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0022-538X/79/04-0014/07$02.00/0

qinlOl: Promoter Mutation Which Allows the Constitutive

Expression of the Late Genes

C.DAMBLY,* M.DELSTANCHE,tAND A. M. GATHOYE

LaboratoiredeGinetique, DepartementdeBiologieMoltculaire, UniversiteLibre de Bruxelles, 1640

Rhode-St-Genese, Belgium

Received forpublication 13 September 1978

We describe the isolation and characterization ofa mutant (XqinlOl) which

rendersX growth Q independent. Wehave shown that this mutation creates a

newpromoter,located between genes P and Q, whichresultsin the constitutive expression of the entire Q late region.

Transcription of the late genes of

bacterio-phage A is assumed to occurby two pathways.

Thefirstis amajor pathwaywhich ispositively

controlled bythe Q product (8, 15, 20). The Q

geneproductappears toact asan

antitermina-tor, allowingtheextension, overthelategenes,

of a short RNA (6S) initiated at the late pro-moter(18) (Fig. 1).

The secondisaminorpathway which is

com-pletely Q independent ("leakiness" of the

Q-mutants) (6). We have shown that this minor

transcriptional pathway operates only in the

presenceoftheNactivator and in the absence

ofimmunity. It consistsof theprolongation,over

the lategenes, oftranscription initiated inthe

immunity region (promoter pR

[Fig.

1]). This

minor transcription occurs to about 5% of the

level ofthe major pathway and is obscured in

the presenceoftheQproduct. Byitself(that is, inQ-mutants), it is tooslowto result inplaque

fornation; however, inliquid medium, Q-

mu-tantsfinally produce nornal phage yields.

Toelucidatethemechanismsofprolongation

andantitermination, wehavelooked, by

differ-entmethods, formutantsabletotranscribe the

lategenes at ahighrate, evenin the absence of

theQ product. Asimilarapproachhas been used

by Herskowitz and Signer (14) and Sato and

Campbell(19).

MATERILS

AND

METHODS

Bacterial strains. All bacterial strains used are listed inTable 1 and are derivatives of Escherichia coli K-12.The strains were used as suchand, for some ofthem, aslysogenderivatives.

Bacteriophages. Xc+, Ximm21c+ (16) c17 (17) is located in region y; it expresses constitutively genes

clI, 0,and P andreplicates even in thepresence of immunity.

tPresent address: Institut National de laSanteet de la RechercheM6dicale,Unit6 de Recherches de Virologie U 102, Lille,FranceF-59045.

Those phageswere used as such or labeled with various amber mutations:Qam2l, Aamll,and Ram5 (4); Sam7 (13);Qam2O3andRam216(22).Most ofthe phages used to infect sensitive hosts carry the cII2002 mutation to diminishlysogenization frequency.

bypE (3) and byp3 (our laboratory) are located between genes P and Q. AQ-h80 (Q- derivative of

Xhy6 (21) is aAX-480 hybridwith the leftarm (A-.

red) from 80 and therightarm (red-- R) from A.

A(QSR)21Kam24 (constructed by L. Desmet and F.

Salomon) is a X-21 hybrid phage with the A -* P

regionfromXand theQ-*Rregionfrom21.Kam24 wasisolatedby Campbell (4) and introducedin the hybrid.

Methods. (i) Infectionexperiments. The infec-tionexperiments weredescribedpreviously by Dam-blyand Couturier(6).Exponentialcultures(lysogens ornot) wereinfectedbyone ortwophages,eachat a

multiplicity of 3, diluted 100-fold, and incubated at

370C. At 100 min afterinfection, the dilutionswere

treated withCHC13, and adequatedilutions were ti-tratedat370C.

(ii)Endolysinassay. Theendolysinassaywas as described by Dambly and Couturier (6) except the indicatorbacteria were treated withCHC13inbuffer (0.05MTris-hydrochloride,pH 7.8).

(iii) Lysogenization experiment. A Lysogens wereselectedandlysogenization frequencieswere de-terminedasdescribedbyGottesman andYarmolinsky

(12) except that the selection was done on EMBO platesseeded with bothAred3int6cIh+A andAcIh+80.

