• No results found

Expression of the cloned bacteriophage phi X174 A* gene in Escherichia coli inhibits DNA replication and cell division.

N/A
N/A
Protected

Academic year: 2019

Share "Expression of the cloned bacteriophage phi X174 A* gene in Escherichia coli inhibits DNA replication and cell division."

Copied!
7
0
0

Loading.... (view fulltext now)

Full text

(1)

0022-538X/85/030807-07$02.00/0

CopyrightC 1985, AmericanSocietyforMicrobiology

Expression of

the Cloned Bacteriophage PX174 A* Gene

in

Escherichia

coli

Inhibits

DNA

Replication

and

Cell Division

JOSEPH COLASANTIANDDAVID T. DENHARDT*

Cancer Research Laboratory and the Department ofMicrobiology and Immunology, University of Western Ontario,

London,

OntarioN6A

SB7

Canada

Received 27September1984/Accepted 26 November 1984

The A* geneofbacteriophage 4X174 has been cloned into the inducible expression vectorpCQV2 under conditions allowing its lethal action tobe controlledby the XcI857repressor. Upon inductionof expression, DNA synthesis inEscherichia coli carrying the recombinant plasmid is severely inhibited;however, thesesame

cellspermit 1-galactosidase inductionataratesimilartothat observed in control cellsattheinducing (for A*) temperature.Cells in which A* is expressed formifiamentsand producemoreRecA protein, indicatingatleast

apartial inductionof the SOSresponse;however, there isnoevidence of damagetothe bacterial chromosome. Itappearsthat the A* protein hasas onefunction theinhibition of cell division and DNA replicationbut not

transcriptionorprotein synthesisduring phage infection.

The functions ofmost of the genes encoded by bacte-riophage 4X174 have been elucidated by genetic

experi-ments, particularly by the isolation of mutations in those genes (see reference 31 for review). For the A* gene, however, this strategy has notbeen fruitful because of the

nature of the relation between A* and the larger A gene. Gene A codes for a polypeptide with anMr ofca. 58,000 whose essential roleas anickaseorligase in the replication

of thephagegenomehas been well documented(4), whereas the function of the A* protein (Mr, ca. 37,000) in vivo is unknown. Unlike the overlappinggenes of(X174, genesA and A*areread in thesamne reading frame; A* results from

a translational restart from a ribosome-binding site (RBS) approximately one-third of the distance from the beginning of the gene A RBS (20). Because of the nested nature of thesetwogenes, anymutation in A*also affectsA, making it difficult to differentiate their respective functions during phage infection.

An early study by Lindqvist and Sinsheimer (19) impli-catedaphage-encoded protein in the phage-induced shutoff of host DNA synthesis during infection. This protein was later found tobe aproduct of the A (or A*) gene (13, 23). Thesestudies, however, didnotreachaclear and unambig-uous position regarding the role of A* protein (7). Amore recent study has shown that A* has the ability to inhibit replication of both replicative form (RF) and DNA single-stranded(ssDNA) XX174 in vitro (11). The purified protein bindsstronglytossDNA and double-stranded DNA(34) and hasendonucleolytic activity with somesequence specificity towards ssDNA (16). In the presence ofMn2+ or the 4X gene A protein, the A* protein may be capable of nicking and binding covalently to XX RF (10, 35). The A* protein boundtothe 5' end ofaDNA strandcanbedisplaced under appropriate conditions by the 3' OH end of the same or another DNA strand, resulting in the formation ofa 3'-5' phosphodiester bond (12, 35, 36).

To elucidate further the function of A*, we used

recom-binant DNA technology to clone the A* gene to study its effects in vivo on Escherichia coli. Initial attempts at this cloning revealed that expression of the A* protein was, as

expected, lethaltocells in which itwasexpressed.

Consist-*Correspondingauthor.

ent with this, van der Avoort et al. (33) were unable to

isolateaclone with the intact AorA*geneincircumstances in which cloned segments of

4X174

RF producing other phage productswere isolated. Forour cloning strategy, we therefore undertook to use plasmid vehicles which allow control ofexpression of clonedgenes. After initial failures with vectorspPLc28 (28), pKC30 (30), and pJL6-8 (18), we succeeded with pCQV2, which contains both the A

PR

promoter and the c1857 repressor (27). This vector was foundtomaintain the A*genein a stable and silent format

30°C andtoallowits expression at 42°C. Upon induction of expression of the A* gene, therateof DNAsynthesis in E. coliwas drastically reduced, and the cellswerekilled.

MATERIALS AND METHODS

E. coli, phage, and plasmid strains. The amber mutants

fX174am16 and

4X174am3

(lysis defective), deficient in geneB and Efunctions, respectively,werepropagated in E. coliCRthy-

Su'

(6). E. coli C Su- and CRweregrownin mT3XD(6). Other strainsweregrownin Lbrothor supple-mentedM9 minimal mediumcontaining 0.2% glucose (22)as

indicated. Theplasmid pCQV2 (27)wasprovided by G. A.

Chaconas; pPLc28 (28) was obtained from W. Fiers along with itshostsK12AHlAtrp and M5219; pKC30wasfrom M. Rosenberg; pJL6-8wasobtainedfrom D. Court.

