JOURNAL OF VIROLOGY, OCt. 1967, p. 948-955Copyright ( 1967 American Society for Microbiology

Lysis Inhibition in Escherichia coli Infectedwith Bacteriophage T4

WERNER BODEDepartment of Physics, Kansas State University, Manhattan, Kansas 66502

Received for publication 8 May 1967

A technique of continuous filtration of T4-infected Escherichia coli has beendevised to study the phenomenon of lysis inhibition. Studies using this techniquerevealed that the length of the lysis delay caused by superinfection can attain onlycertain discrete values, which for low average multiplicity of superinfection isthought to be a reflection of the actual number of superinfecting particles per cell.The time interval between primary and superinfection had little effect on the lengthof lysis delay. With increasing rate of superinfection, the length of lysis delay de-creased. In superinfected cells, the concentration of endolysin exceeded the finalconcentration in nonsuperinfected cells. Superinfection of a lysing culture inducedlysis inhibition immediately. Temperature-shift experiments, with cells primarilyinfected by a temperature-sensitive endolysin mutant, revealed that after the normallatent period superinfection was unable to induce lysis inhibition. Amber-restrictivecells, which were primarily infected by an endolysin negative amber mutant, re-leased adenosine triphosphate (ATP) at the end of the normal latent period al-though lysis did not occur. Superinfection reduced the loss of ATP markedly. Thehypothetical role of the cytoplasmic membrane in lysis inhibition is discussed.

Cultures of Escherichia coli infected by wild-type (r+) T-even bacteriophage do not lyse forseveral hours if the infection is initiated andmaintained at a cell concentration of about 4 X107 cells/ml or higher. This phenomenon wastermed "lysis inhibition" by Doermann (2),who demonstrated that secondary infection orsuperinfection by the phage present in themedium caused the inhibition. One-step growthexperiments at cell concentrations lower than106 cells/ml do not show long-term lysis inhibi-tion, but lysis is delayed for about 10 min if thecells have been superinfected with multiplicitesof superinfection ranging from 2 to 50 (at 37 C).The length of the delay of lysis was found byDoermann (2) to be constant and independentof the time of superinfection. Rutberg andRutberg (7), on the contrary, observed an in-crease of the length of delay when they increasedthe interval between primary and superinfection.Our experiments have been undertaken to

clarify the discrepancy in the published data andto find new approaches to the phenomenon oflysis inhibition in an attempt to understand it.

MATERIALS AND METHODS

Bacterial and phage strains. E. coli S/6/5; T4Dwild type (r+), the late amber mutants am N54, amHil (gene 31), and am 8-82 (endolysin negative),

and ts e 3-90 (temperature-sensitive endolysin) werekindly provided by R. S. Edgar.

Media. Hershey's enriched broth (B) contained:8 g of Nutrient Broth (Difco), 5 g of peptone (Difco),6 g of NaCI, 1 g of glucose, and 1,000 ml of de-mineralized water. PAGFe contained: 18 g ofNa2HPO4- 12H20, 3.0 g of KH2PO4, 1.0 g of NH4Cl,5.0 g of glucose, 0.25 g of MgS04.7H20, 1.0 mg ofFeC13, and 1,000 ml of demineralized water.

Phage assays. Phage samples were plated as de-scribed by Chase and Doermann (1). When largenumbers of samples had to be plated, the amount oftop agar was reduced to 0.7 ml and two samples wereplated in one petri dish.

Standard procedure for experiments on lysis in-inhibition. Aerated overnight cultures of S/6/5 werediluted 1:100 into fresh medium, aerated for 70 minat 37 C, and then concentrated by centrifugation to2 X 109 cells/ml.

Primary infection was performed at 2 X 109cells/ml; 2 min later a 1:100 dilution followed.Superinfection was carried out at 2 X 107 cells/mlwhenever possible in order to distribute the superin-fection events evenly over the period during whichsuperinfection was carried out. During 3 min of super-infection the infection rate decreased by about 30%,from 39% at the start of infection to 28% at the end.(Infection rate = phages adsorbed per min perphages adsorbed during the entire infection period.)In 3 min 28% of the input phage was adsorbed.Superinfection was stopped either by T4 antiserumor by further dilution.

