Bacteriophage SPO1 development: defects in a gene 31 mutant.

(1)

JOURNALOFVIROLOGY, Sept. 1978,p.483489

0022-538X/78/0027-0483$02.00/0

Copyright X1978 AmericanSocietyforMicrobiology

Vol. 27, No.3

PrintedinU.S.A.

Bacteriophage SPOl Development:

Defects in

a

Gene

31 Mutant

ALBERTO N. SARACHU, MARIA C.ANON, ANDOSCARGRAU*

FacultaddeCiencias Exactas,UniversidadNacional de LaPlata,1900 LaPlata, Argentina

Received forpublication21December 1977

SPOl temperature-sensitive mutant ts14-1, located in cistron 31, has a DD

(DNAsynthesis-delayed) phenotypeat370C and producesprogenyin astretched

program. At 440C it behaves as a DO (DNA synthesis-defective) mutant and

shuts off the viral RNA synthesis about 10 min after infection. The thermal

sensitivity of thismutantis duetotheinactivity ofgp-31(the productof gene31)

at440C. However, gp-31 issynthesized at that temperatureandpartlyrecovers

itsactivityat370C. Only5min atthepermissivetemperatureisenoughtotrigger

thecontinuation of thephage program and to produce progeny. Thepartial defect

at37°Candthe expansion ofthemiddleprogramtogether with thepleiotropic

defectsatthe nonpermissive temperaturecould besuitableforthe study of the

controlsinvolvedinbacteriophage development.

SPO1 is a large bacteriophage that infects

Bacillus subtilis. Itstranscriptionprogram has

beendescribed indetail (9, 10). Alimitedsetof

controlsseems toregulate its genetic expression

(8). At5minafter infection the bacterial

speci-ficityof thetranscriptiveapparatusis alteredby

virus-coded positiveregulators of transcription

(1, 12). This action is mediatedbyaproteinof

28,000molecularweightcodedbygene 28(24,

6, 7). A similarsituation, though less clarified

due to the lack of appropriate mutants, was

found for the related bacteriophage SP82 (19).

About 10min after infectionat

370C

several

importanteventsin the viraldevelopmental

se-quence takeplace. The synthesisoftwoclasses

ofviral RNA is shut

off,

athird class isamplified,

andafourth class is turnedon. DNA synthesis

also starts at that time. Thisstage of the

pro-gram is less understood, but some knowledge

has been gained through the study of several

conditional lethalmutants. Nine cistrons have

beenfoundtobeessentialfor DNAreplication

(16), and twowereclassifiedasbeingmaturation

defective (8). Thesetwogenes, 33and 34, were

showntocode fortwopolypeptidesthat bindto

RNA polymerase (7), enabling thatenzyme to

performlatetranscription (5, 20).

In thispaperwepresentthe invivostudyof

apleiotropic temperature-sensitive (ts) mutant

in SPOl gene 31. Under nonpermissive

condi-tions,this mutant is defective in DNA synthesis,

and its transcription is shut off about 10 min afterinfection duetothelack ofatrigger func-tion mediated by this gene. This phenotype is also found in two other gene 31-suppressible mutants, susFl and susHA32.

MATERIALS AND METHODS

Bacteria andbacteriophage.Twostrains of B.

subtiliswere used, 168M and HA-101-B. The latter carries a strong suppressor (15, 18) and was kindly

supplied byS.Okubo.

Wild-type SPOl anditssuppressiblemutantswere the same as inreferences14and 16.

CHT medium(9)wasused for the growth of 168M, and L medium(16)wasused forHA-101-Bgrowth.

The bacteriawerealwaysinfected attheindicated

multiplicity ofinfectionwhen the optical densityat 500nmreached 0.5. Thedoublingtimefor B.subtiliu

168M was 30 to 32 min at either 37 or440C.

Experimental procedures. Labeled RNA was purified from cells thatwerequicklychilled, washed, lysed with lysozymeandsodium dodecylsulfate,and

hot-phenol extracted,asinFujitaetal.(8).

DNA was prepared from step gradient-purified SPOl phage byphenolextractionaccordingto

Man-dell andHershey(13).

