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ANTIMICROBIL AGENS AND CHEmoTHERAPY, Dec. 1975, p. 617-626Copyright 0 1975 American Society for Microbiology

Vol. 8, No. 6Printed in U.S.A.

Inhibitory Effect of Colicin E2 on Transport Systems ofEscherichia coli in the Presence of the rex Gene of

X ProphageTERUHIKO BEPPU,* HIROKAZU YAMAMOTO, AND KEI ARIMA

Department ofAgricultural Chemistry, University of Tokyo, Tokyo, Japan

Received for publication 23 July 1975

Purified colicin E2 was found to cause marked inhibition of the permeationrate of o-nitrophenyl-galactoside (ONPG) in several X-lysogenic strains ofEscherichia coli in the presence of chloramphenicol to prevent prophage induc-tion. The inhibitory effect of colicin E2 on transport systems was analyzed withcells ofE. coli CP78(X). The dose of colicin E2 for the half-maximum inhibitionof the ONPG-permeation rate was about 9 molecules of the colicin per bac-terium under the aerobic condition, which corresponded to about 1 killing unitper bacterium. Kinetics of the transport of ['4C]methylthiogalactoside suggestedthat colicin E2 began to inhibit the influx rate of ,B-galactosides within a fewminutes after the colicin addition, and the maximum inhibition reached morethan 80%. Extensive leakage of intracellular potassium ion and inhibition ofL-proline transport also occurred at the same time. Acid solubilization of cellulardeoxyribonucleic acid by the colicin was apparently delayed to the initiationof the transport inhibition. The extents of the inhibition of (8-galactosidetransport and leakage of potassium ion by the colicin were extensive in cellslysogenic for wild X phage or Xind-, whereas the extent,s were slight in thenonlysogenic cells or cells carrying Xrex- prophage. It was concluded that thesensitization of membrane transport systems of E. coli cells to colicin E2 wasachieved by the presence of the rex gene product of X phage.

Colicin E2 is a protein antibiotic ofEscherichiacoli with a molecular weight of 60,000. Whensensitive E. coli cells adsorb the colicin, damageof cellular deoxyribonucleic acid (DNA) occursat first, whereas other macromolecular syn-theses continue for a relatively long time (18).Colicin E2 is similar in several points, such asreceptor specificities, immunological reactions,and physical parameters, to colicin E3 (13),although the ultimate biochemical effects ofthese two colicins are quite different. Studieson the mode of action of colicin E3 have shownthat the colicin has an intrinsic enzymatic activ-ity to cleave 16S ribonucleic acid and cause theinactivation of the ribosome (4, 5, 21). Severalattempts to find deoxyribonuclease activityassociated with colicin E2 had been negative(1, 18, 22); however, recent studies by Saxeshowed that purified colicin E2 preparationspossess deoxyribonuclease activity to introduceone single-strand scission in supercoiled X phageDNA (24, 25). Although this finding implies thenucleolytic activity of colicin E2 as the basis ofthe in vivo action, the isolation of a mutantin which the colicin stops cell division without

DNA degradation indicates that the primarycause of killing by the colicin is not necessarilyDNA damage (3). In the case of colicins like E,and K, rapid loss of potassium and other ca-tions from the treated cells is observed, and itseems likely that the mode of action involvesstructural alteration of the cell membrane (10,17, 18). However, it has been believed thatcolicin E2 has no such activity to induce loss ofintracellular potassium (19).

In this paper, we reexamined this point withvarious sensitive E. coli strains and found thatcolicin E2 caused strong inhibition of membranetransport systems of the X-lysogenic cells of E.coli without prophage induction. Marked inhibi-tory effects on membrane transport systems forf3-galactosides, potassium ion, and L-prolinewere induced by low doses of colicin E2 in cellsof X-lysogenic cells, although less slight inhibi-tion was also observed in the nonlysogeniccells. Bacteriophage X has been found to conferthe capacity on the lysogenic cells to block themultiplication of rII mutants ofT4 phage, whichmainly involves the product of the rex geneof X (12, 15). Observations had been made that

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618 BEPPU, YAMAMOTO, AND ARIMA

acid solubilization of DNA by colicin E2 waspartially reduced at late times of the colicinchallenge in cells carrying X prophage (16, 20),and this reduction was due to the rex geneproduct (23). This effect may be elucidated bythe marked inhibition of membrane transportsystems observed here. The sensitization oftransport systems of E. coli to colicin E2 is anewly found phenotypic expression of the rexgene. This work gives the first characteriza-tion of the interaction of colicin E2 with therex gene product and the cell membrane.

