Genetic and physiological control of host cell lysis by bacteriophage lambda.

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JOURNALOFVIROLOGY, Sept. 1977, p. 626-636

Copyright01977 AmericanSocietyforMicrobiology Printed inU.S.A.

Genetic and

Physiological Control of Host Cell Lysis by

Bacteriophage

Lambda

BARRY G. ROLFE ANDJOHN H. CAMPBELL*

Genetics Department,Research SchoolofBiological Sciences,TheAustralian NationalUniversity, Canberra,

A.C.T.2601,Australia, andDepartmentofAnatomy,SchoolofMedicine, University of California,Los

Angeles, California90024*

Receivedforpublication23February 1977

The timing of host cell lysisatthe end ofthe lytic cycleofphageX is under

complex control.The ASproteinstimulateslysis.Anotherphysiological system, the lysis regulator, inhibits lysis from occurring prematurely. The effects of a

seriesofphage and bacterial mutations on these controls aredescribed. They

showthat the Xrex geneplaysa role inregulating lysis under suboptimal growth

conditions. In certain mutant cells, and especially under anaerobic culture

conditions, the rexgene aids in thescheduling of host celllysis. The data also

suggestthat thelysis regulator maycontrol the transition of theASproteinfrom aninactiveto anactivestate.

In a series of papers we are reporting our

studiesonthe control of host cell lysis by bacte-riophage X.When this phage lyrically infects its host, Escherichia coli, itreplicates intracellu-larly for aperiod oftime knownasthe latent period. The cells then lyse at a genetically scheduled time. Controloverthe timingof lysis is complex. An important element is the AS gene product, which is a positive effector of

lysis. Itisthoughttoaidinthe sudden

trans-missionof aphage-specified lytic enzyme (endo-lysin) across the membrane to the cell wall, whereithydrolyzescross-linkages (13, 14, 19). The AS protein ismade throughout the second half of the latent period, butisinactive or inef-fective until the scheduled time forlysis.

Dur-ingthistime it canbeartificially activated by

cyanide. Cells infected with wild-type phage but not with AS mutants lyse promptly when exposed to cyanide later in the latent period (14).

Wehave describedaninhibitory system,the

lysis regulator, which prevents lysis from

oc-curringprematurely (5). The lysis regulator is

athermallysensitive systemandrequires

con-tinuous protein synthesis to block lysis. Thus,

infected cellslyse ahead of schedule if the lysis regulator is disrupted by exposing the cells to elevated temperature(480C)or to

chloramphen-icol. Interestingly, these agents cause slow

lysis eveniftheAS gene is mutationally

defec-tive. Also, AS mutants can lyse a variety of

mutant host strains to some degree. These re-sultssuggestthatthereare twopotential

path-ways for triggering lysis. One requires the AS protein to be in an active state. The other is

independent of the S protein, but both are

in-hibitedby the lysisregulator.

Thebacterialmutationsthatmake cells per-missivehosts forAS- phagespresumably inac-tivate genesthatinfluence thelysis regulator. These mutations alsodecreasethe sensitivityof cellstocolicinK, but otherwise are diverse in

phenotype (16). We have suggested that the

Arex geneisalsoacomponentof thelysis regu-latorysystem (5). The Arex gene isnotable for being cotranscribed with the XcI repressor

gene in a Alysogen when the other phage genes

are repressed (7). Its precise fiction is still unknown, althoughrex gene expressionalters the timing oflysis by phage T1 growing inX lysogens(6). Inthe presentstudywe character-ized therexactivityand timing of hostcelllysis after theinductionofaseriesofmutantA lyso-gens to further understand the controls over

lysis.

MATERIALS AND METHODS

Strains. Thephage strains usedinthisstudyare listed in Table 1, together with the properties by which the various phage mutants were originally selected. All of the A phages carry the cI857 ind-allele. TheAach-4phage wasobtained by spotting about 109 cI857phage particles on a lawn of AN259 cells on LB agar (11) at pH 4.5 and isolating the phage from one of the several dozen resulting plaques. Superficially this mutant displays a nor-mal phenotype on wild-type host cells at neutral pH. Weshalldescribe its mutant properties at low pH elsewhere. TheXclo("clock")mutants wereobtained by selecting for mutants ofXcI857 phage with a shortened latent period (5).