DNA purification and manipulation. Plasmid DNA was purified fromalysozyme-Brij 58 clearedlysateby isopycnic centrifugationincesium chloride-ethidiumbromide followed by extraction with n-butanol, dialysis, phenol-chloroform extraction, and a final dialysis and alcohol precipitation. Phage RF DNAwas purified from E. coli CR infected with XX174am16atahighmultiplicity (2to3) when the cells had reachedadensity of 2 x 108/ml; chloramphenicolwasadded

12 min after infection to a concentration of 20 ,ug/ml, and

aeration of the cultures was continued at37°C for5 h. RF

DNA was isolated by the same method used to isolate plasmid DNA.

Allenzymeswereobtained from BethesdaResearch Lab-oratories, Boehringer-Mannheim, orNew England Biolabs and were used as recommended by the suppliers unless

indicated otherwise. To fill in recessed 3' ends, restricted duplexDNAwasincubated with 65 mMTris-hydrochloride

(pH 7.6)-40mM KCl-25 mM MgCl2 in the presence of the 807

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

*PA

pCQV2 - 0X174 StuI A

-GGAGQTTGTATfGATCC

[5

IGGAGGCCTCCACTATGA

-CCTCCAACATACCTAG(3-

bp

CCTCCGGAGGTGATACT

BamHI

FIG. 1. Construction ofpJA*1 and pBCK5. The recombinant pJA*1wasconstructedby inserting the fragmentcontaining the A* gene of

4X174 into thecloning vehicle pCQV2 (see the text). This construction results in placement of the A* gene in the correct orientation for transcription from the A PR promoter. Repressor protein (cI857) prevents transcription from the PR promoter at 30°C; at 42°C, the temperature-sensitive repressor is renderednonfunctional, and transcription occurs, allowing the A* proteintobesynthesized starting from itsownRBS. TheplasmidpBCK5wasconstructed frompJA*1 bydeleting the BamHI-BstXIfragment,blunting withT4DNApolymerase, andreligating. The deletion eliminates A* expression but leaves therest of the plasmid intact, thereby allowing the effects of A* to be separated from those of theaml6geneBproduct and the gene C- and geneK-pCQV2 fusions. The predicted sequence of the

pCQV2-4X174

jointatthe A*Nterminus isshownin detail. Apeptide initiated from thecroRBS and AUG codon terminatesneartheA* startcodon.

fourdeoxynucleoside triphosphates (0.5 mM) and thelarge (Klenow)fragment ofDNA polymerase I (60 to 100 U/ml) for30to60 minat25°C. DNAfragmentswerepurified from 0.7%

low-melting-point

agarosegels by electroelutiononto a

dialysis membrane placed inatrough inthegel (22). Recom-binant DNA was used to transform E. coli RR1 cells to

ampicillin resistance by a modified Hanahan procedure for cells other than X1776 (22). Cells were freeze-shocked in a

-70°C

ethanol-dry

ice bath before addition of dimethyl sulfoxide, which was added only once. Heat shock was

performed at 36°C, and the transformation mixture was incubatedat30°C.

Determination of the rate of DNA synthesis. Cells with various plasmids were growntomid-logphase in L broth in the presenceof carbenicillin (100

p.g/ml)

at 30°C. When the cell density reached 108/ml, half of each culture was trans-ferredto 42°C. Portions (1 ml) were taken at various times beforeandaftertheshift and addedto10,uCi of

[3H]thymidine

andsufficientdeoxyguanosinetoproduce a50 mMsolution. The cultures wereincubated for2 min, and then incorpora-tion was terminated with 2 ml of cold 10% trichloroacetic acid. Trichloroacetic acid-precipitable material was

col-lected onglassfiber filters andwashed,and itsradioactivity was determined.

I-Galactosidase

assay.E. coli RR1 cells

carrying

plasmids

were grown at 30°C in M9 minimal medium supplemented with0.2% Casamino Acidsand 100 ,ugof carbenicillinperml to a density of 5 X

107

cells perml, at whichpoint half of

each culture was shifted to 42°C; 6 min later, isopropyl

,-D-thiogalactopyranoside

was added to a final concentra-tion of 2 mM.Duringthecourseofgrowth, aliquots (0.25 ml) weretaken and added toan equal volume of Z-buffer (25), and the cellswerepermeabilizedwithadropof toluene. The level of 3-galactosidase production was determined as de-scribedbyMiller(25). The apparent

A6Eo

of the cultureswas

alsodetermined atvarious times during growth.

