

the infection

(Fig. 3).

The

washing

procedure

temporarily depressed

the ability of

uninfected bacteria

to

incorporate

[3H]thymi-dine but this

had

normalized

by

min 15.

After

infection by

186,

the

rateof DNA

synthesis

was

initially depressed, increased later

in the

infec-tion to a maximum at about 25

min,

and then

(A

LuA

z

I-z U)

4 z 0

0

Lu

4c

z

EI-z

xz

0 0

o L"

O <

Ji cc

20

40

60

80 MIN.

AFTER INFECT ION

FIG. 3. DNA synthesis incells infected with 186. W3350wasincubatedat37C in TPGCAA,and the rateof DNA synthesisatdifferenttimes after infec-tion with 186cIam53virl (0), or without infection (0), was determined bypulse-labeling (90 s) with

[3H]thymidineasdescribed in the text. The

propor-tionofDNA synthesized that was 186 DNA during a 4-minpulseat20to 24min after infection was deter-minedto be 54% by method A (average of four inde-pendentexperiments)and42% bymethod B (average ofthreeindependentexperiments) and is represented as (0). In these experiments, the linearity offilter hybridization was confirmed when the sample size was doubled. The appearance of progeny phage is plotted (X).

again

fell concomitant with the release of prog-eny

phage. DNA-DNA hybridization

studies

showed that 48% ofthe counts

incorporated

in

a

pulse

at

min

20 were

incorporateh

into 186 DNA

(see legend

to

Fig.

3).

UV

irradiation

of abacterial cell

(uninfected)

dramatically

inhibited

DNA

synthesis (Fig.

4a),

which then recovered at later times of

incuba-tion,

dependent

uponthe

dose. DNA

synthesis

in

the

UV-irradiated cells

infected

with 186was

similarly inhibited,

and

incorporation

at20

min

after

infection

wasless than 1%

(1.4

x 103

cpm/

ml)

ofthe value

obtained

for the

non-irradiated

infected

culture

(1.5

x

105 cpm/ml).

Since 48%

of theDNA synthesized at 20

min

afterinfection

of the unirradiated culture was 186 DNA, one

'SA'-1-sd'

[image:5.497.251.448.262.547.2]

20 40 60 80 100 20 40 60 MINUTES AFTER INFECTION

FIG. 4. DNA synthesis in UV-irradiated cells in-fected withphage. The rates ofDNA synthesis in culturesofW3350, unirradiated (0, 0) or UV irra-diatedat45(A, A)or90Jlm2(LI,*), weredetermined by pulse-labeling with [3H]thymidine at various timesduring incubation at 37°C as described in the text. The open symbolsrefer to uninfected cultures, andthe closed symbols refer to cultures infected with 186cIam53virl or P2vir22. (a and b) Infection with 186. (b) The data of (a) drawn on an expanded ordi-nate.(candd)Infectionwith P2.The dottedlines are a representation ofdata ofthe uninfected cultures from (a) and (b) in (c) and (d), respectively. (d) The dataof(c)drawnon anexpanded ordinate. VOL. 40,1981

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could

conclude

that 186 DNA was

inhibited

in the

UV-irradiated cells.

However,

the fact

that this

inhibition

ap-peared

tobe permanent was

inconsistent with

the

fact that

a

delayed

burst

of

progeny

phage,

necessitating phage

DNA

synthesis

at some

stage,

did

ultimately

ensue

(Fig. 3). When the

counts

incorporated

were

plotted

on an

ex-panded

scale, evidence of

incorporation

oc-curred,

with the maxima

displaced

to

later

times

with

increased dose (Fig. 4b). By calculating the

area

under the

curves to

provide

a

comparative

estimate

of total

DNA

synthesis,

and

assuming

that

half of the

DNA

synthesized during

5 to 35

min (= 1.2 x

106 cpm/ml)

intheinfected

unir-radiated cell

was 186 DNAand all

of that

syn-thesized

during

20 to 55 min in the

45-J/m2-irradiated

cell was 186 DNA

(=

1.9 x 105 cpm/

ml),

we

estimated that

the

drop

in 186 DNA

synthesized

was six- to

sevenfold,

whereas the

drop

in

phage burst

was

only

two- to threefold

(data

not

shown).