DNA-RNA hybridizations were performed in 2x SSC(0.3MNaClplus0.03M sodiumcitrate)at700C for 3 h in a final volume of 200

pl.

DNase-free pan-creaticRNase was added to a final concentration of 12.5 pg/ml andincubated for 15 min at 37°C. The mixture was diluted in 0.01 M Tris-chloride (pH 7.5)-0.5M KCIandcollectedonnitrocellulose mem-brane filters. The filters were slowly washed three times with 10 mlof thesamebuffer, dried, and counted

in aliquidscintillation counter with

Omnifluor-Tolu-ene(New England Nuclear).

RESULTS

Characterization of the SPOl mutant

tsl4-1. Mutant ts14-1 wasisolated from a

mu-tagenized growthofwild-typeSPO-1 in the

pres-ence of 10 yg of

N-methyl-N'-nitro-N-nitroso-guanidine per ml(the isolation wascarriedout 483

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

484 SARACHU, AISIN, AND GRAU

in thelaboratory ofE.P.Geiduschekby I. Lahti

and 0. Grau (1). It is a temperature-sensitive

mutant because its burst sizes at 37 and 44°C

were about 30 and0.2 phages perbacteria,

re-spectively. Itwasbackcrossedtowild-type SPOl three times, giving reversionratesbetween 10-6

and 10-7,whichweretakenasanindication ofa

single mutation.

The DNA-synthesizing ability of tsl4-1-in-fectedB.subtilisatnonpermissivetemperature

was measured in comparison with that ofone

knownDO (DNAsynthesis-defective)mutantin

gene22andwild-type SPOl.Theincorporation of[3H]adenine intoalkali-stable, acid-precipita-blematerialwasnegligible comparedtothe wild

type (Fig. 1). The residual labeling observed for

tsl4-1was nothigherthan that of the DO

mu-tant susF30 (10). From this data it was

con-cluded that ts14-1 is aDO mutant and also is

abletoshutoff the host DNAsynthesis. To determine whether thismutant belonged tooneof thealready knownDO cistrons(16), it

wastestedbyspotcomplementation against

rep-resentatives ofallDO cistrons.Itcomplemented

susF2 (cistron 21); susF30 (cistron 22); susF20 and susO5 (cistron 23); susHA20 (cistron 27); susF21, susO36,susO45,and susO54(cistron 28); susF13 (cistron 29);susF26 (cistron 30); susF38

3

WI x

"I

I'_ 12

2

10 20

Time after infection (min)

FIG. 1. Incorporation of [3HJadenineinto DNAof wild-type-, susF30-, and ts14-1-infected B. subtilis 168Mat44°C. Culturesofwild type(0),susF30(0),

and tsl4-1 (V) SPOl-infected cells (multiplicity of infection, 10)weremadeupto0.5,uCiand 16pgof [3H]adenineperml2.5 min after infection. Atthe indicatedtimes, 0.5-ml samplesweretakenand hy-drolyzedwith 1 mlof1MKOHat45°C for3 h. Each

samplewasprecipitatedwith trichloroacetic acid(5% final concentration),collectedonnitrocellulose

mem-brane filters, washed with 5% trichloroacetic acid, dried,andcountedasdescribed in thetext.

(cistron 32). It did not complement susFl,

susF39, susHA32, and susO8 (cistron 31). In

accordance with these results, ts14-1 was

as-signedtocistron31.

tsl4-1-coded transcription. The ability of tsl4-1todirectthesynthesisof RNAduring its development in nonpermissive conditions was

tested bypulse-labeling B. subtilis-infectedcells

with[5,6-3H]uracilatdifferent times after

infec-tion. The labeled RNAs were purified and

hy-bridizedtoalargeexcessof SPOl DNA. These

data (Fig. 2) give us ameasurementof the

par-titionofRNAsynthesis between B. subtilis and phage DNA templates at different times after

infection (this is notcorrected for the

hybridi-z

0

4.

a.

