Replication of T4rII Bacteriophage in Escherichia coli K-12 (λ)

(1)

Vol. 2,No. 4 PrintedinU.S.A.

Replication of T4rII Bacteriophage

in

Escherichia coli K-12

(X)

I

CLARENCE S. BULLER2 AND L. ASTRACHAN

Department ofMicrobiology, Case Western Reserve UniversitySchool of Medicine, Cleveland, Ohio 44106 Received for publication 9January1968

The defect ofT4rIIreplication in EscherichiacoliK-12(X) canbephenotypically

reversedby various supplementstothegrowthmedium.Arginine, lysine, spermidine,

and a number of diamines allowed varying levels ofrII replication. The best

re-version was obtained with 0.4 M sucrose in 0.002 to 0.005 MCa++. Monovalent

cations severely inhibited reversion. A cell surface site of polyamine action is

consistent with the fact that spermidineinhibits phageghost-inducedcelllysis and

withthefindingthatsufficientpolyamine isavailablewithin thecellstoallow

nor-malpatterns ofneutralization ofphage deoxyribonucleic acid, asdetected by the

polyamine content ofprogeny phage. Intheabsence of effective supplements,

rII-infected cells swelled and lostrefractility.The data indicate thataleakycellenvelop

is involved. No difference inmucopeptides ofuninfected K-12(X) andK-12was de-tected and, because the mucopeptide in r+ infected cellswasfound to be at least

partially hydrolyzed midway through the lytic cycle, it didnotappearthat therIl

defect concernedmucopeptide synthesis.The pattern of cellphospholipid synthesis

changes after phage infection, but no difference was detected between r+ and rII

with regard to biosynthesis of phosphatidylethanolamine and

phosphatidyl-glycerol.

When a population of rapidly lysing T4 bac-teriophage mutants are selected by plaque

mor-phologyon EscherichiacolistrainB,each indivi-dual mutant mapsin one of three separate gene

loci.Mutantsat oneof theseloci,therII mutants,

are readily distinguished because they cannot

replicate on a host

carrying

lambda prophage

[E. coliK-12(X)] and yetthey multiplyaswellas

wild type if the host (E. coli

K-12)

is not

lyso-genized with lambda. Thus, the wild-type rII gene product is required for lysis inhibition in

strain B, is essential for

replication

in K-12 (X),

and is entirely

dispensable

in E. coli K-12 (6).

The gene product and its function remain

un-known,but itsidentityhas beensoughtby

exami-nationof the varied biochemicalconsequences of

infection and by study of growth medium

addi-tions which in some way can substitute for rll

function and allow

replication

in E. coli K (X).

Garen (11) demonstrated that the addition of

highconcentrationsof Mg++tothemedium (0.03

to0.08M Mg++) couldovercometherIIdefect in

IA preliminary account of this work was

pre-sented atthe 1965 Annual Meeting of the American

Society for Microbiology (Bacteriol. Proc., p. 102,

1965).

2Present address: Department of Microbiology,

University of Kansas, Lawrence, Kan. 66045.

K-12 (X). By various criteria-deoxyribonucleic acid

(DNA)

synthesis,protein synthesis, respira-tion, and ultraviolet resistance-itappeared that the reactions of phage development proceed

normally in the absenceofMg++for the first 10

minof infectionbutthereafterare blockedunless Mg++ is added to the medium. Other cations,

suchasspermine, spermidine, anda homologous

seriesofmethylenediaminesfrom putrescine (C4)

and cadaverine (C5) to octamethylene diamine,

canalsophenotypicallycorrectthe rlldefect(2,8) if addedbeforethe 10th minof infection(2).The

different cationsvariedtremendouslyin

effective-ness,bothascomparedtoeachother andas

com-pared fromonelaboratorytoanother.Inthedata

wepresent,acomparison ofeffectivecompounds

indicates that the polyamines' function is not

specific and is related to cationic properties. Although the rII defect may be corrected as

lateas10minafterinfection,adifference between

mutantandwild-typeinfection can be detected at

3 min. Phosphorylation of acid-soluble

nucleo-tides is significantly lower at that time, and as a

result thelevel ofadenosine triphosphate (ATP)

is decreased (27;M.Colowick, personal

communi-cation), which may in turn account for the

de-creased uptake of Mg observed at 6 min (27).

The lowered ATP levels may also have some

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bearing on the fact that monovalent cations strongly inhibit correction of the rII defect (27). Energy would be required to pump out the excess monovalent cations and to pump in Mg++ (27) or polyamines (30).

Even though thelowered phosphorylation rate

isobserved so early after infection, it is not clear

that this is theprimary defect. Investigation of a

number of ATP-generating systems revealed no enzymatic differences between wild type and

mutant (27). It is possible that faulty membrane

repair or synthesis permits significant leakage and thereby leads to a cascading series of defects. Supporting this view are the reports that

pu-trescine (2) andnicotinamide adenine

dinucleo-tide (27) are extensively released from rII infected

cells. We present data that indicate some swelling

and loss of refractility in these cells.