RESULTS

Isolation and characterization. The

qin-101 mutationwasisolated from

XQam2O3Ram-216asaspontaneous plaque-forming phageon alawn of the host HB15.The infecting phage

carries, in addition to a Q- mutation, an R-mutationto be able to further distinguish be-tweentheqinmutants

(XqinQ-R-)

andthe

Q'

revertants (XQ+R-) by transactivation

experi-ments. The HB15 host bacterium was chosen since itcarriesanochresuppressor(supB) able to restore, to a sufficient level, the R function

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Imm. 21

NEAD TA L

int red N ci cro y d O P (byp)Q S R A F K J

tL pL pR tRinl'TERclNATOitA2 LATF

6SPROMOTEA

aoactivation

[image:2.501.89.429.55.185.2]

FIG. 1. Geneticmap of Aprophage.Arrows indicate the origin and direction oftranscription.Theblock represents the region ofA-imn2l nonhomology. Markers given are those relative to this study. Symbols: V, transcription terminationsites; X,promoter sites.

TABLE 1. Bacterial strains

Bacterialstrain Genotype and relevantproperties Source/reference

H F-argHlac amsup+ Ghysenand Pironio(10)

H15 IsogenictoHbut carries theronmutation(defined Ghysenand Pironio (10) bythe number 15)

HB15 supBderivative ofH15 Ghysenand Pironio(10)

W3110 F-sup+ LederbergviaWeigle

594 F-gal- strAsup' Campbell(4)

N100 recAgal- derivative of W3350 Meselson via Yarmolinsky;

Gottesman and Yarmolinsky (12)

RH1413 RH1422 Thermoresistant derivatives of M5020G Castellazzietal.(5) (ANNcI857)

M5020G sup' (lac,pro)strARsgly ProvidedbyJ. Davies

CA244.1 Cured derivativeof CA244 Thislaboratory

CA244 HfrHBr-lac-trp-sup' Brennerand Beckwith(2)

CA244.1recA recA derivative of CA244.1

C600 supII, pernissivefor ambermutations Appleyard(1)

CA161 supHl, usedinsteadof C600(which is resistantto Brenner and Beckwith(2) 480) in the experiments involving phages h+A

andh+80

CA161/A h+A-resistant derivativeof CA161 Thislaboratory

but not the Q function and the ron mutation

whichstrongly decreases the leakiness of the

Q-mutations[compare AQ-productions after

infec-tionsof H (ron+) and H15 (ron-) (Table 2)]. We could isolate only one mutant (XqinlOl) ableto grow onstrain HB15 which plates with the sameefficiencyonsup'hostsand on hosts

carrying an amber suppressor. However, the

plaques formed on the sup' hosts are small,

whereas thoseformedonthe hosts carryingan ambersuppressor arealso

normal

butclear.

Preliminary characterization of the

Xqin-1O1Q-R- phage showedthat it isnot arevertant

orpseudorevertantofthe AQ-R- phage.Indeed,

itfailedtotransactivatetheexpression of the R+

gene of a prophage, showing that it does not synthesize active Q product in detectable amounts. Furthermore, whentheqinlOl muta-tionwascombined with other Qam mutations,

such as t57, Qam2l, Qam501, or Qam73, the

resulting phages behave inthe same way as the qin derivative ofQ203.

Burstsizesof

XqinlOlQ-R+

phageondifferent

sup'

strainsare giveninTable2.Itisapparent

thatXqinlOl

Q-

growsaswellontheH15

(ron-sup')

and the H (ron+

sup')

strains as onthe HB15(ron-supB)strain,theyields

being

about

105 timesgreater than those of

AQ-

platedon

H15. The yield of

XqinlOlQ-

on

sup'

strains

seems, however, to be

impaired by

mutations

conferring streptomycin

resistance,

insofar as

one canjudgefromthelower

yield

onthe strA

strains594and N100.