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LYSIS INHIBITION IN T4-INFECTED E. COLI

The adsorption constant was determined fromthree independent experiments to be k = 5.0 X 10-9ml/min. This value was used to compute the phageconcentrations necessary to establish the desiredactual multiplicities of infection (MOI) or super-infection (MOSI).

For continuous filtering the infected and superin-fected cells were pipetted into a jacketed filter-holdercontaining a membrane filter type B-6 (Schleicher &Schuell Co., Keene, N.H.). Before use, the filters werewashed repeatedly in saline and finally soaked in 30%bovine serum albumin solution. The filtrate waspumped by a peristaltic pump model E (The Zero-Max Co., Minneapolis, Minn.) into test tubes at aflow-rate of 0.5 ml per min. Samples were collectedcontinuously for 0.5 to 2 min periods. Depending onthe phage titer in individual samples, 0.05 or 0.1 mlwas plated on S/6/5. During the experiment, thefilter-holder was heated with water of 38.5 C, whichkept the medium inside the filter-holder at 37 C.Pumped out medium was continuously replaced byprewarmed medium. The pump can accommodatefour filter holders; thus four experiments could be runin parallel. About 1,000 cells were routinely placedonto each filter.

Endolysin assay. The method of Sekiguchi andCohen (9) was employed. One unit of endolysinactivity is defined by a decrease of 0.1 absorbance permin of the substrate in a Spectronic-20 colorimeter(Bausch and Lomb, Inc., Rochester, N.Y.).

Adenosine triphosphate (ATP) assay. The ATPconcentration released from cells into the mediumwas measured by recording the light output of thefirefly enzyme system (5) in a Packard scintillationcounter, set on 54.5 gain, window 0.30 - co. Thismethod was suggested by E. K. F. Bautz. Two-mlsamples containing 2 X 108 cells were pipetted into2 ml of glycine buffer (0.05 M, pH 7.4) with 0.02 MMgSO4, 0.02 M Na2HAsO4 (stock solution adjustedto pH 7.4), and 250 ,ug of firefly enzyme extract(Worthington Biochemical Corp., Freehold, N.J.),and were immediately placed into the counter. Eachsample was counted at least eight times for 0.5 min.The counts were plotted on semilog paper against thenumber of countings. The intersection of the straightpart of the curves and the abscissa was chosen ascharacteristic value of the curves. The ATP concen-tration per sample was obtained by comparing thecharacteristic values of samples and standards. Thecharacteristic value of standards was obtained byadding known amounts of ATP (disodium salt;Sigma Chemical Co., St. Louis, Mo.) to the reactionmixture.

RESULTS

Effect of variation of time and multiplicity oJsuperinfection on the delay of lysis in one-stepgrowth experiments. The time of lysis of an in-fected culture can be described either by the timeat which the rate of lysis (the first derivative of theone-step growth curve) passes through its maxi-mum or by the interval from beginning totermination of lysis. Thus it was desirable to

obtain one-step growth curves which showed well-defined plateaus and inflection points. (One-stepgrowth experiments actually measure release ofphage. Since the rate of phage maturation toburst size is small compared to the average rateof lysis, the kinetics of released phage and oflysis must be very similar.)

It turned out that the shape of lysis curves isdependent on the temperature, the medium used,the state of the bacteria, the multiplicity ofsuperinfection, and the time of superinfection.In PAGFe medium, for instance, the length ofthe delay of lysis increased with decreasing con-centrations of supplementing Vitamin FreeCasamino Acids, whereas the cessation of phagematuration was independent of the type ofmedium used. Optimal lysis curves, with termina-tion of phage maturation and lysis coinciding,were obtained with E. coli S/6/5 in exponentialgrowth phase using Hershey's enriched broth(HB) at 37 C.The main effects of varying time and mul-

tiplicity of superinfection of lysis can be seenin Fig. 1; they are summarized in Table 1. Thecurves we obtained did not permit a reliabledetermination of the time at which the maximalrate of lysis occurred.The results suggest that increasing the MOSI

or the interval between primary and superin-infection, shifts lysis to later times. Doermann(2) obtained the same effect with varying theMOSI, but he did not comment on it. Super-infection at two different times 10 min apart did