Further-more, thefact thatosmotically active compounds

such assucrose and otherpolyols (27) can correct

therII defect also pointsto apermeability

prob-lem.

In this paper, we present our data on the

effectiveness of various compoundswhich correct

the rIIdefectin K-12(X).These andother

experi-ments presented here indicate that a faulty cell

membraneisinvolvedin the rIIdefect. Wefound

that T4 phage infectionhas aprofound effect on

phospholipid biosynthesis, but nodifference was

detected between r+ and rII with regard to

bio-synthesis ofphosphatidylethanolamineand phos-phatidylglycerol. However, we do refer in the

Discussion to preliminary results which reveal

thatr+ andrll-infected cells can bedistinguished

bytheextentof

cardiolipin biosynthesis.

MATERIALS AND METHODS

Bacteria andphage. E. coli K-12 (X) wasobtained

from I. P. Crawford. It is the K-12 prototroph of

Yanofsky and Crawford (34) and has been referred

to asstrainYmel(35). E.coliK-12,itsnonlysogenic

derivative, was isolated after curing with Xi434. E.

coli B, used for titration of bacteriophage, came

originallyfrom R.Herriott.E.coliBBfrom D. Krieg

and K12W1485 from I. P. Crawford were used to

prepare high-titer stocks of bacteriophage because

bothallowlysis inhibition withT4rIIphagemutants.

Wild-type T4r+ andvariousrII mutants were

ob-tained from the Benzer collection. T4r1993 has a

long deletion in the A cistron, r638 has a complete

deletion ofthe Bcistron,andr1272hasbothcistrons deleted. The Xi434anddirections for curingwere ob-tained fromDorothyFraser. T4phage grownonBB or W1485 werepurified by differentialcentrifugation

until they met the optical criterion of more than

2.2 X 1012 phage per optical densityunit at400m,u (14).High-titer stocks ofapproximately1013phage/ml were stored in dilutingfluid containing 0.1M NaCl,

0.001M MgCl92, and 0.0001M CaCl2 over a drop of

CHCl3. Phagewereassayedby the method described

by Adams (1). Phage ghosts were prepared and as-sayed by the method of Herriott and Barlow (15). Media. Tryptone-Na+contained 1% tryptone and 0.1 M NaCl. Tryptone-Mg contained 1% tryptone and 0.08M MgCl2. Soft and hard agar for plating phage and bacteria contained tryptone-Na+ plus 0.55 and1.2%o agar, respectively.

Miscellaneous chemicals. Putrescine, cadaverine,

spermine, and spermidine were purchased from

Nutritional Biochemicals Corp. (Cleveland, Ohio).

Hexa-, hepta-, octa-, and deca methylene diamines

camefromL. Light and Co. (Colnbrook, England).

Reversion ofrIl defect by supplements to medium.

E.coli K-12(X) was grown to 2.5 X 108 to 3 X 108 cells/ml in tryptone-Na. Cells were infected with a

multiplicity of 5 to 8 with T4 phage. After 5 or 6

minofshaking, anti-T4serum wasaddedfor an

ad-ditional 2 to 3 min. At 7 or 8 min after infection

(actual times and multiplicities given with data),

the cells werediluted 104-fold through 1% tryptone and then 10-fold into the final incubation mixture

containing 1% tryptone plus the supplements at the

indicated concentrations. The final mixture was aeratedfor 52minlonger, CHCl3wasadded, and the phage weretitrated on E. coli B. Thenumber of

in-fected bacteria was set equal to the difference in

viable cells before infectionand after 7 to 8 min of

infection. (Survivors ranged from 0.5 to 10% of

starting cells.) "Burst size" indicates the number of

phage perinfectedcell.

Polyamines in phage. Approximately 5 X 1013

to 1 X 10'4phage,purifiedasabove,werehydrolyzed

in 4 NHCl in sealed tubes at 105 to 106 C for 18 to

22 hr. The hydrolysates were evaporated to dryness

overNaOH-CaCl2 ina vacuum desiccator andwere

redissolved in water. After removal and washing of

char at the centrifuge, a sample corresponding to

3 X 1013to5 X 10'3phagewaschromatographed on Dowex 1-Na+ by a method to be described (L. Astrachan and J. Miller, in preparationi). Amines

were quantitated by the ninhydrin method (22).

Phospholipids ofinifected anid uninfected bacteria.