4 ._

L._

:-

._-co

10 20

Time after infection(min) FIG. 2. Viral transcription level in SPOI

wild-type- and tsl4-1-infected B. subtilis 168M at 44°C.

RNAwaslabeledatspecified periods after infection

by2-minpulses of[3H]uracil (20yICi/ml and 0.26-pg/mlfinalconcentration)andimmediatelyisolated andpurifiedaccordingtothetext.From1.5 to2.5 pg

ofeach RNA per mlwashybridizedto50pgofSPOl

DNA per ml as in the text. Symbols: Wild type

(- - );tsl4-1 (-). Multiplicity of infection, 10

bacte-riophages per bacterium.

Backgroundwithout .

.o

. DNA Inputradioactivity

['H]RNA

la- DNA (cpm) beling period (cpm)

(min) Wild tsl4-1 Wild tsl4-1

type type

1-3 10 11 2,000 8,870

3-5 10 16 1,820 4,800

5-7 12 17 1,750 2,370

8-10 13 15 3,240 2,600

15-17 11 16 2,500 7,380

25-27 12 8,800

J. VIROL.

http://jvi.asm.org/

[image:2.501.62.438.245.626.2]

(3)

SPO1 GENE 31 485

zationefficiency of fully

complementary

RNA in

thesameconditions, which is about50%). From

hereon, wewill refertothismeasurement asthe "level of transcription."

Atthebeginning oftheinfectionat440C,the

level of tsl4-1-coded transcriptionis somewhat

lower thanthat of thewild type,but itfallsto

less than 10% ofthewild type at 15 min after

infection and remains at that level thereafter.

Thisshutoffis common toothermutantsinthis

gene. Atnonpermissive conditions susHA32 has

levels oftranscription of12.5and2.8% between

5 to 7and 15 to 17 minafter infection,

respec-tively. Another representative of this gene,

susFl, alsohasaverylowlevel oftranscription

at 25to 27minafter infection(2.2%). This abrupt

shutoff of the phage RNA synthesis must

in-volve the limitation of the synthesis of three

RNAclasses:

m1L

m21,and1(9).

Thelate level of transcriptionints14-1

infec-tionis proportional to thenumber ofinfecting

viral genomes. In Table 1 it is shown that a

variationin themultiplicity of infectionfrom4

to 16bacteriophagesperbacteriumgiveslevels

of viral

transcription

ranging from 0.8 to 4%.

Eventhe highest valueismuchlower than the

30 to 40% usually obtained with the wild type

(multiplicityofinfection, 10).

In vivo

study

of

ts14-1

mutation.

Temper-ature-sensitive phage mutants can be used to

analyze: (i) thenatureof the mutation

itself;

(ii)

the type offunction ofthealteredgene; (iii)the

timespaninwhich the functionisrequired for

the development of the phage (17).The

follow-ingexperimentsweredesignedtoelucidate these

characteristicsinmutantts14-1.

Phenotype

of tsl4-1 at 370C. The ts14-1

burstsize at

370C

is aboutone-half that of the

[image:3.501.259.450.360.519.2]

wild-type SPOl.Inspiteofthisdifference,

370C

TABLE 1. Lateviral transcriptionlevel atseveral multiplicities of infection'

Multiplicity Input radio_ Background % Input

re-of infection Inu ai- without %Intre (phage/ activity DNA tainedon

fil-bacterium) (cpm)

(cpm)

ter

4 13,125 20 0.8

4 7,400 7 0.9

8 6,080 6 2.5

10 5,154 3 2.5

16 4,075 6 4.0

16 1,993 5 3.7

aTwo-minute

pulse-labeled

(25 to 27 min after

in-fection)RNAswerepreparedfromtsl4-1-infectedB.

8ubtilis168M, usingthelabelingconditions inFig. 2. From1.5 to2.5 yg of each RNA per ml washybridized to10jg ofSPO1 DNA per ml(all other conditions andtreatmentasinMaterials and Methods).

wasinitially taken as the permissive

tempera-tureduetothe largevariation in phage

produc-tionamongindividual experiments.