Electroblotting ofproteinsand immunodetection. Cultures ofE. coli RR1 harboring plasmids were grown in L broth with carbenicillin (100 ,ug/ml) at 30°C to adensity of _108

cells per ml. Theculturesweredivided,onehalfwasshifted to42°C,and theincubationswerecontinued foranother 2 h. Aliquots were taken and centrifuged, and the pellets were

suspended in Laemmli sample buffer and boiled for 5 min. Equivalent amounts of each sample (determined by the

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.152.479.71.411.2]
(3)

apparent

A6N

of the cultures) were then run on a sodium dodecyl sulfate-polyacrylamide gel (12.5% acrylamide, 2.25% N,N'-diallyltartardiamide) with a 4.5% acrylamide stacking gel. Immunoblotting was performed essentially as described by Towbin et al. (32) with thefollowing modifica-tions. Afterelectrophoresis, proteins were transferred from the gelontonitrocellulosepaper(Schleicher&Schuell, Inc.) inLaemmlirunningbuffercontaining20% methanol with an E-C Electroblot system. The paper was stained with 0.1% amido black, destained for 1min with 10%acetic acid-10% methanol, washed three times for 5 min each in distilled water, and incubated overnight with Tris-buffered saline (TBS;20 mM Tris-hydrochloride [pH 7.5], 150 mM NaCl). Nonspecificadsorptionwas blocked by incubating the paper with asolution of TBS containing 0.05% Tween 20 (Sigma) for2h atroomtemperature(2). Rabbit antiserum (provided by Shlomo Eisenberg)raisedagainst the 4XX gene A protein was diluted 1:750 in TBS containing 0.05% Tween 20 and incubated withthe blot in a volume of 20 ml for 2 h at room temperature. Rabbit antiserum (provided by Jeff Roberts) raised against E. coli RecA protein was diluted 1:4,000 in the samebuffer and incubatedsimilarly. The blots were washed sixorseventimesfor10min eachin TBSplus Tween 20 and then incubated with 125I-labeled protein A (New England NuclearCorp.), ca. 30,000 cpm per lane, for 2 to 3 h. After extensive washing with TBS plus Tween 20, blots were subjectedto autoradiography.

RESULTS

Controlledexpression of a lethal gene. Phage X regulatory elements have been usedtocontroltheexpression of cloned genes in several systems (3, 24, 29). Shimatake and Ro-senberg (30), for example, used the X PL promoter-c1857 thermolabile repressor system of pKC30to control expres-sion of the lethal X cIIgene. In attempting toclone the A* geneof

4X174,

however,wediscoveredthatseveralvectors bearing the PL promoter apparently exerted insufficient repression of thisgene at 30°C (the restrictivetemperature forexpression)topermittheisolationof stableclones. This was possibly due to an inadequate concentration of repres-sor, since the cI857 gene was located on the host chromo-somein adefective prophage in the systems we tried.

This prompteda search fortemperature-inducible expres-sion vectors that used eitherthe X

PL

or PR promoter and encoded the X cI857 repressor. The plasmid pCQV2 was found tobesatisfactory (seeFig. 1). Itcontainsthe XcI857 repressor gene transcribed from thePRM promoter(which can be stimulated by cI) and the PR rightward promoter (which is repressed by thecIorc1857 protein)just upstream from the RBS and theAUG startcodon ofthe X cro gene.

(These

latterwere notessential in

cloning

the A*

fragment

sinceitcarried its ownRBSand startcodon).

Construction, diagrammed in Fig. 1, of a recombinant with the A*gene in the properorientation relativeto the PR promoter involved cleaving pCQV2 with BamHI, creating bluntends on theduplexwith the KlenowfragmentofDNA polymerase I, and then cutting with SaI. The digestion of

4~X174

double-stranded RF DNAwithNruI, which cleaves 74base pairsupstream fromthe A* start codon, andXhoI, which cleaves 32 base pairs after the A-A* stop codon, yields a 1,142-base-pair fragment containing the A* gene. The normal promoter for the A* gene is the PA promoter, which is just before the start of the A gene and, thus, not present. Consequently, transcription of this fragment is dependentontranscription from the PRpromoter.

Ligation of theprepared pCQV2 DNA with the XX174 A* fragment, followed by transformation of E. coli RR1 to ampicillin resistance, permitted the isolation of the plasmid pJA*1. The flush-ended BamHI-NruI joining results in re-generation of theBamHI site, whereas the SalI-XhoI cohe-sive end joining does not regenerate either site. Given transcription from the PR promoter, the nature of this construction allows synthesis ofapeptide fromthe cro RBS-startcodon into

4XX174

DNA but out offrame with the A* gene andresulting intermination oftranslation upon reach-ing the A* start region. (Expressionof this peptide does not affect E. coli metabolism because clones have been con-structed in which it is absent [by cloning the StuI-XhoI fragment of

4X174

intopCQV2, see Fig. 1]and theybehave as pJA*1 does [data not shown]. These recombinants were not used in this study because the A* produced was altered in that it had five additional amino acids fused in its N terminus.) Since the cloned segment of XX DNA in pJA*1 carried theRBS and initiationcodonfor A*, itwaspossible to obtainexpression of A* protein. Expressionwasinitially evidenced by the inability of the cells to form colonies at 42°C. The presenceof(X174 DNA wasconfirmedbycolony hybridization with labeled (by nick translation) XX174 RF DNA orfragments containing the A*gene.