E. coli K-12(X) was grown with shaking at 37 C in

tryptone-Mg++ to 3 X 108 cells/ml and then was

divided intothree portions of 310 ml each. At time minus1 min, 1 mcof32Porthophosphate wasadded

to each flask. At zero-time, the cells in two flasks

wereinfectedat amultiplicityof sixwithr+orr1993;

and the third flask remained uninfected. All flasks

were aerated continually at 37C. At 5, 10, and 20

minafter infection (6, 11, 21 min after 32p), 100-mi

sampleswere pouredover2.9ml of 11 M HC104and

stirred inanice bath. Aftercentrifugation,

resuspen-sionin coldsaline, neutralizationwith 1 MNaHCO3,

and recentrifugation, the precipitates were extracted

with chloroform-methanol, 2:1 (32). The extracts

were washed once with one-third volume of water, after which thechloroform phasewasdried in vacuo, redissolved in chloroform, filtered through glass

wool, and then chromatographed on silica gel HR

(Brinkmann Instruments, Inc., New York, N.Y.)

thin-layer plates with a solvent mixture of

CHCl3-CH3OH-water (70:25:4). All operations were

per-formed under N2 in the cold wherever possible and

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BULLER AND ASTRACHAN

with distilled, N2-flushed solvents. Phospholipid bandsweredetected by stainingwith I2 vapors and

by radioautography. Eight separate channels were

developedfor eachsample.From fourofthechannels,

the gel bands containing phosphatidylethanolamine

(PE)andphosphatidylglycerol (PG)wereindividually

transferred to Kjeldahl flasks for ashing in 11 M

HC104 and subsequent phosphorus assay (12). Gel

from the other four channelswastransferred tovials

for counting in a liquid scintillation spectrometer. Thus, the specific activities are fromaverage values offour separate determinations each of total phos-phorus and radioactivity. Preliminary experiments

showed that the amounts of silica gel used did not

interfere with either assay. Two additional minor phospholipids were also detected and were clearly

separated on the thin-layer chromatograms. These

werenotfurtherstudied,otherthan toidentifyoneof

them as phosphatidylserine. In addition, therewas

always some radioactive material at the origin, but

this couldwell have been due to the minimalwashing

of the chloroform-methanol extract. PE and

phos-phatidylserine were identified by positive ninhydrin

reaction and extensive incorporation of '4C-labeled

serine. SincePE and PG arethe majorphospholipid

classes ofE.coli (17), theywerereadilynotedbythe

intensity of12 stainand the extent of32p

incorpora-tion.Theyweredistinguishedfrom eachotherbythe

ninhydrinreagent and the relative amounts of phos-phorus in each band (17). Furthermore, our PG co-chromatographedin the abovesolventsystemand

one other solvent system (28) with an authentic

sample of phosphatidylglycerol which was kindly

provided by John Law. The observed RF values

were orthophosphate, <0.05; phosphatidyl serine, 0.24; PG, 0.35; PE, 0.47; (?) (phosphatidic acid?), 0.60.

RESULTS

Growthof T4rHIonK(X) inpresenceo

.^permi-dine, diamines,and basic amino acids. Spermidine

and putrescine are normally foLnd in T4

coli-phage (3, 4) and in bacteria (5, 10, 13, 31). In

bacteriophage,these basiccompoundsappeartobe

involvedinneutralizationof thephosphategroups

of thephageDNA. Inbacteria, polyamineshave

beenimplicatedin the stabilizationoffragilecells

(18, 19) and ofprotoplasts (20, 23). Spermidine

and other polyamines have also been shown to

stabilize ribosomal complexes (9, 36). Since

spermidine in some systems behaves similarlyto

Mg++, it occurredtoanumberofinvestigatorsto

determinewhether thispolyaminecouldsubstitute

for Mg++ instimulatingthegrowthofrIIphages

in L. coli K-12 (X) (2, 8). In our experiments,

spermidine was foundto be effectiveat an

opti-mal concentration of0.03to0.05 M.Theyields of

rII progeny at these concentrations were usually

5to30phageperinfectedcell,representinga

100-to 600-fold increase over the control. These

results were obtained with mutants of eitherthe

A or B cistron and with r1272, an

rII

mutant in

which both cistrons are completely deleted.

In anattempt todeterminewhether spermidine

or

Mg++

satisfiesspecificrequirements,anumber

of other basic compounds were tested for their

ability to stimulate rII replication in K-12 (X).

These supplements were all compared at a

con-centration of 0.03

M,

which may not be optimal

for each one. Theresultsof one such experiment arepresentedin Table 1. In thehomologousseries

of methylene diamines, heptamethylene diamine

and cadaverine were most effective, whereas

decamethylene diamine was inhibitory.

Qualita-tively similar results werereported by Brock (8),

butshereported lower phage yields, presumably,

as we shall see later, because of the presence

of NaClinthe medium.Recently,thestimulatory

properties of putrescineandspermidinehave also

been recorded (2).