Three strains ofE. coli K-12 were used as hosts for 626

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HOST CELL LYSIS BY PHAGE A 627

TABLE 1. Strains used

Strain Pertinent properties Source/reference

E.coli K-12

tolB Pleiotropic, multicolicin tolerance 1

tolA Pleiotropic,multicolicin tolerance 1

tolP Pleiotropic,multicolicin tolerance 1

cyaA Adenyl cyclase negative (GP1allele) 20

uncA Uncoupled oxidativephosphorylation 2

uncB Uncoupledoxidative phosphorylation 3

unc-405 Uncoupled oxidativephosphorylation 3

rep-5 Defective for certain phage and plasmidchromosome repli- 4

cation

mutU Increased mutation frequency 17

uvrD UVsensitive 17

Wild type Isogenic parent or sister strains for each above bacterial mutant

AN259 argH entA strA 5

QD5003 SuIII M. Howe

233 trpE Su- C.Yanofsky

Phage

XcI857 Thermally inducible Sussman and Jacob (18)

XcI857susS7 Lysisdefective Goldberg and Howe (8)

XcI857 ts9B Lysis defective Harris et al. (10)

XcI857rexQ Nonexclusion of phageT4rII Gussin and Peterson (9)

XcI857 rex5a Nonexclusion of phageT4rII Gussin and Peterson (9)

XcI857clo-2 Premature lysis Campbelland Rolfe (5)

XcI857clo-4 Prematurelysis Campbell and Rolfe (5)

XcI857 clo-9 Premature lysis Campbelland Rolfe (5)

XcI857 ach-4 Forms plaques at pH 4.5 This paper

T4+ Wild type C. Fuerst

T4rII rIIrapid lysis mutant C. Fuerst

aAll A phages also carried the ind- allele.

mostof the experimentsinthisstudy:AN259

argH-entA strAr,QD5003 SuIII+, and233trpE-Su-. The

othermutantbacterialstrains, which allowphage

XsusS7toplate(16),arereferredtobytheirrelevant

mutantgene.Theirprincipal phenotypic character-istics (most ofwhich are pleiotropic) are given in Table 1. Lysogens were constructed in a

conven-tionalmannerand tested for levels of Xrex activity

by comparing the plating efficiencies on them of

phagesT4+andT4rll(5).

Measurement of latent periods. Studies on the timing of host cell lysiswere carried out on

ther-mally induciblelysogens carryingthe

temperature-sensitive XcI857 allele of the repressor gene.

Log-phaseculturesweregrownat30'Cto anappropriate

optical density inLBbroth. Aerobiccultureswere

shaken rapidlyinsidearmflasks inanoscillating

waterbath. Anaerobic cultureswere sealed in cu-vettetubes (1by30cm)andkeptforatleast 1hat

300C to use up all oxygen before induction. The cultureswerethenshiftedto40'C,and theiroptical

densitieswere measured periodically in a Unicam SP600 spectrophotometer. Insomeexperiments po-tassiumcyanideorchloramphenicol (Sigma Chemi-cal Co.) was added to induced cultures to a final concentration of 5 mMor250

jtg/ml,

respectively.

Media. The other microbial techniques andmedia used have beendescribed elsewhere (5, 15, 16),

ex-ceptfor theglucose minimalmedium,whichwasM9

(11) supplementedwith 0.5 mMtryptophan and25

mMglucose.

RESULTS

Effect of bacterial mutations on Arexgene

expression.The series ofE. colimutantslisted

in Table 2 have previously been shownto be permissive hosts for AS- phages and

presum-ablyarepartially defectiveintheirlysis

regu-lators(5).Each of thesemutants,togetherwith its isogenic sisteror parent strains, was lyso-genizedwithXcI857rex+phageand tested for its abilitytoplate T4rll phage.Wild-type lysogens completely exclude T4rIIphage growth. Three bacterial mutations, tolA-, tolB-, and tolP-, allowT4rIIphagetoplateontheX lysogensat

the sameefficiencyas onthenonlysogens. The

tol mutations cause a dysfunction ofthe cell

membrane,resulting inmulticolicintolerance, sensitivitytovariousdrugsanddetergents, ab-normal cell shapes,decreasedgrowthyield,and other effects (1; Campbell and Rolfe,

unpub-lisheddata). Six othermutationsdecrease but do not abolish T4rII exclusion. We conclude

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628 ROLFE AND CAMPBELL

TABLE 2. ExpressionofXrexactivity in AS-permissive hostsa

Plating efficiency of

Permissivehost phageT4rII Parentallysogens 10-4

cya- (X) 10-4

uncB (X) 10-3

unc-405 (X) 10-3

uncA- (X) 10-3

rep--5 (X) 10-2

mutU- () 10-2

uvrD- () 10-2

tolP- () 1.0

tolA (A) 1.0

tolB- (X) 1.0

aXisXcI857. Alllysogensplatedphage T4+ at an

efficiency of 1.0. "Parental lysogen" refers to the

corresponding isogenic control strain to each mu-tant.In all cases the behavior of these controls was comparable to one another and to our reference strainAN259.

that thegreatmajorityof, butnotall, bacterial

mutationsthatinterfere with the lysis

regula-tor also depress the expression ofrex activity againstT4rllphagegrowth.