Forabetterunderstanding of the shift exper-iments thatfollow,a moredetailed study ofthe

ts14-1 mutant phenotype at 370C was

per-formed.The kinetics ofDNAsynthesis of

ts14-1-infected B. subtili at 370C were measured.

The results (Fig. 3) indicatethat even at370C

this mutant behaves differently from the wild type. ItsDNAsynthesisstartsveryslowlyand

picks up speed about 20 min after infection,

whereas thewildtypestarts atabout 10 min and

keepsitsslopemore orlessconstantthereafter.

Second, the timecourse ofphage production

isalsodifferent(Fig. 3). The

intracellular

phage curves are

similar,

but the one for tsl4-1 is

delayed for about 10 min, as it is the time of

appearanceofoneintracellularphageequivalent

andbacteriallysis.

Theseresultsindicatethat370Cis a

semiper-missive temperature for the tsl4-1 mutant.

Therefore,thefollowingexperimentsshould be

analyzedinaccordancewith theparticular time

scaleresulting from Fig.3.

Shis fromthepermissivetothe

nonper-missivetemperature.Inanattempt to

estab-0 0

'U

6,~~~~~~~~~~~~~~~~C

<

0

502

4006

[image:3.501.55.248.500.626.2]

Time after infection(min)

FIG. 3. Phage production and DNA synthesis in SPOIwild-type-andtsl4-1-infectedB.subtilis 168M

at370C.Intracellularphages weremeasured at dif-ferenttimesafter infectionaccordingtoGeiduschek

andSklar(11).The resultsareexpressedas percent-ageofthe burst size. Inthreeexperimentsthese burst sizes were36, 82, and50bacteriophagesperinfected bacteriumforthewildtype(0) and60, 9, and24for

ts14-1 (0). The backgrounds (unadsorbed phages)

weresubtracted,theirmaximal valuesbeing0.8and 0.5%forwildtype andts14-1, respectively.

Incorpo-rationof/6HJadenineinto DNAwasdetermined as inFig. 1, except that

rHJadenine

(2.7 yCiand

17-pg/mlfinal concentration) was added 6 min after infection and incubation was performed at 370C. Symbols: Wild type(A);ts14-1(A).

VOL. 27,1978

http://jvi.asm.org/

(4)

486 SARACHU, AISON, AND GRAU

lish theperiodinwhich thefunctionof gene 31

is required, weperformedaseries ofexperiments

involving sudden changes from the permissive

tothenonpermissive temperature.

At different times after infection, B. subtilis

cells infected with ts14-1 at 37°C were

trans-ferred to440Corto amediumcontaining

chlor-amphenicolat370C. The yieldinboth cases was

measured60 minlater. The internalphages at

the time ofshiftwerealso determined (Fig. 4).

Phage production began to be higher than

thatofthe control (internal phages) whenthe

infected bacteriawereshiftedtothe

nonpermis-sive temperature after 20 min postinfection.

Whenthe shiftswereperformed later, the

pro-ductionincreasedconcomitantlytoaverage

val-uesup to 100times thatof thecontrol;thephage

yield obtained 60 min after the shift-up was

always higherthanthe internalphagespresent

atthe momentof shift. Theoppositewasfound

whencells, infected with either wild-type (data

100

0-IV

m

c

4,

4-n

._

mO

0

'I

5

40-C %NU

J. VIROL.

notshown) orts14-1 phage,weretransferredat

the time of shiftto medium containing

chlor-amphenicol; the amount of progeny was never

higher than the internalphages alreadyformed within thecells.

For comparison, several experiments with

wild-type SPOl were performed in the same

conditions.Theyshowedapartial sensitivityof

thewild-typephagetothe temperatureshift-up

from 0 to 25 min after infection. The average value resulted in 26% (standard error of the mean, 4) of the phage yield without shift-up

(average of13 experiments).When the transfer wasdonebetween 25 and35minafterinfection,

thevaluesincreased,although they showed large

variationbetweenindividual experiments.After

35minall the assays (5) gavemaximum yields

(thecellsarelysed), equaltothe ones obtained

when the cells were kept for 60 min at

370C

(value takenas 100%).