Several of the basic amino acids were also

tested. Of these, arginine was the most effective

supplement, allowinginthis experimentthe

pro-duction of41progenyphageparticlesperinfected cell. From one experiment to another, the burst

size with any of the effective compounds varied

as much as recorded above for spermidine, but

qualitatively the compounds were always

effica-cious. Nostimulationof

rII

growth was observed

with nonbasicamino acids. The action ofarginine, ornithine, andlysine is of interest, because they,

incontrast tothe other effectivecompounds,offer

noapparent osmotic advantage over monovalent

cations for the neutralization of fixed anions.

This isrelevant to the site of action of the

effec-tivecompounds andsuggeststhat it is not merely

neutralizationof DNA.

Phage content of putrescine and

speimidine.

Amesand Ames (2)reported that putrescine leaks

out ofacell infected with rII phage and that the

TABLE 1. Effectofdiamines, polyamines, and basic

amino acidsonburst size of

T4rJ993

in

Escherichia coliK-12(X)a

Amine Burst_size Amine Burst_size

Putrescine (C4)... 3.7 Spermine... 0.16 Cadaverine (C5)...54 Arginine....41 Hexamethylene diamine .28 Ornithine ... 9.1 Heptamethylene

diamine... 62 Lysine... 6.1

Octamethylene

diamine... 22 Citrulline... 0.08

Decamethylene

diamine... 0.006Histidine... 0.03

Spermidine... 5.2 Control. 0.03 aMultiplicity of infection = 7. Dilution and additionof0.03 M amine at 8 minafter infection.

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leakage isnotinhibited by

Mg++.

If the effective compounds function by replacing putrescine in

theneutralization of DNA, their presence would

result in a lesser amount ofputrescine packaged

withintheprogenyphage.Totestthispossibility,

T4r+ and rII phagewere grownintryptone-Mg++

medium within K-12 (X) orK-12 bacteria. After

purification by differential centrifugation, the

phagewerehydrolyzed with acid, and the

hydroly-satewaschromatographed onDowex-1 columns.

The data of Table 2show thatmutant and

wild-typephagehavesimilarpolyaminecontents which

donotvarywhether the host is K-12orK-12 (X), or whether the mutation is in the A cistron

(rl993) or B cistron (r638). Comparison of the

last two lines ofTable 2further reveals that the

presence of Mg++ in the medium does not alter

phage polyamine content. It is apparent that

mutant phagegrown either in K-12 (X) or inthe

permissive host K-12 have sufficient polyamine

available toallowanormal pattern of DNA

neu-tralization andincorporationintoprogenyphage.

The datastronglysuggestthat theinabilityofrII

togrowinK-12(X) under standard conditions is

not due to a lethally insufficient availability of

polyamines.

Admittedly,thedata donotsayanythingabout

polyamine concentrations under nonpermissive

conditions. This leaves the possibilitythat Mg++

mayrelieveapolyamine shortage by substitution

elsewhere in the cell. An argument against this

possibility is indicated by the stability of

poly-amine content in the presence or absence of

Mg++,

and withinmutant or wild-type phage.

Inhibition ofghost-induced lysis by spermnidine. Since the stimulatory effect of polyamines does

[image:4.485.252.447.73.184.2]

not appeartobe relatedtoDNA neutralization,

TABLE 2. Piutrescine andspermidine in T4

bacteriophage

Sample

Phage Host AMedium

r1993 K-12

Tryptone-Mg++

r638 K-12

Tryptone-Mg++

rl993 K-12(X)

Tryptone-Mg++

r+ K-12

Tryptone-Mg++

r+ K-12(X)

Tryptone-Mg++

r+ K-12(X)Tryptone-Na

Amtper1013

phage(pmoles)

u

E~~~~~

0.970.36, 3.0 2.7

1.120.37 3 .3 3.0

1.09'0.37 3.3 2.9

1.170.41 3.6 2.9

1.20 0.35 3.4 3.5

[image:4.485.51.244.449.639.2]

1.260.381 3.6 3.3

TABLE 3. Effect ofspermidine

onI

lysis of

Escherichia coli K-12(X) by

rJ993

ghostsa

Optical Incubation mixture ₍₆₆₀density_mpA)

at15min

Cells... 0.34 Cells + ghosts... 0.23

Cells + ghosts + spermidine

(addedat 3 min)... 0.35

Cells + ghosts + spermidine

(addedat end)... 0.28

Final concentrations in incubation mixtures

were 3 X 108 cells/ml, 1.6 X 109 ghosts/ml, 0.16

M NaCl, 0.05 M spermidine, and 1% tryptone. Total volume 4 ml. Mixtures were mechanically shaken at 37 C for 15min and absorbancy was read at that time.

other structures known to bind cationic

com-pounds were considered. It has been suggested

that, in this system, Mg++ may prevent cellular

leakage of essential components (11). To decide

whether a similar function can be ascribed to

spermidine, wemeasured itseffect oncell lysis by

phage ghosts. In Table 3, comparison of lines 2

and 4showsthat spermidine,which hasnoeffect

on the absorbancy of untreated cells, increases

the absorbancy of ghost-treated cells. This

phenomenon dims but does not obscure the

demonstration that spermidine inhibits

ghost-induced cell lysis, behavior consistent with the

idea that the effective siteofpolyamine action is

at thecellenvelope.