Levels ofrex activity of various X phage

mutants.A seriesofXphagemutantssimilarly

weretested forabnormal rex expression when carried asprophagesintwotypesofhostcells (Table 3). In the wild-type host a Xrex+

pro-phage completely prevents T4rII phage from plating, and the two Xrex mutants cause no

exclusion, as expected. The Aclo phages are

intermediateincharacterasthoughthey

spec-ify

reduced levels ofrex activity. The Xach-4 prophage shows no deficiency in T4rII exclu-sion. To test the possibility that this mutant

specifiesanextradegree ofrexactivityinstead ofadeficit,weintroducedit intoatolB- host. A wild-typeAphageisinsufficientto cause exclu-sion inthisbackground. The Xach-4 prophage does excludeand therefore mustexpress a

de-greeofrexactivity above that of thewildtype.

Curiously, the Aclo mutants, which show

re-ducedrex exclusion onwild-typecells, also

ap-pear to havehyper-rexactivity inthetolB host.

Thus, rex gene expression apparently can be

modified by mutation in several directions.

Even mutantsthat appear to havesimilarrex

phenotypes in standard indicator strains may

show demonstrable differences in other cell

backgrounds.

Latent periods of mutant A phages. A

changeinthe durationof thelatentperiod of an

inducedmutantlysogenis asensitive indicator

ofanalteredregulationof hostcelllysis. Figure

1A shows a comparison of the host cell lysis

patternofaArex-lysogenwithaArex+control

lysogen inducedin aerated broth. Cultures of the lysogensweregrown to earlylog

phase

at

30°C and then shifted to40°C to thermally

in-duce theprophages. Theopticaldensities of the cultures continued to increase for a

period

of time after induction while phage

replication

proceeded. They then fell precipitously at 41 minasthecellslysedin

synchrony.

Clearlyan

intact rex gene is notessential for thenormal

schedulingof hostcelllysis undernormal

aero-bic culture conditions. However, the

inactiva-tion of therex gene candelay lysisunder

var-iousspecializedgrowth conditions, suchas

an-aerobiosis (Fig. 1B) and in glucose minimal medium (Fig. 1C).

Figure 2 shows host celllysis curvesfor an

induced

Xclo-4

lysogen. Herelysisispremature under aerobicconditions anddelayed anaerobi-cally. As is the casefor Xrex mutants,

altera-tions in the scheduled time of lysis are not

accompaniedbyasignificant lossinthedegree ofsynchrony of lysis.

Table4summarizesthelengths of latent

pe-riods for the various phagemutantsexamined. Both early anddelayed lysis occuramongthe phagesunder aerobic conditions, whereas all of the alterations observedinthe absenceof

oxy-gen are delays in lysis. In general, anaerobic

conditionsaremoresensitive thanaerobic

con-ditions fordemonstrating changes occurringin

the timingoflysis ofphage mutantswith

al-teredrex expression.

Hostcelllysisby AS- phages:phage AsusS7.

TABLE 3. ExclusionofphageT4rIIby various

mutantAprophagesa

Plating effi- rexalteration of Lysogen ciencyof phage mutant

pro-T4rII phageb

tol+(X) 10-4

tol+ (Xach-4) 10-4

tol+ (Aclo-2) 10-2

-tol+(Aclo-4) 10-2

-tol+(Aclo-9) 10-2

tol+ (ArexQ) 1.0

-tol+(Arex5a) 1.0

-tolB- (X) 1.0

toiB- (Aach-4) 10-4 ++

tolB- (Aclo-2) 10-4 ++

tolB- (Xclo-4) 10-4 ++

tolB

(Xclo-9)

10-4 ++

tolB- (ArexQ) 1.0

tolB- (Arex5a) 1.0

aAlllysogens plated phageT4+atanefficiency of

1.0.

b_,

Indicates lesser exclusion by mutant pro-phage than by wild-type propro-phageinthat cell

back-ground + + indicatesagreaterdegree of exclusion.