Tohave a more

quantitative

measureof the

effect of the qinlOl mutation on late gene

expression,wemeasureddirectlytheexpression

of the late gene Rby endolysin titrations. We found that inW3110(ron+ sup+hostbacterium)

XqinlOlQ- producedtwo tothreetimesasmuch

endolysinasdid

XQ-,

reachingavalue of 10% of

that synthesized by

XQ4

phage

(Table

3). We,

therefore, conclude that the qinlOl mutation

permits growth, in Q-

conditions,

by

only

in-creasingthe lategene

expression

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DAMBLY, DELSTANCHE, GATHOYE

TABLE 2. Growth of XqinlOlQ on different bacterialstrainsa

Yield(phage/bacterium) at 100 min after infection with: Bacterialstrain

AQ-R+ \qinlOlQ-R+

AqQnlOlQ4R+

HB15(ron- supB) 3 75 52 45

H15 (ron- sup+) 5.4x10-4 70.4 58 55

H (ron+sup') 3.5 56.1 50 52

W3110 (ron+sup+) 2.7 66.7 75 60

CA244.1 (ron+sup+) 3 20.4 69 65

594(ron+sup+strA) 2 6.4 33 40

N100(ron+sup'strA) 1.75 7.1 35 29

aTheinfection

experiments

wereperformed as described in the text. Phage productions were measured on the C600 indicatorstrain.AQ-R+isXcII2iooQam2O3, andAqinlOlQ-R- isAcII2emqinlOlQam2O3Ram216;XQ+ andXqinlOlQ+ alsocarry thecII2oom mutation.

2 to 3.

Mapping of the qinlOl mutation. Precise

localization of the qinlOl mutation has been

inferredfrom three types ofcrosses.(i)Ina cross

of Xc+qinlOlQ7R- with Ximm21Q-R- phages,

imm21qinlOlQjR- recombinants [selected on

HB15 (X)] are found with a frequency of 1%,

indicating thattheqinlOlmutation is localized

outside the imm2lregion.

(ii) A moreprecise mappingwasobtainedby

crossing Ac+qinlOlQ-R- phage with various X

prophage deletionmutants.The deletions used

enter the prophage from the left and end at

different points in the P-Q region. For these

crosses,theclear phenotype given bytheqinlOl

mutationwasusedasmarker, and therecovery

ofturbid-plaque Arecombinants indicated that

the qinlOl+ region was present within the

de-leted prophage. The crosses between

Xc+qin-1O1Q-R- and differentdeletedprophages

local-ized qinlOl between the endsof the deletions

RH1417 and RH1415(Table 4).

(iii)We alsomapped the qinlOl mutationin

respect to two mutations located in the P-Q

region:bypE (1) and byp3

(previously

isolatedin

thislaboratory). Thesetwomutations,although

theyhave the sameproperties of N

independ-encefor the relief of the

tr2

terminator sitesand

ofclear-plaque phenotype,aredistinctontheA

map: byp3 is within the RH1415 deletion and

bypE is not. Crosses between

Ac+qinlOlQ-R-phage andXimm2lbypE (Fig. 2) show that the turbid recombinants(v =

5.10-2)

presentalmost all (97%) the imm2l and the Q-R- mutations. On the contrary, the same cross made with

Ximm2lbyp3 (Fig. 2) gives turbid recombinants

withamuchlower frequency (v=6.10-3)which have indifferently immA or imm2l, the am or

am'character. This shows that the qinlOl and

byp3 mutations are very close. The presence withequal probability of one or the other outside marker isprobably due tohighnegative inter-ference. From theseresults,weinferred that the

TABLE3. Endolysinsynthesis byAqinlOl phage infectingasup'straina

Endolysin activity W3110 infectedwith: (units/100ploflysate

at100min)

XQ- 36

XqinlOlQ- 95

XQ+ 595

XqinlOlQ+ 950

aAll

thephagescarry anadditional

cII2moo

mutation. Q- isQam2O3.Endolysinwasassayedas describedby Damblyand Couturier (6).

TABLE 4. DektionmappingoftheqinlOlmutationa Clear Turbid Ratio of

Host amn+ am+ c am

(%)b (%)b (%)/c

can (%)b

RH1422(Q-/R+) 100 lo-l 10-3

RH1415(PI/Q+) 100 1o-l 10-3

RH1417(P7/Q+) 51.8 48.2 0.93

RH1416(PI/Q+) 38 62 1.62

RH1413(PI/Q+) 33.65 66.35 1.97

QR4 3

RH1415

I-RH1417

RH1416

RH14

aThe deleted

strains derive

from M5020G

(sUp+

strA) (see text). The blocks represent the deleted prophageregion.Theam'recombinantsweretitrated on 594 (sup+ strA).Thepercentageof clearam' (or

turbidam+) representsthe fraction ofam' recombi-nantswhichpresentaclear(orturbid)phenotype. Q

isQam2O3;R- isRam216.

bAfter infection with

Xc+qinlOlQ-R-.