We

therefore suspected that

the

added

[3H]thymidine

was

being diluted

two-to

threefold

by cold thymidine during

recovery

after UV

irradiation

or

excluded from

DNA

pre-cursor

pools.

To

investigate this

possibility,

we

reasoned

that

infection

by phage P2,

a

phage closely

re-lated

to 186,

the

latent

period of infection

and

burst

size of which

is insensitive to

preirradiation

of the

host, should

show a

decreased number of

counts

incorporated

into its DNA when

infec-tions of

irradiated and unirradiated hosts

were

compared,

if

pulsing with [3H]thymidine

was

misleading

as a

comparative indicator of

rates

of

DNA

synthesis.

Thiswasindeed

the

case

(Fig.

4c

and

d).

A

total of

3.2 x 105

cpm/ml

were

incorporated

during

a90-s

pulse

at25min

after

P2

infection, and 7.1% (=

2.2 x

104 cpm/ml)

assayed

as P2 DNA

by

DNA-DNA

hybridiza-tion.

(In

contrast to 186

infection,

P2 infection

does not

lead

to an inhibition of

host

DNA

synthesis

[Fig. 3c],

andto

display

P2DNA

syn-thesis,

host DNA

synthesis

has

traditionally

been

depressed by using

a urvA host cell

pre-treated with

mitomycin [3].)

Wheninfection of

aculture irradiated for90

J/m2

occurred,

8.5 x

103 cpm/ml

were incorporated during a 90-s

pulse

at25

min,

and76.0%

(=

6.5 x

103 cpm/ml)

assayed

as P2 DNA

by

DNA-DNA

hybridiza-tion. As the burst sizewas

only

reduced

by 20%

(data

not

shown),

there

appeared

to be

effec-tively

a threefold dilution of the added label. This

figure

of threefoldwasconsistent with the

expectation

from the earlier186data. It was also

significant

that the counts

incorporated during

the first40min after infection of the irradiated

cell (90

J/m2)

were

significantly lower

for 186

infection than for P2 infection and

that

the

maximum DNA synthesis

after

186 infection had

shifted

markedly

to a

later

time.

In these curves, the decrease in rate of DNA

synthesis

later

in

infection

was considered a

reflection of either cell lysis

orthe inhibition of

phage

DNA

synthesis by

a

late

phage gene

func-tion. To

avoid such

a

complication with

186,and to

thereby

optimally display the delay in

186 DNA

synthesis,

a 186

Bam

mutant was used.

The

186

B

gene

controls late

gene transcription,

and cells

infected with

aB mutant

fail

to

lyse,

whereas the

rate

of

DNA

synthesis, presumably

that of

186

DNA, continues

to

increase

indefi-nitely (Hocking and Egan,

in preparation). The

results in Fig.

5ashow

the

rates

of DNA

synthe-sis

for cells infected with

186

Bam, and show

an

extensive inhibition of

DNA synthesis in

irradi-ated cells

during the first

50 min

of infection,

followed

by

a

substantial

recovery.

The striped

bars in

Fig.

5a represent

the proportion of

the

incorporated

countsthat is 186 DNA and

clearly

indicate

a

delay in

186 DNA

synthesis in the

UV-irradiated cell.

During

the

period

of

inhibi-tion,

the

irradiated

cells were capable of

display-ing P2

replication (Fig. 5b).

Effect of the

UV-irradiated cell

on 186

RNA

synthesis.