Replication of

T4rIi

in presence of sucrose:

effect of monovalent and divalent cations. To

determine whetherthecellenvelopeisrelevant to

therII defect, weattemptedto measurethe

effect

of amine supplements oninfected protoplasts. In

the course of theseexperiments, which were

un-successful because of excessive fragility of

rII-infected cells, certain controls revealed that

sucrose alonewould allow growth ofrII in K-12

(X). Table 4 shows that theoptimalsucrose

con-centration forrII growthwas0.4M,atwhich

con-centration average burst sizes in different

experi-ments rangedfrom 5 to 35 with a mode around

10. The averageyield ofr+ wasalsoincreasedby

sucrose, but at a lower optimalconcentration of

0.3 M. It appears from the inhibition of r+ by

sucroseat aconcentration of0.4Mthat anoptimal

concentrationfor rIIinvolves a balance between

stimulation of

rII

growth andinhibition of

host-cell metabolism.Athigher concentrations, growth

of both phages was severely restricted. Control

experiments showedthat the loweredyieldswere

not caused by phage death with high sucrose

concentrations.

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TABLE4. Effect of sucrose on growth of r+ and r1993 in Escherichia coli K-12 (X)a

Burstsize Sucrose concn

r1993 r+

0 0.075 187

0.3 3.6 253

0.4 7.6 140

0.5 7.4 52

0.6 1.2 11

0.7 0.1 0.9

Multiplicity of infection:

r1993

= 12,r+ =6.8.

Dilution andadditionof sucroseat 8min.

In our experiments, which partially overlap

two other reports (8, 27), we attained rII phage

yields intermediate between the results in the

other two reports. Themajor factor contributing

tothedifferentyieldswasprobablythe amountof

monovalent cation in the incubation mixture.

Whenitis considered that 1% tryptone contains

approximately 0.01 M Na+, the shape of the

curvein Fig. 1relatingrIIyieldin sucrosetosalt

concentration suggests that the yields would be

considerablyaugmented in the absence ofinherent monovalent cation. Sekiguchi (27) reported near

wild-type

yields

of rII in sucrose, but these

yields were obtained in a medium containing

only0.2% tryptone andconsequently significantly

lesssalt.This mayalso account for thesurprising factthatSekiguchi found0.15Msucrose tobeso

effective.With respecttospermidine,theinherent

Na+isofnoconsequence, but ifspermidine were

addedtothe usualmedium, which contains 0.05

to0.1 MNaCl for phageadsorption, veryfewrII

progenywould be formed. Theeffect ofMg++is

alsogreatly inhibitedby monovalentcations (11).

Although phenotypic reversion of the rII

defect was achieved with sucrose, the phage

yields inourexperiments didnotapproach

wild-type values. Since low concentrations of Ca++

added to sucrose are known to stabilize

osmot-ically fragile organelles (16), we assessed this

effect in the rll system. Figure2showsthatCa++

or Mg++, at concentrations too low to be of much effect by themselves, greatly augmented

the burst size in the presence of sucrose. The

yields ofrII phage, in sucrose media containing

an optimal 0.002 to 0.005 M

Ca++,

varied from

80to130phage perinfected cell and were always

higher than those attained with

Mg++.

On the

other hand, when Ca++ or Mg++ ions were

present at concentrations (0.05 to 0.08 M) at

which they produced maximal rll yields in the

absence of sucrose, the yields were sharply

reduced. Inanexperiment in which infected cells

were lysed with chloroform at various times

after infection, it appeared that sucrose plus

0.003 M Ca++ had two antagonistic effects. The

mixture slowed the rate of phage synthesis but

1.0u ,-9.

.4

n

.7-

.65-D

.5-.i .3

llJ

.2

.1

-0

\-SPERMMIDINEF

-.-SUCROSE

U-.061 .02 .03 .04 .05 .06 .07 .08 .09 .1

MOLARITY OF ADDED NA+ OR K+ FIG. 1. Effect of monovalent cations on sucrose-or spermidine-stimulated rnI growth in Escherichia

coli K-12 (X). Sucrose:r1993 multiplicity of infection

= 5. Varyingconcentrations ofKCIin 0.5Msucrose.

Dilution intofinalmixtureat8minafter infection. On

ordinate, 1.0 equals a burst size of11. Spermidine:

r1272multiplicityofinfection = 7.3. Varying

concen-trations ofNaCI in 0.03Mspermidine. Dilution at 7

min; 1.0 = burstsizeof32.