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qinlOl: CONSTITUTIVE EXPRESSION OF LATE GENES 17

qinlOl mutation is located to the left ofbypE,

verynearbyp3.

Dominance-recessivity test. To test the dominantorrecessive, cisortranscharacter of the qinlOl mutation, we coinfected strain H15

withXqinlOlQ-h+A (able togrow onH15) and

XQ-h+80 (unabletogrow onH15)and measured

the production of eachphage. The h+80 char-acter wasusedto testfora transeffect of the qinlOl mutation on late gene expression since

thepresence of theh+80 late proteinresults in

ahostrangedifferent from that ofX.

We observed that themixed infection results in high, comparable yields ofboth XqinlOlQ

and XQ- phages (Table 5). The difference in XqinlOlQ- yields betweenmixed andsingle in-fections(comparelines 3 and 1 of Table5) may

beexplained by phenotypic mixing; aswill be-comeapparentlater,there isnosynthesisof late 4)80 proteins, and the coats made by XqinlOlQ-h+X packagenotonlytheirownDNA

butalsoXQ-h+80DNA. Thiscompetitionlimits the amount ofXqinlOlQ-h+X phage packaged.

AcoiNxIQR

A imm2l bypE

XcQINix R[

R-Aimm2lbyp3

c 0

imm21

Indeed,wecansee (Table 5) that,among

prog-eny,the number ofh+80 phages whichpresent

thehostrangeof 480[h+80 (h+80)]remainsvery

low. Asexpected, thisproduction is again high when the qinlOlderivative is Q+ (Table 5).

All of these results showthat, in mixed infec-tion, AqinlOlQ- phage continuestoexpress its ownlategenesbut isunabletoinduce intrans

the late genes of the coinfecting phage. We,

therefore, conclude that qinlOl isacis-dominant

mutation.

Nature of the qinlOlmutation.Asalready described, the qinlOl mutation gives a clear

phenotype in Q+ conditions (phageXqinlOlQ+

orXqinlOlQ- ona sup am host).In Q-

condi-tions(phage XqinlOlQ- ona

sup'

host), plaques are verysmall, and the phenotype isnotaseasy

todetermine. To determine thephenotype, we

measured thepercentageoflysogenization after infection ofa

sup'

hostwith differentderivatives

ofXqinlOl and found that XqinlOlQ- lysogenizes

normally (33%), in contrast to XqinlOlQ+ (<0.05%; Table 6). The observation that the

IINIOI Q0 R

Aimm21C'QR recombinants

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byp3

FIG. 2. Mappingof qinlOl inrespecttobyp3andbypE.

TABLE 5. Cis-dominant effectof the qinlOl mutationa

Phage production (phage/bacterium)at90min

Phage inl Aor Q-hk)r

Total q (h+80)A) (h+80) AQ-h+80(h+80)

AqinlOlQ-h+A 35 35

AQ-h+80 2x10-1 2x10' 2x 10'

XqinlOlQ-h+X+AQ-h+80 24 13 11 2x10'

XqinlOlQ+h+X 28

AqinlOlQ+h+X+AQ-h+80 42 25 17 12

aStrainH15(asensitive hosttobothh+Xandh+80 derivatives)wasinfectedbyeachphageatamultiplicity

of3.Thismixedinfectiongave(4)classes ofphagesfollowingtheirgenotype(h+Xorh+80)and theirphenotype [(h+X) or(h+80)]: the twoparental types,h+A (h+X)andh+80 (h+80), and twotypes duetothephenotypic

mixing, h+X (h+80) andh+80 (h+A). Among the fourprogenytypes,onlythephages which areh+80 (h+80)

should beabletoformaplaqueonastrainresistanttoAandthusmaybe titrated.XQ-h+80isaQ- derivative

ofAhy6 (see text).Totalphage productionwastitratedonCA161, AqinlOlQ-weretitratedonH15,XqinlOlQ+ weretitratedon594,andXQ-weretitratedbythe difference betweenthe total andXqinlOl yields.XQ-h+80

(h+80)productionwastitratedonCA161/X.All thephagescarrythecII2002mutation.

imm21 bypE

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DELSTANCHE,

frequency of lysogenization by XqinQ+ is im-proved(5%) in thepresenceofmutationsS and

R- (Table 6) and almost reaches the normal level(26%) withanSR-A- triplemutant(Table

6) shows that these functions areexpressed by

XqinlOlQ+ under immunity andthus indicates that theqinlOlmutationconstitutesapromoter

abletoinitiate,evenunderimmunity,the

con-stitutive expression ofgeneQ (in Q+ conditions)

and ofallthelate genes. InQ- conditions, this

additional transcription does not provoke the killing of thelysogens but sufficestoallowphage

growth.