MATERIALS AND METHODS

Organisms. E. coli K-12 CP78 (F-, thr-, leu-,arg-, his-, thi-, mal Xr, supE, rel+, X+) was mainlyused as sensitive strain. E. coli K-12(A) wild,W2252T- (Hfr, met-, thy-, X-), and B were alsoused. Nonlysogenic derivatives of CP78 includingstrain 353 were isolated by plating the ultraviolet-irradiated CP78 cells with X-antiserum. SpontaneousXs revertants of the strain 353 were obtained byselecting mal+ colonies (29). Series of lysogens forwild X phage and its mutants were reconstructedby reinfection of the strain 353Xs, and their lyso-genies were confirmed by their immunities againstXcI and ultraviolet inducibilities.

Wild X phage was obtained by induction of K-12(A).Several mutants of A phage, i.e., kind-, Ximm434,and XcI, were supplied by Y. Sakakibara, NationalInstitute of Health of Japan. Two Rex mutants,Xrex-5a and Xrex-Qam, were originally isolated andsupplied by G. N. Gussin, the University of Iowa(12). Bacteriophage T4B and its rI mutant, r221,were supplied by H. Uchida, the University ofTokyo.

Growth media and treatment with colicin E2.Nutrient broth (Eiken, Tokyo) and C medium con-taining 0.78 g of NaH2PO4 2H20, 3.57 g of Na2HPO4'2H20, 0.2 g of MgSO4 7H2O, 4.0 g of CasaminoAcids (Difco, vitamin free), 5 mg of thiamine-hydro-chloride, and 4.0 g of glycerol in 1 liter of distilledwater were used for almost all experiments. Toinduce S-galactosidase and 8-galactoside permease,0.25 mM isopropylthio-fi-D-galactoside (IPTG) or 1mM methylthio- 8-D-galactoside (TMG) was added tothese media. M medium for the experiment of L-proline transport contained: 8.8 g of NaH2PO4 2H2O,3.0 g of KH2PO4, 1.0 g of NH4C1, 20 mg of MgSO4_7H20, 0.8 mg of FeCl3 6H20 and 2.0 g of glucosein 1 liter with supplements of required amino acidsto 20 ,g/ml, and thiamine-hydrochloride to 5 itg/ml.Cells were grown aerobically in 10 ml of mediumcontained in 30-ml L-shaped tubes with shaking at37 C, and growth was monitored by measurementsof the optical density at 550 nm or total cell countingby Coulter counter (Coulter Electronics Inc.). Chlor-amphenicol (100 jAg/ml) was added to the cultureat a cell density of 5 x 108 to 8 x 108 cells/ml. Thenthe cultures treated with chloramphenicol weredivided into 5-ml portions in the L-shaped tubes andtreated with different amounts of colicins 15 minafter the addition of the antibiotic under the aerobic

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condition with constant shaking at 37 C. Nutrientbroth containing 1.5% agar was used for countingviable cells.

Preparations of colicins and assay in their killingactivities. Colicin E2 was produced by E. coliW3110(E2) and purified mainly according to themethod of Herschman and Helinski (13), except forslight modifications described below. Extraction ofthe colicin from cells was carried out with 0.01 Mphosphate buffer (pH 8.0) containing 0.01 M ethyl-enediaminetetraacetate and 0.1 M NaCl instead of1 M NaCl. The colicin was precipitated from theextract with 0.3 to 0.6 saturation of ammoniumsulfate. After successive chromatographies of di-ethylaminoethyl-cellulose and CM-Sephadex C-50columns, the final preparation was concentrated byultrafiltration with Diaflo UM-10 membrane (Ami-con) to give a solution of 2 mg of colicin proteinper ml and stored at -80 C. Almost all experimentswere carried out using this preparation after appro-priate dilution with 1 mg of bovine serum albuminsolution per ml. The specific activity of this prepa-ration was 1.09 x 1015 killing units/mg under theaerobic condition described above.