Shifts from the

nonpermissive

tothe

per-20

40

[image:4.501.116.405.301.594.2]

Time

at

37'C(min)

FIG. 4. Shift-up experiments. B.subtilis 168Mwasinfectedwith ts14-1 at37°C (multiplicity of infection, 0.04). Samples ofthe culture werediluted at various times into bubblers at44°C, where incubation was

continuedfor60 min. At the timeof shift-up, othersampleswerediluted into medium containing chloram-phenicol (0.1 mg/ml). Internalphages present at time of shift were determined by lysis, according to Geiduschek andSklar(11). The results areexpressedaspercentageoftheyieldwhen theculturewasnot shiftedup(burstsize at37°C).Infour experimentsburst sizeswere60,24,5,and 9bacteriophagesperinfected

bacterium, respectively. Thehighest experimental background, given bytheextracellularphages,was0.5%of

the burstsize at37°C. Symbols: Shift-up (0); chloramphenicol shift (V);internalphages (0).

Q0

/

0

0~~~~~~~

00

0 ₀ ₀ v

0

- - 0-o_4-&--°--°80°f

v88A

#V-A a A A

http://jvi.asm.org/

(5)

SPOl GENE 31 487

missive temperature. Atdifferent times after

infectiontsl4-1-infectedB. subtilis were

trans-ferred from 44 to 37°C, and the progeny was

determined 60 min later. The culture can be kept at440Cfor15 min withoutanyimpainnent of the phage yield (Fig. 5). However, more ex-tended periods caused an abrupt decrease in phage production: at 20 min it was less than 50% anddecayedrapidly thereafter. The wild-type-infected cells gave an average of 60 (standard errorofthe mean, 4)phages per bacterium in 25

experiments in which the shift was performed

between 0and 30 min after infection. They did notshow any trend towards a decrease in the

yield at the later times; for example, when

shifted down 30 min after infection, the yield

was 63phagesperbacterium.

Experimentsinvolvingacombination of

shifts.Ananalysisof theabove-mentioned

re-sults led to the conclusion that there was a

favorable window between min 15 and30 after infection to furtherstudythe nature ofthe tsl4-1mutation and theaction of the gene 31 product. The experiments performed involvedthe com-bination ofshift-ups and shift-downs with the addition and removal of chloramphenicol. To establish the feasibility ofthe experiments, it was necessary tomake surethat theextra