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HOST CELL LYSIS BY PHAGE X 629

0.3

E

0

0 0

a,

u 0.2

-o

a C01

0.1

0.0 minutes after heat induction

0 20 40 60 80 100 120

minutes after heat induction

0 10 20 30 40 50 60 70

minutesafter heat induction

FIG. 1. Host celllysisprofiles of induced Xrex+ and Xrex-lysogens.(A), Aerobic culturesin Lbroth; (B) anaerobiccultures inLbroth; (C)aerobic culturesinglucose-minimalmedium. Symbols: 0,233(XrexQ);

*,233(XcI857).

The XsusS7 phage hasasuppressiblemutation

inthe S gene. Itisunable to form plaqueson

Su- host cells, but will plate on the SullI+

strainQD5003 (8).

Even in thepermissive host,S geneactivity

isnot normal. Figure3 shows acomparison of

the host cell lysis patternsof XsusS7 and XS+

phages in strain QD5003. The XsusS7 phage

has a distinctly shortened latent period.

An-other indication that the S gene product

syn-thesized in QD5003 cells is not normal is its

inabilitytocauselysisat48TC(Fig. 4). Shifting

thetemperature ofwild-type lysogens to48TC

at 23 min after prophage induction does not

delay lysis. In fact, it stimulates lysis (5). In

contrast, shifting the temperature of induced

0.4

0.3

E

0

-o

u

o 0.2

-oE

0

-o

a

0.1

0.0

0.3

E 0.2

0 0 10

~0

° 0.1

0

0.0

L

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0.3L

0 0

0.2

-o

C

0

0.1

oco

0 10 20 30 40 50

0.5 c 0.4

C

0 0

w 0.3

0

-o

° 0.2

0.1

0.0

0 10 20 30 40 50 60 70

minutes afterheatinduction

FIG. 2. Host celllysis profile ofAN259 (Xclo-4) lysogen. (A) Aerobic cultures; (B) anaerobic

cul-tures. Symbols: 0, AN259 (AcI857); 0, AN259

(Aclo-4).

QD5003 (XsusS7) lysogenspreventslysis.

More-over, ifthe inhibited cellsarethenreturnedto

4000,they promptlylyse. Thus, high

tempera-ture converts the suppressed S7 protein to a

reversible inactivestate. Leavingthe cells for

anextendedlength oftimeat48°C resultsina

diminution of the completeness of lysis upon

returnto40°0. Thismayindicateaslow,

nonre-versible denaturation of theS7geneproduced,

or it may merely reflect a slighttemperature

sensitivity ofsomeotherstepinlysis.

Induced QD5003 (XsusS7) lysogens also

re-spondabnormallytochloramphenicol and

cya-nide. Addingchloramphenicol towild-type

ly-sogensat 23 minafterinductioncauses a

syn-chronous lysis 5 to 10 min later. In contrast,

chloramphenicol stimulates only a very

asyn-chronouslysisof theinducedQD5003 (XsusS7) lysogen (Fig.5).Approximately half of the cells lyse earlier than they would ifchloramphenicol hadnotbeen added, butasignificant fractionof

TABLE 4. Latentperiod of various Xphagemutantsa

Latentperiod (miAb Phage strain

Aerobic Anaerobic

A 41 45

Xclo-2 39 43

Xclo-4 34 52

xclo-9 37 47

Xach-4 41 50

XsusS7 300 NTc

Xts9B 125 NT

XrexQ 39 56

Xrex5a 40 48

aAisXcI857, and the hoststrain is either AN259 orAN233.

°Lengthoftimeat40'C foralog-phasecultureof thelysogentofalltoone-halfof its maximaloptical density after thermal induction of the prophage. Measurements areaccurate towithin 2min aerobi-callyand 5minanaerobically.

cNT, Not tested.

0.3

c 0.2

0 0

u

C

-0I

0

9A 0.1

0

0.0

0 10 20 30 40 50

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minutes after heat induction FIG. 3.Latentperiod of XsusS7 phage in a

per-missive SuIII+ host. Symbols: 0, induced QD5003

(AsusS7)lysogen;0,inducedQD5003 (AcI857) lyso-gen.

A

aerobic induction

/N

*

IN~~~~~~~~~

-.

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HOST CELL LYSIS BY PHAGE A 631

0.5

0.4

0.3

E

c

0 0 o4

:0.2 \ \ :\ 48

(20').

40

02

0

48°(10').