Fractionation of the purified colicin E2 preparationby isoelectric focusing electrophoresis was performedin a 100-ml Ampholine column containing 0.5%Ampholite (pH 7-9) stabilized with sucrose densitygradient. The dialyzed preparation containing 14 mgof the colicin protein was applied in the columnand electrophoresis was continued for 72 h. Frac-tions of 50 drops were collected and monitored forkilling activity, permease inhibiting activity, opticaldensity at 280 nm, and pH.

Colicin El was produced by E. coli JC411(EI)and purified as described by Schwartz and Helinski(27) except for omitting the final chromatographwith CM-Sephadex. The specific activity of thepreparation was about 8 x 1012 killing units/mg.

Colicin E3 was produced by E. coli W3110(E3)and purified according to the method of Hersch-man and Helinski (13). The specific activity ofE3 preparation was not assayed exactly but itseemed to be comparable with that of E2 prep-aration judging from titration by the spot test.

Killing of the bacterium was measured bytitrating viable cells on nutrient agar plates. Todetermine killing units of colicins, cells of CP78which had been treated with chloramphenicol wereincubated with different dilutions of colicin prep-aration for 30 to 40 min at 37 C. Each samplewas then assayed for viable bacteria. The numberof killing units was calculated from the killingmultiplicity obtained from the equation m = - ln(S/SO) where S/S0 is the survival ratio of sensitivebacteria. The presence of 1 mg of bovine serumalbumin per ml in dilution medium for the colicinand the aerobic condition of cells by shaking wereessential to obtain the higher killing activity ofcolicin E2. In the case of fractionation of colicinE2 by Ampholine electrophoresis, killing activitiesof fractions were assayed by serial dilution and byspotting on nutrient agar plates freshly seededwith about 107 cells of CP78 as an indicator.

Assay of P-galactoside permease. Galactoside

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TRANSPORT INHIBITION BY COLICIN E2 WITH X-rex

permease was assayed by two types of experiments.In the first of these, the rate of hydrolysis ofo-nitrophenyl-8-D-galactoside (ONPG) by intact cellswas measured as follows. Cells were grown with 0.25mM IPTG for more than three generations at 37 Cto a cell density of about 8 x 108 cells/ml and100 ,ug of chloramphenicol per ml was added tostop the growth. Then colicin E2 was added 15 minafter the addition of the antibiotic. Samples (0.3 ml)were removed at intervals and mixed with 3.0 mlof ONPG solution (0.5 mg of ONPG per ml in 0.1M phosphate buffer, pH 7.4) at 37 C. After incuba-tion for 3 min, the reaction was stopped by adding 1ml of 1 M Na2CO3, and the production of o-nitrophenol was measured at 410 nm after theremoval of cells by centrifugation at 3,000 x g for15 min. Almost all incubations with the colicin forthe assay except for those of Ampholine elec-trophoresis were performed under the aerobic condi-tion.

Galactoside permease was also tested by meas-uring the accumulation rate of ['4C]TMG by shortperiod incubation followed by rapid filtration. Cellswere grown with 1 mM TMG in C medium to acell density of about 5 x 108 cells/ml. Colicinchallenge was carried out similarly to the aboveexperiments in the presence of chloramphenicol.Samples (0.25 ml) were rapidly mixed with 0.05ml of the growth medium containing 1 mM radio-active TMG (2.5 ,uCi) at 37 C. After 30-s incuba-tions, 0.2 ml of the incubation mixtures was re-moved to filter cells on membrane (MilliporeCorp.) filters (0.45-,xm pore size), and cells wererapidly washed twice with 2 ml of the freshmedium prewarmed at 37 C. Radioactivity accumu-lated in cells on the membrane filters was countedin a dioxane scintillator solution by a liquidscintillation counter (Aloka LSC-655, Nihon MusenCo.).To measure leakage of intracellular TMG by

colicin E2, cells of CP78 were grown in C mediumcontaining 1 mM TMG and treated with chlor-amphenicol as described above. Then ['4C]TMG(0.5 ACi/ml) was added and the culture wasdivided into 5-ml portions. Colicin E2 was added 15min after the addition of radioactive TMG andsamples (0.2 ml) were taken to filter at intervals,washed, and counted as described above.