It

was

possible

that

the

tran-sient inhibition of

186DNA

synthesis

seen

when

the

phage infected

a

UV-irradiated cell

reflected

a

delay in

the

transcription of the

186gene

A,

which is essential for its replication.

We

there-fore investigated

186

transcription

in the

UV-irradiated cell.

To assaytranscription of gene A, we

used

RNA

hybridization

to the DNA of

pEC13,

which

is the

vector

pBR322

carrying

a

3.2-kilobase insert from the early region of

186

that

encodes

gene A

(186 coordinates

81 to

92%

[10]). For comparison,

weassayed hybridization

to

the

DNA

of

pEC11,

which

is

pBR322 carrying

a

6.5-kilobase insert from the late region of

186

encoding

the

tail

genes

G, H, I,

J, and

K

(186

coordinates

37 to

59%

[10]).

Total

RNA

synthesis

wasreduced some

50%

by

aUV

dose of

90

J/m2 and

wasthus

relatively

insensitive to UV irradiation compared with

DNA synthesis (data not shown). Also, total

RNA

synthesis

was extensive in 186-infected

cells,

both irradiated and non-irradiated,

al-though

the

patterns

varied

(Fig.

6a,

inset).

When

samples

were

analyzed

by RNA-DNA

hybridization

186,

specific

mRNA was

dramati-cally

depressed

inthe irradiated

cell

(Fig. 6a),

which

would

be expected, given that phage

rep-lication is needed forlategene transcription (11).

Accordingly, hybridization

to

pEC11

DNA of RNA isolated after infection was reduced for the

UV-irradiated

cell compared

with the

non-irra-diated

cell

(Fig. 6c). However,

what was more

significant

was the fact that the extent of hy-J.VIROL.

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(7)

E

C; CD

z

2

0 b

II

.

3--iI

N

22 EI

to

IL

20

40

60

80

100 MIN.

AFTER

INFECTION

FIG. 5. DNAsynthesis in UV-irradiatedcells

in-fected with the 186 B mutant. The rates ofDNA synthesisafter186Baml7vir2infection of W3350 str tonA were determined by pulse-labeling with

[3H]thymidine

atvarious timesduringincubationat 37°Casdescribedinthetext.Thecultureswereeither unirradiated

(0)

orUVirradiatedwithadoseof90

Jlm2 (U)

before infection. (b)Theordinate has been

expandedtenfold, and alsoplottedaretheratesof

DNAsynthesisfor UV-irradiated bacteria after P2

bridization

to

pEC13

DNA

for the

two cases was

not so

dramatically

different

(Fig. 6b).

We

con-cluded

that

transcription

ofthe

early region

was not

significantly restricted

in

the

186

infection

of

a

UV-irradiated

cell,

and therefore it

was not

the

lack of

transcription of

some

phage

gene

essential for phage

replication

that

led

to

the

inhibitory effect of the UV-irradiated cell

on

phage

replication.

DISCUSSION

We have found that when

186

coliphage

in-fects

a

bacterial cell

previously

UV

irradiated,

then the

appearance

of

progeny

phage is delayed

compared with

the

infection of non-irradiated

bacteria. For the doses studied here

(s90

J/m2),

UV-irradiated bacteria

do not

extend the latent

periods of

coliphages

Aor

P2,

although

Kellen-berger and

Weigle (15) did

report some

effect

on

X

infection for doses

exceeding

90 J/m2.

We

have

shown that the

onset

of

186DNA

replication is

delayed in the irradiated

cell,

which

probably

is a

sufficient

explanation for the extended latent

period.

Transcription of the

early region of

186,

which encodes

a

known

replication

gene

(Hock-ing and

Egan, in

preparation), is efficiently

tran-scribed, and

we

conclude

therefore that the

UV-irradiated

cell

has a

relatively

direct effect upon 186DNA

replication.