PropertiesofpBCK5, a deletion mutant of A*. Because of thepresence ofoverlapping reading frames in this segment of XX174 DNA, it isimpossibletoclone the A* gene without cloning all or partsofothers, in this case the entire B gene andthe 5' ends ofgenesC and K. Tominimize interference

M

a

b

c

d e f

g

h

i

j k

--_

-_

=

=_~-

58k

_~~

--37k

A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

FIG. 2. Electroblotting and immunodetection of gene A* pro-tein. Equivalentamounts of cell extracts (E. coli RR1) were elec-trophoresedon a sodium dodecylsulfate-polyacrylamide gel, elec-troblottedontonitrocellulose paper, and probed first with antibody to XX gene A protein and then with 1251I-labeled protein A as described in thetext. Lanes athroughccontain2,0.4, and 0.04 ,ug, respectively, ofa purified preparation of

4X174

gene A protein provided by S. Eisenberg. Lanes d and e are extracts of strain RR1(pCQV2)at 30 and 42°C (2 h),respectively; lanes f and g are extractsof strainRR1(pJA*1)at30 and42°C(2 h),respectively;and lanes hand iareextractsof strainRR1(pBCK5)at 30and42°C (2 h), respectively.Lanesjand k are extracts of E. coli Cuninfected(lane j) and infected(lane k)with 4X174am3for1.5 h at37°C. Molecular weight markers (Bio-Rad Laboratories) onthe leftare phosphor-ylase b(Mr,92,500), bovineserumalbumin (Mr,66,200),ovalbumin (Mr, 45,000), carbonic anhydrase (Mr, 31,000), soybean trypsin inhibitor (Mr, 21,500)andlysozyme(Mr, 14,400).

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.612.320.555.379.565.2]
(4)

A

E

0.

I

B

700-600.

0)

.-:

500-0)

U)

0 'o

O' 400.