400-

e300-

a~~~~~~~~~~

-0 10 20 30 40 50 60 70 80 90Minutes after primary infection

FIG. 1. Effect of varying time and multiplicity ofsuperinfection on the delay of lysis: A, no superinfec-tion; 0, superinfection 5 min after primary infection,MOSI = 2; O, superinfection at 5 min, MOSI = 20;*, superinfection at 15 mini, MOSI = 2; *, superin-fection at 15 min, MOSI = 20.

949VOL. 1, 1967

Beginning and end of lysisa

infection) MOSI AIOSI

5 26b to 35c 30bto 42C15 28b to 40C 30b to 48 c

a Multiplicity of primary infection = 10. With-out superinfection lysis commenced at 20 min,stopped for the first time at 28 min, started again,and stopped definitely at 40 min.

I Beginning of lysis defined as the time when a

burst size of 20 was reached.c End of lysis determined by the intersection of

the extension of the slope and the upper plateau.

1000 O

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600

400

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25 30 35 40 45 50Minutes after primary infection

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FIG. 2. Continuous filtering of infected cells, super-infected with varying multiplicities 15 min after primaryinfection. Cultures of infected (and superinfected) cellswere pipetted onto membrane filters in jacketed filterholders (for details see Materials and Methods). Thefiltrate was continuously collected, and the samplingtubes were changed every 1 or 2 min. The figure showsthe actual number of plaques scored per sample, togive an idea of the statistical reliability. (A) MOSI =

0.4; (B) MOSI = 1.6; (C) MOSI = 7.6; (D) n1osuperinfection.

J. VIROL.

not shift the time of lysis for the same length,but only for 2 to 3 min.

Continuous filtering of infected cells. The reso-

lution power of one-step growth experimentscould be increased substantially by using a

method which directly yields the number ofreleased phage per unit of time. It consistsmainly in continuous filtering of infected cellsthrough a membrane filter, which is permeableto phage but not to cells, and sampling thefiltrate at appropriate intervals.

Superinfection with varying multiplicity. Inthe following experiments, the multiplicity ofprimary infection was purposefully kept at only0.5 in order to minimize the fraction of multiplyinfected cells. Without intentional superinfection,the release of phage started at about 22 to 23min (Fig. 2); the rate of lysis reached a maximumat about 27 min. Superinfection at 15 min withvarying multiplicities created a number ofadditional peaks. With increasing multiplicityof superinfection, the peak at 27 mn becamesmaller, whereas, later peaks became larger.The first and second peak are about 5 minapart (27.5 min and 32.5 min); later peaks are

spaced by 2 to 3 min. In Table 2 the areas underindividual lysis maxima are compared with termsof the Poisson distribution, assuming that thefirst superinfection maximum (at 32.5 min) iscreated by lysis of cells which all have beensuperinfected by one phage, the second superin-fection maximum by superinfection with twophage, and so forth. The comparison yieldedgood agreement except for MOSI = 0.4. It mustbe expected that at low MOSI the fraction ofcells superinfected during primary infection can

distort the data more than at higher MOSI.

TABLE 2. Comparison of the relative size of the areas

under lysis maxima in Fig. 2 with terms ofthe Poisson law

No. of lysis maximum and raMOSI

and na

0.4 0.67 0.27 0.05 0.007 Ab0.4 0.5 0.1 0.03 Bc

1.6 0.22 0.35 0.28 0.15 Ab0.28 0.35 0.24 0.14 Bc

7.6 0.07 0.26 0.67 Ab0.065 0.43 0.49 Bc

a Variables of the Poisson law: pr = nr e-n/r!b pr(r = 0, 1, 2, 3)/po + Pi + P2 + P3-c Areas under lysis maxima 0, 1, 2, 3 in Fig. 2

(corrected for increase of the burst size with thetime of lysis, rate of phage maturation = 14phage/min was determined from Fig. 1) dividedby the sum of these areas.