ma-*

100

S~~~~S

L I

U,,\

.o

~

~~~

0

L

\

6) ~ ~ r-~

20 40 60

[image:5.501.51.246.376.558.2]

Time at 44'C (min)

FIG. 5. Shift-down experiments. B. subtilis 168M

wasinfectedwith tsl4-1at44°C(multiplicity of infec-tion, 0.22). Sanprles ofthe culture were diluted at various times into bubblersat37°C,where incubation

wascontinuedfor60 min. Theresultsareexpressed aspercentages, in reference to theyieldwhen the culturewasshifteddown between 5and 15 minafter

infection.In twoexperiments,100%was30(standard

error, 2) and 69(standarderror, 4) bacteriophages

perinfectedbacterium, respectively. Thehighest

ex-perimental background was 0.8% (0.24

bacterio-phagesperinfected bacterium).

nipulations involvedwouldnotalter the behav-iorofthe system.

The positive control, that is, the growth of

tsl4-1at

370C,

was notaffectedbythedilutions and transfersrequiredby the experimental

de-signand gave 60phages per infected bacterium (Fig. 6,line 7). The experimental conditions were

adjustedtolower the backgroundgivenby the

unadsorbed phages; the value of 0.02 to 0.04 phages per infected bacterium obtained was sat-isfactory. Chloramphenicol added 15 min after infection gave a highlevel of inhibition, confirm-ing previousresults,and when it was diluted off at 30minafterinfection,about 10% of the phage

yield of the controlwasobtained(Fig. 6, lines8

and 9).

Fortheanalysisof theexperiments, theyield

of a shift-down at 15 min was taken as 100%

(Fig.6,line 2), and the unadsorbedphageswere

determined and subtracted in all cases before

the ratioswere calculated. The combination of shift-down and chloramphenicol gave the

ex-pected results. When chloramphenicol was

added atthe time of shift-down and remained for therestof theexperiment (line3), therewas

no phage production. When chloramphenicol

wasdiluted 5, 10,or15minafter theshift-down,

thephageproduction recovereduptoabout 25%

(line 4). The experiment shown in line 5 gave

about a 15% yield,

indicating

that 5 min was

enoughtosynthesize and/or permitthe action

of gp-31 to produce progeny. The

experiment

shown inline6gaveabouta2.5%

yield,

which is

10times higherthan the controlat 44°C (line

1). Thisproduction indicates that gene31 had been activebefore min15,producing gp-31,that

the product was

partly

reactivable at the

per-missive temperature, and that5minofactivity

(atmost) wasenough toproducesomephages.

DISCUSSION

TheSPOl temperature-sensitivemutant

tsl4-1isarepresentativeof cistron31.In accordance

with thephenotypeof thiscistron,it isnotable

toreplicateits DNA(16).Atnonpermissive

con-ditions tsl4-1 as well as susHA32 and susFl

transcription is severely impaired 15 min after

infection. This effect is similarto the one ob-served insus mutantsF30, F4,and F14 (8) but

is more intense. In this case the low level of

transcription is proportional to the number of

infecting genomes. This isclearly not the only factorinvolvedinthe shutoff ofthesynthesis of

RNA, because even at large multiplicities of infectionthelevel of viraltranscriptiondoesnot

exceed 10% of that of the wild type.

Thetemporal parameters of the lytic cycle of the tsl4-1 mutant at

370C

are different from

VOL. 27,1978

http://jvi.asm.org/

(6)

488 SARACHU, ANMSN, AND GRAU

EXPERIMENTAL

DESIGN

PHAGE YIELD S

°

15

T

T+60

TIM OF

SECN

SHIFT MIN

_

20

25

30

1 .

,

.

W

.

27

17 .24

2 I DO

liO

100 _

cm cm

.0o

4

27

0

2

5 II

17

18

6

2.4

3.7

7 100

8 * c c

.__

FIG. 6. Experimentscombining temperatureandchloramphenicol(CM) shifts. B. subtilis 168M wasinfected

withtsl4-1at440C (linesIto6;multiplicity ofinfection,0.39) or370C(lines 7 to 9;multiplicityofinfection,

0.33); 15min laterthecultures were diluted 10 times into thefollowingmedia: CHT at 44°C (line 1); CHT at

370C (lines2,5,and 7), andCHTat370Ccontaining 0.1 mg ofchloramphenicolperml (lines 3,4,6,8, and 9).

Five, 10, or 15min later(Tminutesafter infection),the cultures wereagaindiluted: 10 times into CHT at