0

0

0%

3100-\/

1

2X1

4X'I00

21

~~~~t

10' 10 20 30 40 50 0 15 30 45 60

Time (min) Time (min)

FIG. 3. Effect of A* protein on DNA synthesis and 3-galactosidase induction in E. coli. (A) DNA synthesis was determined by measurementof[3H]thymidineuptake intoacid-precipitable material. CellsweregrowninLbroth(containing100,ugofcarbenicillin perml) to108cells per ml at 30°C, and 1-ml portions were taken(0min)and added to 10,uCiof[3H]thymidinefor2min (see the text). At20 min after thefirst sampling, half of each culture wastransferredto420C, and the measurements werecontinued. (B) Cells weregrownin minimal medium(containing100,ugof carbenicillin per ml) to5x 107cells per ml at30°C, atwhich point half of each culture wastransferredto42°C (O min). Isopropyl 3-D-thiogalactopyranoside was added six min laterto a 2 mM finalconcentration, and 0.25-mlsamples were takento determine levels ofP-galactosidaseinduction. Strains used:E.coliRR1(pCQV2)grownat30°C(0)andat420C(0),E.coli RR1(pJA*1) grown at30°C

(E)

andat42°C (E), andE. coliRR1(pBCKR) grownat30°C (A)and at42°C (A).

fromgeneB,anambermutant,4X174am16,wasused in the cloning, and studies were done in a nonpermissive host. However,onecouldstillarguethatitwasthetruncatedgene B product or gene C-pCQV22 or gene K-pCQV2 fusion products whichwereresponsible for the effects attributedto

the A*protein. DNA-directedtranscription-translation reac-tions performed in vitro with recombinant plasmids (not shown) indicated thepresenceofputative fusionpeptides of theapproximate molecular weight predicted for the XX174-pCQV2construct.

To prove that the lethal effects were indeed due to A* expression, we constructed a mutant, pBCK5, by deleting theBamHI-BstXI fragment from pJA*1 (Fig. 1). This dele-tionremovesthe A* startsitealong with the first half of the genebut stillallows transcription ofgeneB and thegene C and Kregions asbefore. The cro start siteisjoined to the remaining 4X174sequencesuch that thepeptide produced is in adifferent reading frame from A* andterminates almost

immediately after it starts. This is borne out by immuno-blotting results(see Fig. 2). The ability of cells bearing this plasmid to form colonies at42°C indicates that these other polypeptides did notkill the cell and thatexpression of the A* proteinwas necessaryfor the lethal effect.

Anadditional argumentthatgene B,geneC, and geneK

products are not lethal to the cell can be based on the

existenceof the

PB

promoter. Thispromoterprecedesgene B(Fig. 1) and presumably permits constitutive transcription of thisregion in the cell. Since the cellssurvivedat30°C, this transcription was notlethal. Inaddition, vander Avoort et

al. (33) showed that cells thatwere expressing theB and C genes (and, hence, able to complement 4X gene B and C mutations) grew well.

Detection of the A*protein.In casesin whichgenes are put undercontrol ofAPL or PR promotersand

c1857, induction

at 42°C often results inthe

production

ofalargeamount of protein (27, 30). Unfortunately, thiswas notobserved with induction ofE.colicarrying

pJA*1.

TheA*protein (Mr, ca. 37,000) did not stand out on Coomassie blue-stained

polyacrylamide

gels or in autoradiographs of

gels

of

[35S]methionine-labeled

cell extracts induced at 42°C (not shown). However, major E. coli

proteins migrated

in this region and could have obscured moderate amounts of an

inducedprotein.

Theavailability ofananti-A antiserummadeitpossibleto usethe more sensitive immunoblotting procedureto detect the A* protein. Figure 2 shows the presence of the A* protein after induction of expressionat42°C,whereas none at all was detectable at 30°C. Cells

carrying

pBCK5 pro-duced neither A* protein nor a truncated product after induction.

Interestingly, the anti-A antiserum seemed to react spe-cificallywith anotherE. coliprotein intheregion ofthegel indicating an Mr of ca. 56,000. The nature ofthis bacterial protein is unknown; however, since at

420C

heat shock increases itsexpression, it could be the B56.5protein (also called the A protein and the groE protein [26]). The cross-reactionwith anti-+X174gene Aproteinserummayindicate a shared

epitope.

The other(less intense) bands on thegel

0

0 a

0

o

XZ/

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.612.127.501.73.342.2]
(5)

A

B

FIG. 4. Induction offilamentsby A* protein.E.coli RR1 cellsharboringeitherpCQV2orpJA*1weregrowntomid-log phaseinLbroth (with 100 ,ug of carbenicillin per ml)at30°C, transferredto Lagarplates(withcarbenicillin), and allowedtogrowateither30 or42°Cfor1.5 h.Blocks of agarcontainingcellswerethen fixed with 1%Os04;the cellsweretransferredtoglassslides and then stained withHCl-Giemsa. This procedure stains the nuclear regions ofE. coli darkly. Strains shown: E. coliRR1(pCQV2) at 30°C (A) and42°C (B) and E. coli RR1(pJA*1)at30°C (C) and42°C(D).

appeared with nonimmune rabbit serum also (data not shown).

Effect ofA*protein on E. coli DNA synthesis. One ofthe effects ofA* protein in vitro is inhibition of(X174 DNA synthesis (11). Induction ofA*expressioninvivoallows one to determine its effects on E. coli replication

directly.

Measurement oftherateofDNAsynthesis via[3H]thymidine uptake revealed a marked decrease in synthesis in cells in which A* expression had beeninduced (Fig. 3A), whereas cellsat 30°C and those

carrying

theparent plasmid atboth temperatures continued

synthesizing

DNA. Similarly, cells harboringpBCK5 showed noinhibitionof DNAsynthesisat 42°C, providing further evidence that A* protein directly inhibitsE. coliDNAreplication. The residual DNA synthe-sis in the E. coli