90a 80a 70-ii 60

N lI

50

ar

40

-,\

10;₂₀M\mg

.01 .02 .03 .04 .05 .06 .07 .08 .09 .I

MOLARITY OF ADDED MG++OR CA++

FIG. 2. Burst size in 0.5M sucrose of T4rJ993

grown in Escherichia coli K-12 (X). Effect of Ca++

and Mg++. Forboth experiments, multiplicity of

in-fection = 8and dilution intofinalincubation mixture

was at 7minafter infection.

302 J. VIROL.

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u

a

u

I

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delayed lysis of both r+ and rll-infected cells so that increased yields were eventually obtained.

The sucrose-Ca++ mixture thus appears to

functionbypreservingtheintegrityof theinfected

cells.

Swelling and absorbancy changes of infected

cells. Two other lines of evidence indicate that

the rll defect involves a

leaky,

defective cell

envelope. Figure3shows how

absorbancy changes

with time after infection with r+ or r1993.Cells,

infected with rll in the presence ofMg++orwith

r+in the presence or absenceof

Mg++,

followed

the same pattern of increasing absorbancy until

thetimeoflysis. Therll-infected cells in the

ab-sence of Mg++ followed the pattern for only 4

min,afterwhich timeagradual declineoccurred.

A decrease in absorbancy has been used as an

indication ofmitochondrial

swelling

(25);

in the

presentexperiments, suchadecrease isconsidered

toindicatethatthecellsswellandlose

refractility.

A more direct indication ofswellingis

provided

in Fig. 4, where it can be seen that rll-infected

cells swell more thanr+-infected cells. Forthese

experiments,densecellsuspensions of

centrifuged

log-phase cells were infectedwith T4r+ or r1993

atmultiplicities greater than 12. Throughout the

.6

,o,~ s,r+-Na

r]-4-,

en \

0

4 8 12 16 20 24 28 32 36 40 44 50 MINUTES AFTER INFECTION

FiG. 3. Absorbancy changes ofK-12 (X) with time

after infectionwithT4r+orT4r1993:effectofmedium.

Cellsat2.5 X 108 in tryptone-Na- ortryptone-Mg++

were infected with T4r+or T4r1993 at a multiplicity

of6. Infectedcultures wereaeratedat37C, and

sam-ples wereremovedat 2-minintervals. Absorbancy was

read at 540 myi in cuvettes thermostatted at 30 C.

Lii E 20

J

0 >

15-z

Lii 10

c-)

Lii u- 5-z z o Lii lii 0.