950 BODE

TABLE 1. Effect of ti/ne and nmultiplicity of super-

infectionI on1 the timing of lysis

Time of superinfection(min after nrimary

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LYSIS INHIBITION IN T4-INFECTED E. COLI

1200 / :'e

i1000 I:

800

600-

400

200

0 20 25 30 35 40 45Minutes after primary infection

FIG. 3. Continuous filtering of infected cells, superinfected with varying multiplicities 5 min after primary in-fection. The cultures were pipetted onto filters, filtered, and sampled as described in the legend to Fig. 2. Symbols:0, MOSI = 0.3; O, MOSI = 0.4; A, MOSI = 1.6. The control without superinfection was left out for clarityof the graph; it is similar to (D) in Fig. 2.

Superinfection at 5 min after primary infec-tion yielded curves with the same trend, namely,increasing multiplicity caused the peaks toappear later (Fig. 3). The time of appearance ofcorresponding peaks is different in the experi-ments of Fig. 2 and 3. After superinfection at5 min, the first superinfection peak appearedat about 30 min; this is about 2.5 min earlierthan with superinfection at 15 min. Thus, chang-ing the time of superinfection did not shift thetime of lysis by a comparable amount. Weconclude that superinfected cells can lyse onlyat certain discrete times with constant intervalsbetween. The MOSI per cell determines at whichtime an individual cell lyses.

Superinfection for varying lengths of time.From unpublished experiments, we learned thatraising the multiplicity of superinfection beyond4 to 5 phage per cell was only partially effectivein further delaying the time of lysis. This satura-tion of the system might be due to a limitingmaximal rate of superinfection; i.e., beyond somenumber of superinfecting phage per unit time,additional phage cannot add to the length ofthe delay of lysis. To check this possibility, cells

25 30 35 40 45 50Minutes after primary infection

FIG. 4. Continuous filtering of infected cells super-infected with constant multiplicity = 10 for differentlengths of time. The cultures were pipetted onto filters,filtered, andsampledas described in the legend to Fig. 2.Superinfected from 15 to 16.5 min (1.5 min), 0;superinfected from 14 to 17 min (3 min), CJ; superin-fectedfrom 13 to 17.5 min (4.5 min), A.

were superinfected around 15 min for varyinglengths of time keeping the MOSI constant(Fig. 4). Inspection of Fig. 4 reveals that theshorter the period of superinfection the earlier

VOL. 1, 1967 951

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FIG. 5. Endolysin activity in once-infected and insuperinfected cells. Cells were primarily infected witham N54 (MOSI = 10). Superinfection was stopped byadding T4 antiserum. Final cell concentration was2 X 108 cells/ml, 35 C. Samples were filtered throughmembrane filters B-6 (Schleicher & Schuell). Thisfiltrate is termed "primary filtrate." The loaded filterswere then placed upside-down on the filter holder, andthe cells on the filter were washed off with fresh mediumofthe same volume as the sample (5 ml); this suspensionis termed "cells." Endolysin activity in "primary fil-trate" and in "cells" was determined separately afterchloroform had been added. Primary infection only:"cells," 0; "primary filtrate2" LI; sum of both, AL.With superinfection: "cells," *; "primary filtrate,"*; sum of both, A.

the lysis maxima appeared. In an attempt toquantitate the effect, we compared the numberof phages released after a certain, rather ar-bitrarily chosen, time to the sum of all thephages released through the entire lysis period.Forty-two min was chosen as "line of demarca-tion" because at this time curve b (the experi-ment with intermediate MOSI) has dropped toabout half of its maximum. The figures were:0.42 (4.5 min adsorption), 0.17 (3 min adsorp-tion), and 0.19 (1.5 min adsorption). The resultsof another experiment with the MOSI = 6,were: 0.34 (6 min adsorption), 0.25 (3 min ad-sorption), and 0.17 (1.5 min adsorption). Thesedata suggest that the rate of superinfection canlimit the efficiency of superinfection. Moreinformation has to be compiled before a quan-titative evaluation can be made.