RR1/(pJA*1)

culture at42°Cmay be due to a subpopulation of cells lacking the ability to express A* protein.

Effect of A* on

I(-galactosidase

induction in E. coli. To ascertain theeffect ofthe A*proteinoncellulartranscription and translation, we monitored ,B-galactosidase production.

Cells carrying either pCQV2 or pJA*1 were grown toearly log phase at 30°C, shifted to 42°C, and then induced to produce ,3-galactosidase with isopropyl ,,D-thiogalactopy-ranoside. The effect of A* protein on enzyme production was assayed colorimetrically. There was not a noticeable difference in

P-galactosidase

levels incellscarryingeither of the plasmids at either temperature (Fig. 3B). This may indicate that A* acts specifically to inhibit host DNA syn-thesis and does not significantly affect transcription or translation. Consistent with this conclusion is the fact that the cells bearing pJA*1 form filaments (Fig. 4) and continue to increase in mass at42°C several hoursafter induction of A* synthesis.

Are SOS functions induced? It is usually the case that when cellular DNA is damaged orcell DNA replication is inhibited or both, the cell responds by expressing a set of genes under the control of the lexA repressor (21). The phenomenoniscalled the SOSresponse and inlargepart is directed atfacilitating repairof thedamagedDNA. Wehave investigated whether thisresponse occurs afterinductionof

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.147.469.71.467.2]
(6)

ja

b

c

d

ellf

g

h

i

la"elIf'

FIG. 5. Quantification of RecA protein by immunoblotting. EquivalentamountsofE.coliRR1carrying pCQV2(lanesathrough e)orEcoli RR1carryingpJA*1 (lanesfthrough j)were

electropho-resed on an sodium dodecyl sulfate-polyacrylamide gel and im-munoblotted withantiserum raisedagainstE.coliRecAprotein (see the text).Cells in early log phase eitherwereexposedtomitomycin C(1.5 ,ug/ml) for30min (lanes bandg) and 90min (lanescandh)

or wereshiftedto42°Cfor 30min (lanesd andi)and 90min (lanes

eandj). Lanesaand frepresentcellskeptat 30°Cfor 90min.The fourlanes onthe right representtwofold dilutions ofthesamples

seeninlanesa,e,f,andj. Arrowsonthe left indicate thepositions

of themolecular weight markers (Bio-Rad) phosphorylase b (Mr, 92,500), bovine serum albumin (Mr, 66,2000), ovalbumin (Mr, 45,000),andcarbonicanhydrase(Mr, 31,000).

A* expression. One of the diagnostic features of the SOS responseis increasedsynthesis of the RecA protein, and the experiment presented in Fig. 5 reveals that this occurred when A*wasinduced,although theaugmentation of

synthe-siswasnotasgreataswasfoundwhen the cellsweretreated

with mitomycin C,a DNA-damagingagent. This is

consist-ent with a partial turn-on of the SOS function and could

accountfor the filamentationobserved, since LexA repres-sion of the sfiA gene, whose product inhibits septation, wouldbe lifted.However, thecomplete explanation is likely

more complex, since A*-induced filamentation was also observed in recA56 and lexA3(Ind-) strains in which the SOSresponse cannotbeinduced, and in whichmitomycin C

treatmentdidnotcausefilamentation (datanot shown). DISCUSSION

Earlyattemptsbyourlaboratory and others(33) indicated that successful cloning ofgene A* required that its lethal action berepressed. To accomplish this,weused thevector

pCQV2 (27), which uses the A

PR

and cI857 controlling elementstoregulate the expression of clonedgenes. Several

PL-containing

vectors(pPLc24,pKC30,andpJL6-8)used in earlierexperiments apparently provided insufficient repres-sionofA* synthesistoallow isolation ofstableclones;this may be because these vectors rely on c1857 repressor provided byasingle-copy defective lysogen, whereas pCQV2

carries a copy of the repressor gene itself, thus increasing thegene copynumberof therepressorgene.

When expression of A* protein in cells carrying pJA*1

was induced by shifting the cellsto 42°C, large amountsof theproteinwerenotmade, raising thepossibility of

autoreg-ulation of synthesis. Detection of expression required the immunoblotting technique (Fig. 2) to visualize the A* pro-tein, which was otherwise obscured by E. coli proteins.

Upon expression ofA*, cellularDNA synthesiswas

inhib-ited,and thecellsstoppeddividing.Howeverthey increased in mass,formedfilaments, and,wheninducedto

synthesize

a newprotein (here by isopropyl

,,D-thiogalactopyranoside

induction of

P-galactosidase

synthesis), responded as did control cells

(Fig.

3). Ghosh and Poddar (14) found that 4X174 infection caused inhibition of

P-galactosidase

produc-tion in E. coli, and they speculated it was caused by a phage-encoded protein. Ourresults suggestthat it isnotA* thatisreponsible for this shutdown. Apparently, the mech-anism that A* uses to shut down E. coli DNA

replication

doesnotresult ina

generalized

breakdownof the bacterial DNA, and doesnotinterfere with

transcription

and transla-tion.

The greatersensitivity of ssDNAto cleavage bythe gene A*

protein

(12, 16, 17) encourages thethought thatthe A* proteinmay actspecificallyatsingle-stranded regions in the E. coli growing fork, breaking the DNA at that point and blocking furtherreplication. Other

possibilities

include phys-icalobstruction merelyasthe resultofA*

binding

toduplex DNA(11)or totheRep

protein,

ahelicase

normally

involved in separating the strands of the bacterial chromosome. Inhibition ofDNA replication causes induction ofthe SOS response, which includes the increased

production

ofthe SfiA

protein,

an inhibitor of

septation

(9, 15). However, since

septation