40-0.1

548'(5_)

40

400

0.0 I L

0 20 40 60 80 100

FIG. 4. Temperature sensitivity of the

SuIII+-sup-pressedXsusS7 geneproduct. At 23 minafter

induc-tionat40°Ccultures ofQD5003 (XsusS7)lysogens

wereshiftedto48°Cfor theindicatedperiodsoftime before being shifted back to 40°C. Symbols: A, at

48°Cfor 5 minbeforebeing returnedto40°C; 0,at

48°Cfor10minbefore being returnedto40°C; 0,at

48°C for20 min before being returned to40°C; *,

controlleftat48°C;*,control40°Cculture.

the cells actuallyaredelayedinlysis by

expo-sure to it.Chloramphenicolalso is ineffectiveat

stimulatinglysisof these cellsat480C. Cyanidecausesimmediate lysisof

wild-type

lysogens when added at anytime after20min postinduction. It slightly hastens premature lysis of induced QD5003

(XsusS7)

lysogenslate

inthelatentperiod (28min orlater),buttends toblock, more than stimulate, lysis whenadded

at 23min (Fig. 6).

Clearly, the SuIII+ suppressor does not

re-store anormalphenotypetotheXsusS7phage.

Itseems unlikely that the remaining defectis

simply a lower quantity of AS gene product synthesized (since lysis is premature and not delayed). Instead, the

suppressed

XsusS7 phage-infected cell seems to be deranged in

various aspects of the temporal control of S protein activity.

Hostcell lysis by AS- phages: phageAts9B. The phage Xts9B has a temperature-sensitive mutation in the S gene and will not form plaques at elevated temperatures (13, 14). When aninduced culture of a Xts9B lysogen is maintained at 400C, the latent period is ex-tended for at least several hours. If the temper-ature isreduced to 100C during this period, the cellslyse immediately.Remarkably, a shift to a low temperature even before the normal sched-ule time for lysis initiates an immediate asyn-chronous lysis (Fig. 7). If the temperature is dropped at 23 min after induction, it takes 17 minfor half of the cells to lyse. Lysis is more synchronousifthe temperature is dropped later in the latent period, and the cells lyse simul-taneously by 41 mm, the normal scheduled time of lysis by phage XcI857. Dropping the temperature ofaculture of aninduced control XcI857lysogendoes not cause lysis.

Adding cyanide to induced wild-type lyso-gensduringthesecond half of the latent period stimulates a lysis to occur prematurely, pre-sumably by activating the S protein ahead of schedule (13). Cyanide can also trigger the lysis of induced Xts9Blysogens even atanelevated, nonpermissive temperature (Fig. 7 and 8).This effectisseenonly ifcyanideisadded after the normally scheduled time for lysis (41 min). Adding cyanide earlier than this time does not havea significant effect, even ifthe

tempera-04

0.3 E

c

0

02

'.2

C

01

00

0 10 20 30 40 50 60 100 110 minutesofter heot induction

FIG. 5. Decreased sensitivity of inducedQD5003

(AsusS7) lysogens to premature lysis by

chloram-phenicol. Symbols: 0,after23 min at40°C, cultures

wereshiftedto48C; A,exposedtochloramphenicol

at40°C; 0, exposedtochloramphenicolat48°C;or

*,leftat40°Cas acontrol. VOL. 23, 1977

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632

0.25

30 0.20

0 0.15

0

-~0.10

0.05

0.0

0 10 20 30 40 50 60

FIG. 6. Stimulation of lysis of induced QD5003 (XsusS7) lysogen by cyanide. A culture ofQD5003 (XsusS7)lysogenwasmaintainedat400C untillysis

wascomplete (a).Parallel cultureswereexposedto5

mM cyanide at 23 min (0) or 27 min (0) after inductionasindicated.

ture is droppedimmediately after itsaddition (Fig.9). Evenincubatingtheinducedlysogens

withcyanideat40'Cfor 30 mindoesnotaffect

the kinetics of lysis when the temperature is

subsequentlylowered (Fig. 9). Thus, theXts9B mutant displays a complex abnormal

pheno-type at a low temperature as well as athigh

temperature.Atalowtemperaturethe induced

lysogens are defective inthe mechanism that

normallydelays lysisuntil the scheduled time.

Also, cyanide willnotstimulate the lytic

proc-essata lowtemperature, although it still will

atahigh temperature.

Effects of certain bacterial mutations on

thephageXlatent period.The seriesof

bacte-rialmutationsthat interfere withXrex

expres-sion were tested for influences on the latent

period ofphage X (Table 5). Themoststriking

effectswere seenwithtwo unc-mutations

(un-coupled oxidation phosphorylation).