To obtain more direct evidence supporting

thisconclusion,we measured the expression of

twolate genes (R and K) by aAqinlOl phage

infectinganimmune host. In the R experiment,

theprophage only provides the immunity, and

we tested whether, despite this immunity, the

superinfecting XqinlOlQ+ phage is ableto

syn-thesizeendolysin. Resultsareshown inTable7,

part1.We observeda smallbutsignificant

pro-duction of endolysin when the homoimmune phage carries the qinlOl mutation, production whichdisappears entirely iftheAqinlOl phage isQ- (Table 7). This production ofendolysin is low(11 units), aswould beexpected in view of

the low dosage ofgene R+ present in the cell. Indeed, thepresenceof thec17 promoter

muta-tion,which enables thephagetoreplicateeven

in the presence ofimmunity, greatly improves

the endolysin production (Table 7). Moreover, the Xcl7qinlOl phage gives high phage bursts under immunity and is Nindependent for its development (data notshown). We also

exam-ined the trans effectofthe Q product synthe-sizedbythe homoimmuneXqinlOlQ+ phageon an R+ gene located on the prophage. In this

experiment, the strain islysogenic forAR+

pro-phage, and the AqinlOl infecting phagecarries

an R- mutation (Table 7,part 2).Itwasfound

that AqinlOl Q+R- phage completely fails to elicit endolysin expression in trans. However, thepositiveresult obtained with the c17 deriv-ative shows that this trans expression is

[image:5.501.266.461.285.663.2]

com-pletelydependentonthe numberofXqinlOlQ+

TABLE 6. Lysogenization by XqinlOlderivativesa CA244.1infected with: Lysogeny(%)

Ac+orXc+Q- 35

Xc+qinlOlQ- 33

Ac+qinlOlQ+ <0.05

Ac+qinlOlS-R- 5

Ac+qinlOlS-R-A- 26

aTheprocedureused isdescribed in thetext.The

percentage oflysogenyrepresentsthe numberof

ly-sogensdividedbythenumber ofinfectedcells.Q- is Qam2O3,S- isSam7,R- isRam5,and A-isAamll.

copies present. The fact that this transactivation isnotseenwith the productof a single Q+ gene shows thatQactspreferentially in cis, in agree-ment withprevious observations(7, 9).

In theKexperiment, the prophage constitu-tively provides the K gene product, and we tested the growth characteristics of a heteroim-mune superinfecting phage which was neither able to synthesize the K product itself (being

K)norabletotransactivate theK+ gene of the Aprophage (since its Q product has a different specificity: Q21). Thus, we infected the

Xinm21c+qin1O1Q+SR7A-K`

lysogen with a

hybridphage A(QSR)21K7 and looked for

com-plementation of the Kdeficiency by the K+ gene ofthe prophage, inAK progeny. This comple-mentationbyauniqueK+gene iseasily detect-able in view of thecatalytic character of the K function. The yield of the superinfecting

X(Q)+21K-

phagewasgreatlyimproved when the

prophage

carriedthe

qinlOl

mutation

(Table

8)

TABLE 7. R experimenta

Endolysin activity Strain (units/100 ug of ly-sate) atloomin Part 1: CA244.1 recA (Ac'R)

in-fected with:

Ac+ <2

Ac+qinlOlQ+R+ 11

Xc+qinlOlQ- <2

Acl7qin1O1Q+R+ 570

Ac17 <2

Part 2: CA244.1 recA (Ac+R+) in-fectedwith:

XqinlOlQ+R- <2

Acl7qinlOlQ+R- 8

aInfection

experiments

were performed as de-scribed in the text. Q- isQam203,and R- isRam216.