Superinfection at the end of the latent period.Endolysin appears about one-third of the waythrough the latent period and increases in con-centration until the cells lyse (8). It is natural toinquire whether lysis inhibition might be due to

an effect of superinfection on endolysin synthesis.Several experiments were carried out to testthis possibility. We found that superinfectionslowed down the initial rate of endolysin syn-thesis (Fig. 5) as has been shown by Rutbergand Rutberg (7). Subsequently the rate ofendolysin production increased and the endolysinconcentration exceeded the concentration innonsuperinfected cells. Since this experiment wasperformed at high cell concentration, superin-fection resulted in a long period of lysis inhibi-tion. This is also the case with cultures whichhad been primarily infected by a late ambermutant, as has been done in this experiment,and to which T4 antiserum had been added toinactivate the few progeny phage. The increaseof endolysin activity in the primary filtrate ofsuperinfected cells, and the constant activityafter the end of the normal latent period in thecells which stayed on the filter, might be due tocell lysis as a result of stresses arising from thefiltration procedure. Measuring the recovery ofsuperinfected cells from filters directly supportedthis explanation (unpublished data). Since theconcentration of endolysin in lysis-inhibitedcells exceeds the final concentration in cellswhich are not superinfected, we conclude thatthe endolysin concentration does not deter-mine the time of lysis.We next did an experiment to determine

whether there existed a point in time beyondwhich superinfection would be incapable of

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0 20 40 60Minutes after primary infection

FIG. 6. Superinfection ofa lysing culture. The opticaldensity of two cultures at 37 C primarily infected witham Hll (MOSI = 4) was recorded by measuring theabsorbancy at 520 mA.s. One culture (@) was superin-fected at the time indicated (SI) with am HI I (MOSI =5). Dilution from superinfection caused the opticaldensity to drop by 0.01 absorbance.

SiI,

BODE J. VIROL.

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LYSIS INHIBITION IN T4-INFECTED E. COLI

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A I~~~

TS

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I4I I

p0 10 20 30 40 50nimary infection

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0 10 20 30 40 50 0 10 20Minutes after primary infection

30 40 50

FIG. 7. Temperature shift (40 C - 27.5 C) experiment with cells prinarily infected by a temperature sensitive

endolysin mutant (ts e 3-90). Optical density was measured at 520 m,. All cultures were primarily infected at 40 C(MOI = 4). The cultures represented by filled symbols were superinfected (MOSI = 10) shortly before they wereshifted to 27.5 C. The time of the temperature shift is indicated in the graphs (TS).

arresting the lytic process. Optical densitymeasurements of cultures infected by a lateT4 amber mutant (to prevent autosuperinfec-tion) indicated that even after lysis in a culturehad started, superinfection could still inducelysis inhibition (Fig. 6). The almost instantresponse to superinfection suggests that thefraction of cells which has passed the criticaltime is very small or that the period betweenthe critical time and actual lysis is very short.

Independent of the mechanism of lysis, onemight assume that there has to be a critical timeat which the cells become sensitive to endolysin.

It might be possible to separate this criticalmoment from actual lysis by reducing the endo-lysin concentration in the cells. When a tem-perature-sensitive endolysin mutant was usedfor primary infection, incubation at 40 C sup-pressed the endolysin concentration below ameasurable level, and cells did not lyse at theend of the latent period. (T4 antiserum was addedto exclude autosuperinfection.) Shifting to27.5 C (the temperature sensitive endolysin isstable at this temperature), even after the endof the normal latent period, would result inaccumulation of some active endolysin if protein