Aerobi-cally, the latent period isslightly shortenedby

uncA- and uncB- mutations. Anaerobically,

E

a)

E

0

>

a)

E 25 [

20 L

15

10

5

0

0 20 40 60 80 100 120

FIG. 7. Time course of sensitivity of induced Xts9B lysogens to lysis by a low temperature and

cyanide.At thetimesindicatedafterprophage

induc-tion, cultures ofinduced AN259 (Xts9B) lysogens

were shifted from 40 to 15'C (U) or given 5 mM

potassium cyanide(0), and the length oftime for the

optical densitytofalltoone-half its maximal value is

indicatedbytheordinate.

60' 06

40'

05 23 4 E

0.4 ~0

0.2 0.1 00

0 20 40 60 80 100 120 140 160

[image:7.503.63.257.58.368.2]

minutesafterheotinduction

FIG. 8. Lysis of induced Xts9Blysogensby

expo-suretocyanideat400C. Atthe indicated timesafter thermal induction of cultures ofAN259 (Xts9B) lysogens,potassiumcyanidewasaddedtoa concen-trationof5mMand thetemperaturewasmaintained at40TC. Symbols: control maintainedat400C ( 0); cyanideaddedat23 min (0), 30 min (*),40 min (0), and 60 min(c).

lysis isdelayedsomuchthat the latentperiod

cannotbemeaningfullymeasured.Incontrast,

a lack ofoxygen delays the lysis ofwild-type

lysogens by onlyafewminutes.

,a1

0f,

. . * .-8 *

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HOST CELL LYSIS BY PHAGE X 633

Mostof the other bacterial mutations tested also affect the duration of the latent period. Underaerobic conditions, some promote earlier lysis and others delay lysis. Under anaerobic conditions, only delays in lysis were observed. The rep--5 mutation is unique among those tested in that it affects rexexpression, but does not noticeably alter the latent period of A

0.6 0.5

* 60'

E 0.4

-0

0 0O.3

A. 0.2

-o a

I . .a

0 20 40 60 80

[image:8.503.51.237.77.365.2]

FIG. 9. Effect ofcyanideonlatentperiodofXts9B

phageat100C.A cultureofAN259 (Xts9B) lysogen

wasincubatedat400Cfor30 minanddividedinto

fourparts.One partwasshiftedto10C(*);asecond

wasexposedtocyanideandplacedat100C (C>),and

thethirdwasincubated withcyanideat409Cfor30 minand thenshifted to100C (0). The control was

maintainedat40"C (0).

03

20.2 *X

0

0.

phage. Thus, abnormal rex expression is usu-ally, but not always, associated with the abnor-mal scheduling of host cell lysis in mutant bac-teria.

Host cell lysis of tolB- cells by A phage mutants.The mutations in the tolB, -A,and -P genes are the most extreme onesat abolishing rex activity, sensitivity to colicin K, and the requirement for S protein for Aplating.Induced tolB- (X) lysogens also have anodd pattern of lysis. Lysis begins early andcontinues gradu-allyfor 100min.Two of theAmutantsthat are able to express rex activity in tolB- cells

(Xach-4 andXclo-2) partiallyovercomethe tolBeffect onhost cell lysis(Fig. 10).InducedtolB- (Xach-4)lysogensgive anessentially normallysis pro-file, whereas induced totB- (Xclo-2) lysogens give a compound lysis pattern. A slow asyn-chronouslysis begins earlyinthelatentperiod, similar to that occurringin theinduced toEl-(X) lysogens. This lytic process is then inter-rupted by a more synchronizedlysis of the

re-TABLE 5. Latentperiod ofphageXcI857in various mutanthost cells

Latentperiod (min) Lysogen

Aerobic Anaerobic

Wild type (x) 41 45

rep-5 (A) 41 42

mutU (A) 52 73

tolB (X) 65 77

uvrD (A) 54 82

uncA (X) 36 >1,000

uncBh

(_)

37 >1,000

aXiskcI857.

0 10 20 30 40 50 60 70 80 90 100 110

FIG. 10. Hostcelllysisprofiles ofvariousinduced tolB-lysogens. Symbols: *,tolB- (Xc1857); U,tolBE

(xclo-2); O,tolB-

(Xach-4);

0,

tolBs

(XcI857).

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634 ROLFE AND CAMPBELL

maining cells as occurs in the tolB- (Xach-4)

cultures.

DISCUSSION

Previousphysiologicalstudies have indicated that host cell lysis is controlled by a double regulatory mechanism. The XS gene product is

apositive regulator that promotes the lysis of wild-type cells.Thelysis regulator is the second

componentwhose activity blocks lysis.