a

~~~/

0 0r

2 4 6 8 10 12 14 16 18 20 22 24

MINUTES AFTER INFECTION

FIG. 4. Volumechanges upon infection ofK-12 (X)

withr+ orr1993phage. Cells,grown to3X 108/mlin

tryptone-Na+, were harvested, washed, and

resus-pended in the same medium at 4 X 1010 cells/ml.

A 1-ml amount ofsuspension in a 25-ml Erlenmeyer

flask wasinfectedwithr+ orr1993at multiplicities of 13.5. Infected cells were shaken vigorously at 37 C.

At2-minintervals,sampleswere withdrawninto

hema-tocritcapillaries, sealed with plasticene, and heldinan

ice bath until the end of the experimenzt. Capillaries

werecentrifuged, and the volume of packed cells was

measuredas apercentage of the totalsample volume.

courseofinfection, sampleswerecentrifugedand

thevolumeofpacked cellswas measured. It was

necessary to shake the infected cells vigorously

to prevent too early lysis. When the cells were

infected with

multiplicities

lower than 10, no

swellingwasobserved.This mayindicate that the

observed differencesathighermultiplicities repre-sent some lysis from without. It is nevertheless

significant that r+-infected cells were better able

to withstand such lysis than were cells infected

withrII. In someexperiments,thecell volume of

r+-infected cells also increased, but always less than a parallel culture infected with rII. When

Na+ was replaced by Mg++, the volumesof

rll-infectedcells were,asexpected, thesame asthose

ofwild type.

Cell-envelope components.Al thedatapresented

thusfarwarrant

investigation

ofthe cell

envelope

as a possible site ofthe rII defect. More in the

nature ofeliminatinga possibility, we examined

thehost-cell mucopeptides becausetheyare

gen-erallyconsideredtoplayanimportantrole in the

structuralintegrityof the cell. Since the rlldefect

is innocuous inE.coliK-12,themucopeptides of

this organism (uninfected) were extracted,

purified, andcomparedwith those ofE.coli K-12

(X) (21). The reason for this comparison is that

K-12 doesnotneed therIIproduct wheninfected

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because it must differ in some way from K-12

(X). Whatever the difference, it was not detected

inacid hydrolysates of mucopeptides from thetwo

organisms whichcontained nearly identical molar

ratios of alanine, glutamic acid, lysine,

diaminopimelic acid, muramic acid, and

hexose-amine. Attempts were made toextendthis

com-parison to mucopeptides of K-12 (X) infected

with r+ and rII, but these experiments were

abandoned when it was found that the

muco-peptides ofr+-infected cells had already suffered

some hydrolysis at 10 to 12 min after infection.

(The mucopeptides were solubilized during a

formic acid extraction step, which does not

solubilize intact mucopeptides.) Since

muco-peptide is hydrolyzed early in wild-type phage

infection, itis probablynotthe specific structure

that differentiatesbetween mutant and wildtype

in the maintenance of cell integrity. This was

perhaps predictable because the rll-infected cells

do not swell excessively nor lyse but appear to

havea permeability defect whichwould be more

reasonablyassociated with the cell membrane.

Phospholipids, found in high concentrations

in the cell membrane, are also known to bind

polyamines. We examined the biosynthesis of

phospholipids ofK-12 (X) after infection withr+

and rII and in uninfected bacteria. Log-phase

cells of E. coli K-12 (X) intryptone-Mg++ were

exposed to 32P-labeled orthophosphate. One

minute later, twoofthree samples were infected

with wild-type or mutant phage. At the times

indicated in Table 5, samples were taken for

phospholipid extraction, and the phospholipids

were subsequently separated by thin-layer

chro-matography. Thematerialintheseparated bands

was analyzed for radioactivity and total

phos-phorus. The results presented in Table 5 reveal

that infection with T4 phage causes a marked

inhibition ofPEsynthesisandaslightstimulation

of PG synthesis. However, nodifferencewas

ob-served between r+- and rll-infected cells. Similar

results have been reported recently in

experi-ments with E. coli B (M. H. Furrow and L. I.

Pizer, Bacteriol. Proc., p. 26, 1967).

Inlaterexperiments, we found that 20to25%C/=

ofthephosphorusin the PGband is in the form

of cardiolipin (diphosphatidylglycerol). This

could introduce a great error in the specific

ac-tivities recordedfor PG if32Pwere incorporated

exclusively into either PG or cardiolipin. From

preliminary measurements of the distribution of

radioactivity between PG and cardiolipin, we

could calculate that the recorded PG specific

activities are 10%' too high for the rll-infected

cells and approximately 10%I too low for the other two. This does not change the conclusion

that T4infectionhas slight effect onPG

biosyn-thesis,whereas itmarkedly inhibits PEsynthesis.

DISCUSSION

Duringinfection ofahost cell, a hole is made

inthe cellenvelopetoallow penetrationby phage

DNA.Puck andLee(24) haveshownthat leakage

of cellular substances occurs inT-eveninfections

but eventually stops. With T4rII infection of E.

coli K-12, (X) however, loss of putrescine (2),

[image:7.485.55.451.437.577.2]

nicotinamide adeninedinucleotide,and ATP (27)

TABLE 5. Intcorporationt of32p i/itophospholipids ofuninflitedan1dr+- antdrII-inifected

Escheric/ia coli K-12(X) 't

Uninfected r+-infected rll-infected

Min

Mp

after_afe I'}lospho-

iPopho-

Cotunts

_Counts _Counts

32p lipid

'Counts/min

Atofp permin CountsC'minper Counts Amt of per

mi.i

(X105) Amt perpmole (X 1O5) Amtof perAmole mi p pier,emole

pmitoles u,ioltes inoloes

6 PG 0.21b 0.1 lb 1.9 0.37b 0. 9lb 1.9 0.64 0.38 1.7

11 PG 1.06 0.38 2.8 1.51 0.32 4.7 1.61 0.34 4.7

21 PG 2.43 0.48 5.1 2.97 0.44 6.8 2.56 0.40 6.4

6 PE 0.30b 0.47b 0.64 0.41b 0.58b 0.7 0.53 1.03 0.5

11 PE 1.80 1.15 1.6 0.94 0.91 1.0 1.02 0.91 1.1

21 PE 4.65 1.14 4.1 1.53 1.02 1.5 1.62 1.01 1.6