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953VOL. 1, 1967

J. VIROL.

L10-

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aC--

-20 0 20 40 60 80Minutes after primary infection

FIFG. 8. Leakage of adeizositie triphlospliate frominfected and superinfected cells into the medium.Symbols, O*, primarily infected with am 8-82(endolysin negative), MOI =5; F-1, primarily in-fected with am HI], MOI =5; ON, superinfectedwith am HlIl at MOSI = 5. Temperature, 33 C;medium, PAGFe + 0.5% peptone.

synthesis is not shut off but continues as longas templates, precursors, and energy are avail-able. Unpublished experiments showed thatlysis can be induced by shifting to low tempera-ture after the end of the normal latent period.When the infected cultures were shifted later,the rate of lysis decreased. Now we can askwhether superinfection after the end of the normallatent period can still induce lysis inhibition.Results of the experiment to answer this ques-tion are presented in Fig. 7. Superinfection at15 and 20 min (before the end of the latentperiod) did induce lysis inhibition, whereassuperinfection at and after the end of the latentperiod (25 and 30 min) could not cause lysisinhibition. We conclude that at the end of thelatent period cells can be lysed by subnormalconcentrations of endolysin, and that at the sametime ther supernhe ability to respond to super-infection.Leakage of ATP from infected cells. ATP is

the main energydtetFig. in E. coli, as itis in other organisms. It is reasonable to assumethat availability of ATPuiinhibitionsite forinfected cells to stay intact. One step in the proc-ess which finaWly leads to lysis might be therelease of ATP from the cells into the medium.We werenergy inte studying the behavior of

ATP before the cells lyse, and therefore choseconditions which precluded cell lysis. To achievethis, primary infection was performed with anendolysin-negative amber mutant infecting E. coliS/6/5, a restrictive host. Upon primary infec-tion, cells commence to discharge ATP into themedium (Fig. 8). Thereafter, throughout thelatent period, the cells continuously leak ATP ata moderate rate; near the end of the latent periodthe rate changes rapidly to a much larger value.Superinfection reduces the rate of leaking inthis final stage by a factor of 5. Cells infectedby an endolysin producing late amber mutantyielded similar results.

DISCUSSION

The resolution power of one-step growth ex-periments has been substantially increased in thepresent work by using a technique which meas-ures directly the rate of phage release. With thistechnique, it was possible to resolve the lysiscurves of superinfected cultures into several dis-crete subclasses of lysis maxima (Fig. 2 to 4).At low multiplicity of superinfection, individuallysis maxima result from simultaneous lysis ofcells which very probably have been superinfectedby the same number of phage. It became clearthat the length of lysis delay is determined pri-marily by the actual MOSI per cell, whereas thetime of superinfection influences the lysis delayonly to a small extent. Experiments with con-stant MOSI and varying duration of super-infection revealed that the faster subsequentsuperinfections follow each other the less ef-fective they become (Fig. 4). This result couldbe interpreted in at least two ways: (i) thenumber of superinfections per cell, or (ii) theamount of DNA or some other material of thesuperinfecting phage determines the length oflysis delay. The fraction of 32P from super-infecting DNA which leaks into the medium issurprisingly constant (3). Applying the firsthypothesis, the explanation of the rate effectwould be as follows. The system which receivessuperinfections as lysis inhibition-inducing stimulicannot recognize two subsequent superinfections(of one cell) as descrete stimuli if they followeach other too closely. According to the secondhypothesis one must assume that, with increas-ing rate of superinfection, the maximal lengthof lysis delay which can be displayed by cellsshortens, or that more superinfecting materialgets lost. The second assumptions seem to usless plausible than the earlier one. The smalleffect of varying the time of superinfection onthe length of lysis delay (Fig. 2 and 3) suggestsa model in which the process of lysis delaystarts at a constant time (reference point).