44°C (line1);100times into CHTat37°C (lines2, 4, 7,and9);10times into CHT at37°C containing 0.1 mg

ofchloramphenicolperml(lines3and 8); and 100 times into CHT at44°C (lines5and 6). Totalphages were

determined60min later, lysingthe cells as described by Geiduschek andSklar(11).Resultsinlines 1 to 6

correspondtoexperimentsperformed simultaneously. Theyareexpressedaspercentageoftheyieldin line 2 (71 bacteriophagesperinfected bacterium). Results in lines 7 to 9correspondtoexperimentsperformedin parallel. Theyareexpressedaspercentageoftheyieldin line 7(60bacteriophagesperinfected bacterium). Theexperimental background (extracellularbacteriophages)was 0.04and0.02bacteriophagesperinfected bacteriumforlines 1 to 6 and 7 to9,respectively.

those ofthe wildtype. Theeclipse,the onset of

replication, and lysis take place about 10 min

laterthaninSPOlwild-type infection.

These results could be interpreted as ifthe

ts14-1 program at 370C were stretched out.

There isnoindication ofasubstantial alteration

in the known sequence of events leading to

phage production.

In theshift-up experiments the effect of the

gene 31function,in tsl4-1, beginstobe

notice-able atabout 20min afterinfection. After this

time it could be interpreted that gp-31 is

re-quired all alongthephage cycletoobtain

maxi-mumyield. However, this could not be

deter-mined definitelybecause the sensitivity ofthe

wild type to shifts-up up to 25 min could be

extendedtolongertimesbythetsl4-1 stretching of theprogram.

Itwassurprisingtofind thatts14-1 and

wild-typeSPOLare notassembledwithout

concomi-tantproteinsynthesis,asitwasobservedduring

thebacteriophageT4lyticcycle (17). The

pres-enceof

chloramphenicol

inthemedium reduces

the finalphage

yield

tothe amountofinternal

phages present at the time of addition of the drug and sometimestovalues lower than those. This is not due to a direct effect of

chloram-phenicolupon thephage,becausematurephages

arenotaffectedby thedrugatthe concentration

used.

The cell machinery,

partially

controlled by the viralgenome,canspare itscapacityto

pro-duce phage in the absence of gp-31 for some

time.

Forexample,afullamountofphageswas

obtained when tsl4-1-infected B. subtiliswere

kept at thenonpermissivetemperature for upto 15 min and then shifted down to

370C.

After thattimethesystem isnot

stable,

andat30min J. VIROL.

http://jvi.asm.org/

[image:6.501.144.371.79.344.2]

(7)

SPOl GENE 31 489

it cannotrecoveritsabilitytoproduceprogeny.

This time interval, 15 to 30 min, was used in

studying thenatureof the ts14-1 mutation and thetime of action and synthesis of thegp-31.

The window experiments (Fig.6)indicatethat gp-31is inactivatedat440C butcanbe

synthe-sizedatthattemperatureand that its activity is atleast partly recovered when it is transferred to370C. Part of this synthesis takes place before

15min at440C, and its action between 15 and

20min is enoughtoproducesomephages. This

phage production takes place after thewindow

is closed(see Fig.4and line6ofFig. 6), indicat-ing that the action of this gene generates

pre-cursors orotherwise triggers intermediate

con-trols that will allow itssynthesis.

The data in Fig.6, although showingasmall

correlation between the length of the window and the phage yield, are not conclusive. How-ever, the amounts of phage DNA and RNA

synthesized are proportional to the time the window is open (Sarachu, submitted for

publi-cation).

The results of shifts and window experiments just mentioned, and the peculiar behavior of

ts14-1 at370C,suggestthatgp-31 issynthesized early in infection (before 15 min) but that its action isspecially delayed. This could arise from

an abnormally long processing time (if any is

involved) or fromthe existence ofa threshold

for its activity, which is attained after a long

time due to poor specific activity ofgp-31 or

some othermorecomplex (or indirect)

mecha-nism.

It istobenotedthatpartial damage ofgp-31

activity of tsl4-1 leads at 370C to aparticular