aPhospholipidswereextractedfrom2.7 X 1010bacteria(viableassayjust before32Paddition and in-fection with phage) after various durationsof 3pincorporation. Thephospholipids were separatedby thin-layerchromatography beforeassayof totalPandradioactivityin the PG and PE. Theamountsof

PE and PG perbacterial cell mayappeartobe ratherhigh. This isprobably relatedtothe factthat the

cells were grown intryptone-Mg++,inwhichmedium thecellsaremuchlargerthannormal.

b We lost partof thelipidextracts from the6-min r+-infectedand uninfectedsamples. Thisdoes not

affectthespecificactivitydatapresented.

304 J. VIROL.

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continue. The cell is fragile, low in ATP, unable

totolerate monovalent cations, and incapable of

producing progeny phage. Some of these faults are reversed sufficiently toallowphage

synthesis

when any of a number of chemicalsareaddedto

the medium. The rII block can be partially

relieved by arginine, spermidine, and certain

diamines. These substances, like Mg++, operate in a diversity of biochemical systems and,

there-fore, donotallow apreciselocalization of where

or howtheyactto overcometherIIdefect.

How-ever,they are not needed torelievea deficiency

of polyamines within the cell, since sufficient

putrescine and spermidine are available for

normal neutralization of DNA and

packaging

into progeny phage. The fact that spermidine

inhibits ghost lysis suggests that the polyamine action is not localized in the cell interior. The

efficacious action of sucrose, which does not

cross the

permeability

barrier ofthe

cell,

points

to anosmotic

problem

and

thereby

a

faulty

cell

envelopeassociated with therIIdefect. This

inter-pretationis furthersupported

by

the

finding

that

rII-infected cells lose

refractility

and swell.In

fact,

all

of

thedata

presented

hereareconsistentwith

the idea that the

rII

defect is somehow

asso-ciated with altered permeability. Nearly all the

effective additives have, in one system or

an-other, been shown to stabilize

fragile

cells or

protoplasts. Furthermore, cations which affect rII

multiplication

have direct effects on the

plasmamembrane. Exposure ofamoebaetoNa+

causes

swelling

and lowered electrical resistance

of the

plasma

membrane,

coincident with

in-creased

permeability

(7).

The addition of Ca++

or sucrose hasthereverse effect.

Thus,

an agent

which increases

permeability

inhibits rII

multi-plication, and agents with the reverse effect are

stimulatory.

Finally,

another indicationthat the

rII product is involved with the cell

envelop

is thephenomenon of

lysis

inhibition in E. coliB. This phenomenon occurs when the cell is first

infected withr+ phage and then reinfected with

either mutant or

wild-type phage.

Since the

second infection is not

specific

and also since it can occur so late that the

superinfecting

phage

DNA cannot provide progeny genes, action at

the surface is

indicated,

presumably

with a

product whose nature is determined

by

rI, rII,

and rIIIgenes.

There seems little doubt that in someway the

rll defect inE. coli K-12

(X)

results ina

faulty

cell envelope. But theunanswered

questions

are

why is the

envelope

defectiveandhowdoes

wild-type rII product maintain it. We have

presented

evidence that

phospholipid

synthesis

is altered

after phage

infection,

as indicated

by

the

de-creased synthesis of PEand increased

specific

activity in the PG band. It was disappointing, if not surprising, tofind no difference in

phospho-lipid synthesis between r+- and

rhl-infected

bac-teria. It is clear that phage infection has a

pro-found effect on phospholipid metabolism, and

this would appear to be an appropriate area of

study for detecting a defect in cell-membrane

synthesis. Ofcourse, even if the rll defect were

concerned with some aspect of membrane bio-synthesis, the study of 32p incorporation into PE and PG could only be a crude start at delineating

the nature of the defect. The answer could as

wellcome from a study ofminor phospholipids,

the proteins(s) of cell membrane, or the lipid

componentsofthephospholipids. Recent results

in this laboratory (M. Aghdashi and L.

Astrachan, unpublished data) reveal that rII may

be distinguished from r+ by the fact that rII infection causes a much greater stimulation of

32p incorporation into cardiolipin. This phenom-enon was not detected in the presently reported

experiments because the thin-layer solvent

system we used does not separate PG from

cardiolipin. Theeffect oncardiolipin synthesis is

detected after separation by column

chromatog-raphy ofthelipids (26, 29) or of the deacylated

backbones (33). Even though a difference

be-tween r+- and

rhl-infected

cells can be observed,

this does notnecessarily point directly to the rll

defect. The difference could occur if the specific

defect were inadequate synthesis of ATP or

some other essential substrate, with the

conse-quence that a reaction involved in membrane

synthesis does not occur. For the time, the best

that can be said is that achemicalphenomenon,

of

possible

interestin thesynthesisof membrane

phospholipids, has been detected in association

with the rII defect. It is interesting that ATP

formation and cardiolipin synthesis are both

associated with the

rII defect,

because ATP

for-mation is so often associated with membranous

structures. The identification of which

phenom-enon is causative

(if,

indeed, eitherone is) must

await identification ofaparticularenzyme activity

present in thewildtype andabsentin the mutant.

Ourefforts willbedirectedto thecharacterization

of the

phospholipid

difference and, if successful, tothestudy ofitsmetabolism.

ACKNOWLEDGMENTS

Thisinvestigationwassupported by Public Health

Service researchgrant 5 ROI AI-04884from the Na-tional Institute of Allergy and Infectious Diseases and by Public Health Service Research Career

De-velopment Award 5-K3-GM-4673 to L. Astrachan.

The technical assistance ofJoan Miller is

grate-fullyacknowledged.

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