954 BODE

LYSIS INHIBITION IN T4-INFECTED E. COLI

The reference point might be identical with theend of the normal latent period. Cells changedrastically at this point even if lysis does notoccur because of lack of endolysin. Oxygenuptake stops (6), ATP is released (Fig. 8),and the ability to respond to superinfection bylysis inhibition is lost (Fig. 7).We favor a model in which the site of lysis

inhibition is located in the cytoplasmic mem-brane, but at this stage of our knowledge oflysis inhibition other models cannot be excluded.In primarily infected cells, the membrane changes(conformational change) at the end of the latentperiod in such a way that ATP is released andrespiratory enzymes become inactive. Becauseof lack of available energy, the membranedisintegrates and thus permits endolysin toattack the cell wall. A more direct possibilityis that depletion of usable energy terminatesrepair of endolysin-induced cell-wall damage(in this case endolysin would have to penetratethe membrane before the end of the latentperiod).

If certain prerequisites are met, superinfec-tion interferes with this chain of events. The pre-requisites are primary infection by r4 phage andprotein synthesis after primary infection, i.e.,expression of r4 function (unpublished data).In successfully superinfected cells at the end ofthe normal latent period (the reference point),the very first changes of the membrane mightalso occur, but not the key one which is re-sponsible for release of ATP (Fig. 8). At leasttwo hypothetical mechanisms can be envisagedby which superinfection could change the be-havior of the membrane after the end of thenormal latent period: superinfection causes (i)a conformational change, or (ii) a change in theenvironment of the membrane (probably onlyin the interstitial space between membrane andwall). Both mechanisms must either slow downthe normal lytic process, which starts with someminor initial change in the membrane and endswith lysis, or activate a different process which istypical for superinfected membranes. This processis affected in some way by the multiplicity ofsuperinfection. The number of possible hypo-thetical explanations is still too large to be dis-

cussed here. An interesting possibility is suggestedby Lehninger's studies (4) of swelling cyclesof mitochondria in which the mitochondrialmembrane and ATP play a central role.

ACKNOWLEDGMENTS

We are grateful to R. S. Edgar for provoking ourinterest in lysis inhibition. We thank E. K. F. Bautz,J. D. Friesen, and K. G. Lark for stimulating discus-sions and J. D. Friesen for indefatigable help in pre-paring the manuscript. We thank also L. W. Phillipsand P. J. Garcia for building the jacketed filter holdersand Susan Zielie and Barbara Stout for technicalassistance.

This investigation was supported by grant GB-2674 from the National Science Foundation.

LITERATURE CITED

1. CHASE, M., AND A. H. DOERMANN. 1958. Highnegative interference over short segments of thegenetic structure of bacteriophage T4. Genetics43:332-353.

2. DOERMANN, A. H. 1948. Lysis and lysis inhibi-tion with Escherichia coli bacteriophage. J.Bacteriol. 55 :257-275.

3. FRENCH, R. C., S. M. LESLEY, A. F. GRAHAM, ANDC. E. VAN ROOYAN. 1951. Studies on the rela-tionship between virus and host cell. III. Thebreakdown of p3alabelled T2r+ bacteriophageadsorbed to E. coli previously infected by othercoliphages of the T group. Can. J. Med. Sci.29:144-148.

4. LEHNINGER, A. L. 1965. The mitochondrion. W. A.Benjamin, Inc. New York.

5. McELRoY, W. D. 1963. Crystalline firefly luci-ferase, p. 445-448. In S. P. Colowick and N. 0.Kaplan [ed.], Methods in enzymology, vol. 6.Academic Press, Inc., New York.

6. MuKAI, F., G. STREISINGER, AND B. MILLER. 1963.Cited by S. P. Champe. Ann. Rev. Microbiol.17:87-114.

7. RUTBERG, B., AND L. RUTBERG. 1965. Role ofsuperinfecting phage in lysis inhibition withphage T4 in Escherichia coli. J. Bacteriol. 90:891-894.

8. SECHAUD, J., AND G. STREISINGER. 1962. Cited byS. P. Champe. Ann. Rev. Microbiol. 17:87-110.

9. SEKIGUCHI, M., AND S. S. COHEN. 1964. The syn-thesis of messenger RNA without protein syn-thesis. II. Synthesis of phage-induced RNA andsequential enzyme production. J. Mol. Biol.8:638-659.

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