Phage-infected cellscanbetriggeredtolyseahead of schedule by blocking protein synthesis or by briefly exposing them to a high temperature. An interpretation of thesedataisthat thelysis regulator is a temperature-labile protein, whose continuous synthesis is necessary for blockingprematurelysis. Accordingly, phageX

should havetwopotentialavenuesfor

trigger-inglysis (activating the S system and blocking the action of the lysis regulator). By using var-ious phage mutants in a tolB- host, we con-firmed that induced lysogens indeed canshow

twotypesof control over lysis. These two

proc-esses aredesignatedlysis I and

lysis

II inTable

6. LysisIoccurs ata genetically scheduled time andresultsinasynchronous lysis ofthe cells. It requiresafunctional S gene. Lysis II can occur

in acellat anytime after20 minfrom

induc-tion, is insensitivetothelysisItiming mecha-nism, and causes cells to lyse asynchronously. Presumably lysis II isbroughtaboutby abolish-ing the inhibition of the lysis regulator. This inhibition requires the tolB+ geneproduct and probablyto alesserextentthe normal activities ofthe various other bacterial genes, whose

mu-tational inactivation allows XS-phagemutants toformplaques(5).Lysis IIisindependent ofS. The two patterns of lysis control exhibit a degree ofindependencein thattheycan occur

individually ortogether in aculture of phage-infected cells. For example, wild-type phage lyse wild-type host cells exclusively under S

TABLE 6. Patternsof host celliysisexhibited by various mutantXlysogens

Occurrence of lysis type: Lysogen

I II

toiB- (Xclo-2) + +

tol+(XcI857) +

-tolB- (Xach-4) +

-tolB- (XcI857) - +

tolB- (XsusS7) - +

tol+(XsusS7) -

-tol+(Xts9B) -

-control andlysetolB- cellsexclusively by lysis II. tolB- cells infected byXclo-2 phage actually

are susceptibletoboth patterns oflysis. At 45 min after induction, both lytic processes are

actually occurring simultaneously among the remaining cellsinthe culture(Fig. 10). Despite this evidence ofindependence, thetwocontrol mechanismsareclosely relatedphysiologically. For example, the tolB mutation of the host

drastically affects both control mechanisms, and theach mutation ofXreverses both of these

effectssimultaneously. Also, most of the other

hostmutationsthat enhancelysis IIto alesser

extent also affect thetiming of lysis mediated

by the S system (Table 5). Thus, althoughthe

two systems can be dissected by abnormal

ge-netic orphysiological factors, theyprobablyare

integrated into a single control system in the wild-type-infected cell.

We have suggested on the basis of circum-stantialevidence that theXrexgeneproduct isa

component of the lysis regulator system. The data presented above reinforce this model by bearing out the following three predictions aboutrex geneactivity.

(i) If rex canaffect the timingoflysis, then

one should be able to isolate phage mutants

that lyse early, and some of these mutants

should have altered levels of rex expression. Selecting mutants with ashortened latent

pe-riod has provedtobestraightforward. We have thermallyinducedaculture of a XcI857 lysogen andcollected the free phage particles thatwere

present at 13 min before the normally sched-uled time oflysis (5).

Individual phageswerepickedasplaqueson

sensitive cell lawns and designated Xclo (for "clock" phage). Threeout often phages

exam-ined had an altered rex expression as a pro-phage. Two of these had altered latent periods

in awild-typebacterial host. The third hadan

alteredpattern of host cell lysis of tolB- cells. The other seven phages were normal in both their timing oflysis and rex expression. Pre-sumably, theyweremerely XcI857 phages that passed throughourselection procedures.

(ii) A variety of bacterial mutations allow

AS- mutant phage to form plaques. We have

previously ascribed their permissiveness to an

impaired expression of the lysis regulator (5).

Aspredicted, most of these mutations also af-fect thetimingofhost celllysis byXcI857phage

at least under some conditions and interfere

withthe exclusionofT4rIIphage plating bya

Xrex+prophage (Tables2and4). Thediversity

of these mutations strongly suggests that the

joint impairment ofrex andlysisregulator

ac-tivityismore thanacasualcoincidence.

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HOST CELL LYSIS BY PHAGE x 635

(iii) rex- X mutants have been isolated by

Gussinand Peterson (9) asprophagesunableto

interfere with the replication of T4rII phages. The latent period of one of these mutants, XrexQ, is shortened under aerobic conditions

andlengthened underanaerobic conditions and

inglucose-minimal medium(Fig. 1). The other

possibly shows a marginally delayed host cell

lysis anaerobically (Table 4).