is inhibited under conditions in which the LexA

repression

of the

sfiA

geneis notlifted

(in

recA56or

lexA3 mutants), it may be that inhibition of cell division occurs astheresultofan SOS-independent

coupling

of cell divisionto DNA

replication

(5).

The role of the

4X174

gene A*

protein

in the viral life

cycle

is unknown.That it is made

by

alltheisometric

phages

examined and that it

seemingly

has a counterpart in the filamentous phages(8) implies thatit isadvantageous ifnot necessaryforsomeaspectof the viral life

cycle.

Itsactions

on ssDNA suggest a role in

superinfection

exclusion

(7).

Other

possible

roles include

shutting

off host cell DNA

synthesis

and

facilitating

the transition fromRF

replication

toDNA

synthesis

(10, 11, 13, 17, 23). Earlier

research,

the

more recent studies of

Eisenberg

and Ascarelli

(11)

and

Langeveld

etal.(16),and our owndataargue

strongly

foran

effect on host DNA

synthesis;

presumably

a cessation of host DNAsynthesis increasesthepool of nucleotide precur-sors available forphage DNA

synthesis.

Evidence

against

adirect roleforthe A*

protein

inXX174 ssDNA

synthesis

comesfrom in vitrostudies

indicating

that it is not

required

forRF

replication

orssDNA

synthesis

in vitro (1). One

explanation

fortheinferredessential function of the A*

protein

could be the existence of a host cell

protein,

required

for

replication

ofthe bacterial

DNA,

that interferes with viralssDNA

synthesis (1).

Therole of the A*

protein

would be topreventthis interference.

Oligonucleo-tide-directed in vitro

mutagenesis

of the A*

coding

sequence in

plasmid pJA*1

will make

possible

incisive

experiments

into A* function.

ACKNOWLEDGMENTS

Thisresearchwassupported byagranttoD.T.D. anda Student-ship to J.C., both awarded by the Medical Research Council of Canada.Supportwasalsoprovided bytheNational Cancer Institute of Canada.

We thank those individuals who provided us with strains and

plasmids. WearegratefultoC.F.Robinowfor thephotographyin

Fig.4,toShlomoEisenbergforthegiftsof antiserumagainstthe4OX

AproteinandasampleofAprotein,andtoJeff Roberts for thegift

of anti-RecA antibody.

[125I]protein

Awas agiftfromSam Dales. Wealso thankDylanEdwards andNancyNgfor usefuldiscussions,

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.612.70.298.73.223.2]
(7)

Sharon Wilton and Sven Beushausen forhelp withimmunoblotting, andGeorge Mackie for doing the in vitro transcription-translation reaction. The figures and manuscript were skillfully prepared by Dale Marsh and Linda Bonis.

LITERATURE CITED

1. Aoyama, A., R. K. Hamatake, andM. Hayashi. 1983. Invitro synthesis of bacteriophage 4)X174 by purified components. Proc. Natl. Acad.Sci. U.S.A. 80:4195-4199.

2. Batteiger, B.,W.J.Newhall V,and R. B.Jones. 1982.Theuse of Tween 20 as ablocking agent in theimmunological detection of proteins transferredtonitrocellulose membranes. J. Immun. Methods 55:297-307.

3. Bollen, A., R. Loriau, A. Herzog, and P.Herion. 1984. Expres-sion of human a1-antitrypsin in Escherichia coli. FEBS Lett. 166:67-70.

4. Brown, D. R., J. Hurwitz, D. Reinberg, and S. L. Zipursky. 1982. Nuclease activities involved in DNA replication, p. 187-202.InS. M. Linn and R. J. Roberts (ed.), The nucleases. ColdSpring Harbor Laboratory, Cold SpringHarbor,N.Y. 5. Burton, P., and I. B. Holland. 1983. Twopathways of division

inhibition in UV-irradiated E. coli. Mol. Gen. Genet. 190: 309-314.

6. Denhardt, D. T.1969. Formation of ribosylthymine in Escheri-chiacoli.Studiesonpulselabeling with thymine and thymidine. J. Biol. Chem. 244:2710-2715.

7. Denhardt, D. T. 1977. The isometric single-stranded DNA phages, p. 1-104. In H. Fraenkel-Conrat and R. R. Wagner (ed.),Comprehensive virology, vol.7.PlenumPublishing Corp., NewYork.

8. Denhardt,D.T. 1978.Overview:acomparisonof the isometric and filamentous phages, p. 645-655. In D. T. Denhardt, D. Dresslerand D.S. Ray (ed.), The single-stranded DNA phages. ColdSpringHarborLaboratory, ColdSpringHarbor, N.Y. 9. Devoret,R. 1983. Workshop summary: inducible responsesto

DNAdamage, p. 571-576.In E.C.Friedberg andB.A.Bridges (ed.), Cellular responses toDNAdamage. Alan R. Liss, Inc., NewYork.

10. Dubeau, L., and D. T. Denhardt. 1981. The mechanism of replication of4)X174.XVIII.Gene A and A* proteins of

+X174

replicative formDNA. Biochim.Biophys. Acta 653:52-60. 11. Eisenberg, S.,and R.Ascarelli. 1981. The A*protein of4)X174

is an inhibitor of DNA replication. Nucleic Acids Res. 9:1991-2002.

12. Eisenberg, S., andM.Finer.1980. Cleavage and circularization ofsingle-stranded DNA: anovel enzymatic activity of

+X174

A*protein. Nucleic Acids Res. 8:5305-5316.

13. Funk, F. D., and D. Snover. 1976. Pleiotrophic effects of mutantsin geneAofbacteriophage4)X174.J.Virol. 18:141-150. 14. Ghosh, A., and R. K. Poddar. 1977. Reduced synthesis of ,B-galactosidase inEscherichiacoli infected withphage

+X174.

Can. J. Microbiol.23:1069-1077.

15. Huisman, O.,R. D'Ari, andS. Gottesman. 1984. Cell-division control in Escherichia coli: specific induction of the SOS function SfiAproteinis sufficienttoblockseptation. Proc. Natl. Acad. Sci. U.S.A. 81:4490-4494.

16. Langeveld,S.A.,A.D. M. vanMansfeld, A. vander Ende, J. H. van dePol, G. A. van Arkel, and P. J. Weisbeek. 1981. The nuclease specificity of the bacteriophage

+X174

A* protein. NucleicAcids Res. 9:545-562.

17. Langeveld,S.A.,A. D. M. vanMansfeld, J. M. de Winter, and P.J.Weisbeek.1979.Cleavage of single-stranded DNA by the A and A* proteinsof bacteriophage 4)X174. Nucleic Acids Res. 7:2177-2189.