DD (DNA synthesis-delayed) phenotype (Fig. 3), which,incontrast tothe known DDmutants (16), is abletoproduceprogeny.

Thissituation, together with the already men-tionedstretchingof the program,could be

par-ticularlysuitable forstudyofthe multiple

con-trols involved in SPOl middledevelopment.

ACKNOWLEDGMENTS

Wewarmlythank E. P. Geiduschek and G. Favelukes for advice,support, andencouragement.

This researchwassupported by grantsfrom theConsejo NacionaldeInvestigacionesCientfficasyTecnicasde la Re-publica Argentina,Comisi6n deInvestigacionesCientificasde

laProvinciade BuenosAires,and Universidad Nacional de LaPlata.

A.N.Sarachu and M. C.Afi6nwerefellows ofthe Comi-si6n deInvestigacionesCientificas de la UniversidadNacional deLaPlata andConsejoNacional deInvestigaciones

Cienti-ficasy Tecnicas de la Republica Argentina. 0. Grau isa

recipientofaresearch careeraward from the Comisi6n de Investigaciones Cientificas de laProvincia de Buenos Aires.

LITERATURE CITD

1. Afi6n, M. C., and0. Grau. 1973.Transcripci6n en el crecimientoyla diferenciaci6n de SPO1, p. 109-112.In F. BarbieriandA. Legname (ed.), Progresos en biolo-gia.Tucuman, Argentina.

2. Duffy,J. J.,andE. P.Geiduwchek.1973. Transcription specificity of an RNA polymerase fraction from bacte-riophageSPO 1 infected Bacillus subtilis. FEBS Lett. 34:172-174.

3. Duffy,J.J., and E. P.Geiduschek.1975.RNA polym-erase fromphage SPO 1 infected and uninfected Bacil-lus subtilis. J.Biol.Chem. 250:4530-4541.

4. Duffy,J.J., R.L.Petrusek, and E. P.Geiduwchek. 1975. Conversion of Bacillus subtilis RNA polymerase activity in vitro by a protein induced by phage SPO 1. Proc. Natl. Acad.Sci. U.S.A.72:2366-2370.

5. Fox, T. D. 1976.Identificationofphage SPO 1 proteins codedby regulatorygenes 33and 34. Nature (London) 262:748-753.

6. Fox, T. D., R. Losick, andJ. Pero. 1976. Regulatory gene 28 ofbacteriophage SPO 1 codes for phage-induced subunit of RNApolymerase.J.Mol. Biol. 101:427433. 7. Fox,T.D., andJ. Pero. 1974.New phage SPO1 induced polypeptides associated with Bacillus subtilis RNA polymerase.Proc.Natl.Acad. Sci. U.S.A.71:2761-2765. 8.Fujita,D.J.,B. M.Ohlsson-Wilhelm,andE. P. Gei-duschek. 1971. Transcription during bacteriophage SPO 1development:mutationsaffecting theprogram of viraltranscription.J.Mol. Biol. 57:301-317. 9. Gage,LP., andE. P.Geiduschek.1971.RNAsynthesis

during bacteriophage SPO1development:sixclassesof SPO1RNA.J. Mol.Biol.57:279-300.

10. Gage,LP., andE. P.Geiduschek.1971.RNAsynthesis during bacteriphage SPO1development.II.Some mod-ulations andprerequisitesofthetranscriptionprogram. Virology44:200-210.

11. Geiduschek,E. P.,andJ. Sklar. 1969.Continual re-quirementfor a host RNApolymerasecomponent in a bacteriophage development. Nature (London) 221: 833-836.

12. Grau, O.,B.M.Ohlsson-Wilhelm,and E. P. Geidus-chek. 1970.Transcription specificityinbacteriophage SPO1development.ColdSpringHarborSymp. Quant. Biol.35:221-226.

13.Mandel,J.D.,and A. D.Hershey.1960.Afractionating column foranalysis ofnucleic acids. Anal. Biochem. 1:66-77.

14. Okubo, S.,M.Stodolsky, and B. Strauss.1964.The possiblerole ofrecombinationintheinfection of com-petent Bacillus subtilis by bacteriophage deoxyribo-nucleic acid.Virology24:552-562.

15. Okubo, S., and T. Yanagida.1968.Isolationof suppres-sor mutant in Bacillus subtilis. J. Bacteriol. 95:1187-1188.

16. Okubo, S.,T.Yanagida,D.LFujita,and B. M. Ohls-son-Wilhelm. 1972. The genetics ofbacteriophage SPO1.BikenJ.15:81-97.

17. Pulitzer,J. F. 1970.Functionof T 4gene 55. I. Charac-terization of temperature-sensitive mutations in the "maturation"gene 55. J.Mol. Biol. 49:473-488. 18. Shub,D. A. 1975.Nature of the suppressor of Bacillus

subtilis HA101B. J.Bacteriol. 122:788-790.

19.Spiegelnan,G.B.,andH. R.Whiteley. 1974. In vivo andin vitrotranscription byribonucleic acid polymer-asefrom SP82infected Bacillussubtilis.J. Biol. Chem. 249:1483-1489.

20. Tjian,R., andJ.Pero. 1976.Bacteriophage SPO 1 reg-ulatory proteins directing late gene transcription in vitro.Nature(London) 262:753-757.

VOL. 27,1978

http://jvi.asm.org/