Although there can belittle doubt that the

rex geneisintimately relatedto the lysis

regu-lator, the nature of this relationship remains

obscure. Most mutations, both bacterial and viral, that affect the timing ofhost cell lysis also affectrex activity, but the reciprocal

rela-tion is not as strong.It ispossibletomutatethe

rex gene to a variety of forms without grossly

affecting the length of the latent period. The Xach-4, Xclo-2, and XrexQ phagesall lysetheir hosts at the normal time under usual

condi-tions.

One complexity of the rex mutants is that

they appear to represent more diversity than

simply variationsinthe level ofrexgene

prod-uct or "rex activity." For example, the Xclo

mutationsdecreasethe level of T4rII exclusion

inwild-typeE.coli lysogens, butincrease it in

tolB- lysogens. If the rex gene is capable of mutatingtoqualitativelydifferent alleles,even

ttrex-negative" mutants (selectedascausing no

phageT4rll exclusion) mayhavesome form of

rex activity. In fact, the two Xrex- mutants

studied here,

XrexQ

and Xrex5a, show

differ-ences in their latent periods. These mutants

presumably are isogenic, although they were

isolated after exposure to nitrosoguanidine, a

chemical thatfrequentlycausesmultiple

muta-tions (9).

The simplest role for rex consistent with these observations so far is that the rex gene

helpstoinsulate thelysisregulator from envi-ronmental or genetic factors that otherwise would disturb the timing oflysis. Under opti-mal growth conditions, Xrex activity is there-forecompletelydispensible.Thisview

rational-izestheinability of Gussin and Peterson(9) to

detect any effect of rex- mutations on the growth cycle of the X phage in theircarefully conductedstudy.

TheXclo mutantsshowthat thelengthof the

latentperiodisgenetically determined andcan

bealteredbymutations.Therefore, the

charac-teristic ofthe wild-type phage mustrepresent

anoptimalvaluesetbyadaptiveevolution. The

primary function ofthe rex gene might be to

ensurethat thisoptimal valueoccurs

irrespec-tive ofoxygen tension. Alternatively or

addi-tionally, a rexfunction may betopromote the

optimal latent periodacross an enlarged range

ofE. coli strainsoccurring in its native anaero-bic environment.

The complexity ofcontrol over host cell lysis

byXphage makesitimpossible at this point to

present more than a plausible model for the

control ofAS protein activity. The lytic

behav-ior of both S+ and S- X strains is compatible

with the following hypothesis. The S protein is

capable ofexisting intwo states, one active and

the otherinactive. During the latterhalfof the

latent period, Sprotein issynthesized and

accu-mulates in the inactive state due to the pres-enceof the lysis regulator. At41min an altera-tionin thephysiology of the cell either

inacti-vatesthelysis regulator ormakes theSprotein

insensitive to the presence of the lysis regula-tor. Poisoning the cell with cyanide can also

convertthe Sprotein totheactive state

prema-turely. The active S protein insults the cell membrane and allows theendolysintodestroy the cell wall.

The S protein of the Xts9B is altered in its

activation properties. A high temperature

causes it tobe more difficultto activate.

Nei-ther thepassageof41 min northeexposure to

cyanide alone will triggerinduced Xts9B

lyso-gens to lyse, althoughthe combination of the

twowilldo so (Fig. 8). A low temperature

fa-vors the active state. The S protein becomes

activeafterashorterspanoftimethannormal,

but no longer demonstrates activationby

cya-nide. Possibly, at alowtemperaturetheS-ts9B protein spontaneously occurs in the activated

state that the wild-type protein assumes in a

cellexposedtocyanide.

TheS-7 proteinsynthesized in anSuIII+ cell has a derangement in its activity similar to

that of the S-ts9B protein. It isreversibly

con-vertedto aninactive statebyanelevated

tem-perature, in which it cannot be activated by

agents that normally trigger the mechanism

(Fig.5). At alowtemperature itsactivityisless

sensitive torestraintby thelysisregulatorand

tostimulationbycyanide and chloramphenicol. These parallels between twoindependentAS

mutants strongly suggest to us alterations in

the conformational states ofan allosteric

pro-tein. Both theS-ts9B and the suppressedS-7

proteinsmight be described aspartially

"desen-sitized" (12) to agents that affect the

equilib-riumbetweenan activeandinactive state. It is

possiblysignificantthat theS7 andts9B

muta-tionsmapcloseto oneanotheratthedistal end

oftheSgene (13).

ACKNOWLEDGMENTS

Wethank PeterFokkerforhistechnicalassistance.

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636 CAMPBELL

This study wassupported by Public Health Service grant AI 13089from the National Institute of Allergy and Infec-tious Diseases.

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