18. Lautenberger, J. A.,D.Court, and T. S. Papas.1983. High-level expression in Escherichia coli of the carboxy-terminal

se-quences of the avian myelocytomatosis virus (MC29) v-myc protein. Gene 23:75-84.

19. Lindqvist, B. H., and R. L. Sinsheimer. 1967. Process of infection with bacteriophage

+X174.

XIV. Studieson macromo-lecularsynthesis during infection withalysis-defectivemutant. J.Mol. Biol. 28:87-94.

20. Linney, E.,and M. Hayashi. 1974. Intragenicregulationof the synthesis of

+X174

gene A proteins. Nature (London) 249: 345-348.

21. Little, J. W., and D. W. Mount. 1982. The SOS regulatory systemof Escherichia coli. Cell 29:11-22.

22. Maniatis, T.,E. F.Fritsch,andJ. Sambrook. 1982. Molecular cloning,alaboratory manual. Cold Spring HarborLaboratory, ColdSpring Harbor, N.Y.

23. Martin,D.F.,and G. N. Godson.1975.Identification ofa

+X174

codedprotein involved in the shut off of host DNA replication. Biochem.Biophys. Res. Commun. 65:323-330.

24. Meilado, R. P., and M. Salas. 1982. High level synthesis in Escherichiacoli of the Bacillus subtilis phage+29proteins p3 and p4 under the control of the phage lambda PL promoter. Nucleic Acids Res. 10:5773-5784.

25. Miller, J. H. 1972. Experiments in molecular genetics, p. 352-355. ColdSpringHarborLaboratory,ColdSpring Harbor, N.Y.

26. Neidhardt, F. C., T. A. Phillips, R. A. VanBogelen, M. W. Smith,Y.Georgalis,and A. R. Subramanian. 1981. Identityof theB56.5protein, theA-protein, and thegroE geneproduct of Escherichiacoli. J. Bacteriol. 145:513-520.

27. Queen, C. 1983. A vectorthatuses phagesignals for efficient synthesisofproteins in Escherichia coli. J. Mol.Appl. Genet. 2:1-10.

28. Remaut, E.,P. Stanssens,and W. Fiers. 1981. Plasmidvectors forhigh-efficiency expressioncontrolledby the PL promoter of coliphagelambda.Gene 15:81-93.

29. Remaut, E., P. Stanssens, and W. Fiers. 1983. Inducible high levelsynthesis ofmaturehumanfibroblast interferon in Esch-erichiacoli. Nucleic Acids Res. 11:4677-4688.

30. Shimatake, H.,and M.Rosenberg. 1981. PurifiedX regulatory protein cIIpositively activates promoters for lysogenic devel-opment. Nature(London) 292:128-132.

31. Tessman, E. S., and I. Tessman. 1978. The genes of the isometric phages and their functions, p. 9-25. In D. T. Den-hardt, D. Dressler, and D. S. Ray (ed.), The single-stranded DNA phages. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

32. Towbin, H.,T.Staehelin,andJ. Gordon. 1979.Electrophoretic transfer ofproteins from polyacrylamide gelsto nitrocellulose sheets:procedureandsomeapplications.Proc.Natl. Acad.Sci. U.S.A. 76:4350-4354.

33. van derAvoort,H.G.A.M.,R.Teertstra,R.Versteeg,and P.J. Weisbeek. 1983. Genes and regulatory sequences of bacterio-phage

+X174.

Biochim. Biophys.Acta741:94-102.

34. vanderEnde, A., S.A.Langeveld, G. A.vanArkel,and P. J. Weisbeek. 1982. Theinteraction of the A and A* proteins of bacteriophage

+X174

withsingle-stranded and double-stranded 4)XDNAin vitro. Eur. J.Biochem. 124:245-252.

35. vanderEnde, A., S.A.Langeveld,R.Teerstra, G. A.van Arkel, and P.J. Weisbeek. 1981. Enzymatic propertiesof the bacterio-phage

+X174

A*proteinonsuperhelical

+X174

DNA: amodel for the terminationof the rolling circle DNA replication. Nu-cleic Acids Res. 9:2037-2053.

36. vanMansfeld,A.D.M.,H.A. A. M. vanTeeffelen, J. Zandberg, P. D.Baas,H.S. Jansz, G.H. Veeneman,andJ. H. van Boom. 1982. A*protein of bacteriophage+X174carriesan oligonucle-otidewhich itcantransfertothe 3'-OH ofa DNA chain. FEBS Lett. 150:103-108.

on November 10, 2019 by guest

http://jvi.asm.org/

Figure

FIG.1.joint4X174transcriptiontemperature-sensitiveanditsseparated own Construction of pJA*1 and pBCK5
FIG. 2.j)tein.trophoresedtroblottedtodescribedprovidedrespectively,RR1(pCQV2)extractslanesrespectively.inhibitorweight(Mr,ylase and XX Electroblotting and immunodetection of gene A* pro- Equivalent amounts of cell extracts (E
FIG. 3.measurementtothedetermine(Oatmedium 30°C min). 108 Effect of A* protein on DNA synthesis and3-galactosidase induction in E
FIG. 4.ThisRR1(pJA*1)h.(with Blocks Induction of filaments by A* protein. E. coli RR1 cells harboring either pCQV2 or pJA*1 were grown to mid-log phase in L broth 100 ,ug of carbenicillin per ml) at 30°C, transferred to L agar plates (with carbenicillin),
+2

References

Related documents

The first aim of this study is to explore the role of sensitivity to reward and sensitivity to punishment in examination test settings in terms of the elicitation of positive

Gradient elution at ambient temperature with the mobile phases as (A) H 2 O containing 0.1% volume/volume (v/v) formic acid and (B) acetonitrile con-. taining 0.1% (v/v)

In order to provide a direct comparison between this TiO 2 -coated and traditional metallic PEF treatment cells, the PEF treatment procedure was identical to

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania

head formation, it would appear that core structure is affected in some way which leads to increased aberrant assembly of the capsid pre- cursor. This suggests that correct

In our model, higher nutrient loads acted to increase the biomass of all trophic groups, which buff- ered the effects of AC predation on lower trophic levels, decreased competition

By examining the views of the clinical decision makers (anesthesiologists and surgeons) in a theory-based systematic manner, we have identified the theoretical domains we

c) reaction time, d) reaction temperature, and e) level of incorporation.. Determined against integral at δ 3.90. Determined against integral at δ 3.85.