TABLE 8. Kexperimenta

Yield(phage/bacterium)after infectionwith

A(QSR)2,K-phage

Bacterial strain

re-Super- combinants infecting or

type revertants

CA244.1 0.1 0.02

CA244.1(Ximm2l) 0.5 0.3

CA244.1 (21c+) 7 0.3

CA244.1 9 0.28

(Ximm21qin1O1S-R-A-)

A K J immA Q S R

aSuperinfectingphage productionwastitrated on C600(21c+),andXK+ productionwastitratedon594

(21c+).Heterologoussubstitutions of the superinfect-ing phage DNA withADNA are given by the dark area.

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and even reached theoptimal values obtained

afterK' transactivation (Table 8).

The results of these experiments show that the late functions tested are expressed under immunity in the presence of the qinlOl muta-tion, confirming the promoter character of this mutation.

DISCUSSION

qinlOl isapromoter mutation located in the

P-Q regionwhich allows expression of Q and of

the late genes. The levels of endolysin

synthe-sized by XqinlOlQ-phage suggest that late gene

expression, in the presence of this promoter,

results in a two- to threefold increase in the level of expression over that observed with Q- phage (residual levels of endolysin are synthesized by phage as a result of the leakiness of the

Q-mutation). Although the increase of late

tran-scription broughtabout by the qinlOl mutation

isrelativelysmall, it is nevertheless sufficient to

make qinlOlQ- phage plaque formers, in con-trast to Q- phage. Moreover, the fact that the

XqinlOlQ- phagecontinues tomake plaques in

a ron host, which completely eliminates the

leakiness of the Q- mutation, shows that the

level of late transcription, resulting from the

presenceofthe qinlOl promoter, is sufficient to allow the growth ofaQ- derivative. This

con-stitutive late expression does notalterthe

via-bility of qinlOlQ- lysogens. Late expression,

which isnormallyamplifiedbyactive replication inthe absence ofimmunity, is notamplified in

theXqinlOlQ- lysogenas aresultofthe presence

of immunity.

Endolysin titrations with XqinlOlQ+ phage

showed that, after superinfection ofnormal

ly-sogens (and transactivation experiments), this

phage produced significantamountsofQ

prod-uct.It is thereforenotsurprisingthatqinlOlQ+

lysogens are nonviable. However, qinlOlQ+

ly-sogenswithmutationally inactivated S,R, and A genesareviable.We have also shown that the

qinlOlQ+phage under immunity constitutively

expressesthelategene K.

Howcan weexplainthe molecular mechanism

bywhichtheqinlOlmutation

gives

risetolate

gene expression? We propose that the

qinlOl

mutation, by promoting a reinitiation of

tran-scription before gene Q, increases

Q

mRNA

synthesisto alevelwhichresultsin the

produc-tionofasmallamountofQ productdespitethe presence of the Qam mutation. These few Q

moleculeswould then allow thepartialrelief of

the lateterminator,resultinginlate gene

tran-scription.

This explanation is consistent with the

ab-sence of growth of qinlOlQ- phage on strA

hosts. Indeed, sinoe the strA mutation greatly decreases the degree of misreading of codons in general and of amber codons in particular (11),

onewould expectXqinlOlQamphageto synthe-sizeless Q product in the strA host and conse-quently fewer late proteins. Furthermore, our explanation is consistent with the absence of growth ofXqinlOl phage containingtwoamber mutations in gene Q (data not shown). The presence of such adouble mutation in gene Q should reduce the amount ofQ proteins synthe-sized.

ACKNOWLEDGMENTS

We aregratefultoR.Thomas forhelpfulsuggestionsand constructive criticism of themanuscript.We also thank M. Couturier and F. Salomon forstimulatingdiscussions,L. Des-metforconstructingdifferentphagederivatives,and 0. Dou-bleday for correcting the paper.

ThisworkwascarriedoutundercontractEuratom-U.L.B. 156-761BIOB and an agreement between theBelgian Govern-mentand theUniversiteLibre de Bruxelles "Actions de Re-cherchesConcertees."

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Figure

FIG.aoactivationrepresentstranscription 1. Genetic map of A prophage. Arrows indicate the origin and direction of transcription
TABLE 2. Growth of XqinlOlQ
FIG. 2. Mapping ofqinlOl in respect to byp3 and bypE.
TABLE 7. R experimenta

References

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