One

explanation could

be

that

186

replication

needs concomitant host DNA

replication

and

that its inhibition

by UV irradiation thereby

prevents 186

replication. Such

a

dependence

on

the host

DNA

is reminiscent of

phage

Mu

rep-lication (18) but in

the

limited

literature of 186

replication there

are no

parallels. Viable

int

mu-tants

of

186

exist (3),

in contrast to

Mu

(23), and

monomeric circles

(8) rather than

heterogeneous

circles (22)

appear

during infection.

In

fact,

186

replication

appears to

be

unexceptional. It is like

P2

in that it

replicates unidirectionally

(8),

ac-cumulates monomeric circles

(8), and requires

host

rep

function

(6).

The substrate for

DNA

packing

in

the

caseof P2

is the

closed

covalent

monomeric circle

(5), and the existence ofhybrid

phage,

possessing

P2

head

genes and 186

repli-cation genes

(10), indicates

that P2 heads will

recognize the end

product of

186

replication.

However,

only

one phage replication gene

has

been

identified for 186

(Hocking

and Egan, in

preparation),

whereas two have been described

for

P2

(12).

Furthermore,

in a short

communi-infection (X; redrawnfrom Fig. 4d) and for uninfected UV-irradiated bacteria

(5;

redrawnfrom Fig. 4b). The bars representthefraction of labeledDNA at that time that wasphage DNA, as determined by DNA-DNAhybridization (methodB, triplicate sam-ples).

VOL. 40,1981

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[image:7.497.59.240.70.578.2]

(8)

E E

v-J

02 g

20 40 60

*041

pEC

13

A--a

-

-U-*02F

*08

_pEC

₁₁

.06

*04

-U-w

.021F

20 40 60

p

_z_z__m

Ie

N-IL*-A- -4irI I

20 40 60

MINUTES AFTER INFECTION

FIG. 6. RNA synthesis after186infection of UV-irradiated and non-irradiated cells. A culture of W3350

waseither UVirradiated (90 J/m2)ornotandthen infected with186cIam53virl. Atintervals,portions of the cultureswerepulse-labeled with[3H]uridine,and the RNAwasextracted andhybridized with DNA from 186

(liquid RNA -DNAhybridization)andfrom pEC13 and pEC11 (filter RNA -DNAhybridization).pEC13 carries DNAfromtheearly region ofthe 186chromosomeandpEC11fromthelateregion. Theinsetshowsthe rates of RNA synthesisatvarioustimesafterinfection,measuredastrichloroacetic acid-precipitablecountsafter 2-minpulsesof[3H]uridine. Symbols:*, non-irradiated cells; *, UV-irradiated cells.

cation (13),we showthat 186 needs host DNA

initiationfunctions,whereas P2 does not.

If186replicationdoesnotrequireconcomitant

host DNA replication, then weappear tohave

a situation in which UV irradiation inhibits

DNAreplicationofatemplate that hasnotitself

been irradiated. A study ofthe reasonfor this

inhibition could afford us the opportunity to

characterize aspects of the UV inhibition of

DNAsynthesisother than thephysicalblockage

to themovement ofthepolymerase by the

py-rimidine dimer.Inaformalsense,thepyrimidine

dimers in the host cell DNA, generated by UV

irradiation of the cell before infection, have a

trans effectonthereplication ofanotherDNA

molecule, implying the involvement ofa

diffu-sible component(orlack ofit).Asamajor

inter-est ofour laboratory is induction of SOS

func-tions (25), wehopeto identifyanysoluble

mol-eculeappearingafterUVirradiation ofthe cell. For the present, our aim is to identify the

reason 186 replication isdelayed ina

UV-irra-diated host, and with that in mind, in a short

communication (13) wecommence a

character-ization of the host functions necessary for 186

replication.

ACKNOWLEDGMENT

We gratefullyacknowledge support from theAustralian Research GrantsCommittee.

LITERATURE CITED

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3. Bradley, C.,0.P.Ling,andJ.B. Egan. 1975.Isolation

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