Measurements of intracellular potassium ion.About 10 to 1 ACi of 42KCI per ml was addedto the cells growing in C medium supplementedwith 1 mM KCl at 37 C. After growth for two orthree generations, radioactive KCl in the mediumwas removed by filtration and cells were re-suspended into the fresh potassium-free medium.Chloramphenicol was added 10 min after the re-suspension, and the colicin challenge was madewith 5 ml of the chloramphenicol-treated cultures.Samples (0.3 ml) were filtered on membrane(Millipore) filters and cells were washed with 2 mlof cold C medium. Radioactivity on the filtermembrane was counted by a GM counter.

Measurement of L-proline transport. Cells ofCP78 grown in M medium were treated with 100,ug of chloramphenicol per ml. Colicin E2 was added

10 min after the addition of the antibiotic andL_['4C]proline (final 20 ,uM, 20 mCi/mmol) wassubsequently added 1 min after the colicin chal-lenge. Radioactivity incorporated into the cells wasmeasured by the rapid filtration of 0.3-ml samplesand washing as described above.

Measurement of DNA degradation by colicinE2. DNA of E. coli CP78 was labeled by growingfor two generations in C medium containing 1.7,uCi of [14C]thymine (2.5 mCi/mmol) per ml and 0.2mM deoxyadenosine. IPTG (0.25 mM) was alsoadded to induce ,-galactosidase and its permease.Radioactive thymine in the medium was removedby filtration and resuspension of cells in the freshmedium, and the colicin challenge was carried outas described above. Samples (0.5 ml) were takeninto equal volume of 10% trichloroacetic acid andcells were filtered by membrane (Millipore) filters.Radioactivity in the cold acid-insoluble materialson the filter membranes was measured by liquidscintilation counting.

Chemicals. IPTG, TMG, and thiodigalactosidewere products of Sigma Chemical Co. (St. Louis).ONPG was purchased from Wako Pure ChemicalsCo. (Osaka, Japan). [Methyl-14C]TMG was ob-tained from New England Nuclear Corp. (Boston).42KCl was supplied by the Japan Atomic EnergyInstitute (Tokai, Japan). [2-14C]thymine and L-[U-14Clproline were purchased from the Radio-chemical Centre (Amersham, U.K.). Crystallinetrypsin was from Mochida Pharmaceutical Co.(Tokyo, Japan).

RESULTSInhibition of ONPG permeation by colicin

E2. When purified colicin E2 was added toactively growing E. coli K-12(X) previouslyinduced with IPTG in nutrient broth, a distinctdrop of the ONPG permeation rate occurredafter a short lag period. In the presence of 100ug of chloramphenicol per ml to avoid prophageinduction and net increase of the permeationactivity due to growth, a more rapid decreasewas observed and final inhibition by about 50%was obtained 20 min after the colicin addition.

Various strains of colicin E2-susceptible E.coli were tested for the inhibition. The ONPGpermeation system of E. coli CP78, which waslysogenic for X phage, was found to be themost susceptible to the colicin (Fig. 1). On theother hand, transport in E. coli B and severalstrains of K-12 such as W2252T- was consider-ably less susceptible to colicin E2, althoughtheir cells were susceptible to the lethal effectof the colicin. Most of the further experimentson this permeation inhibitory effect of colicinE2 were performed with E. coli CP78 in thepresence of chloramphenicol.The inhibitory effect of colicin E2 on the

ONPG permeation system of CP78 in nutrientbroth was compared with those of other E-groupcolicins, E, and E3. As shown in Fig. 2,

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FIG. 1. Inhibition of ONPG permeation rates ofvarious E. coli strains by colicin E2. Colicin E2 (final0.4 pg/ml, about 500 killing unitslcell) was added attime 0. Under the employed condition, the initialrates of permeation of cells without the colicin werethose producing 74.4 nmol of o-nitrophenol for 3min by 4 x 108 cells of CP78, 89.3 nmol for K-12,and 62.4 nmol for W2252T-, respectively. Symbols:*, CP78; 0, K-12; A, W2252T-; A, CP78 withoutthe colicin.

colicin E2 induced a gradual decrease of thepremeation rate which started immediatelyafter the colicin addition, but colicin E1 causeda rapid drop without an induction period. Theinhibition of the permease by colicin E2 wasnot reversed at all by adding trypsin (0.5mg/ml) to the inhibited culture 10 min afterthe colicin challenge. Colicin E3 did not show adistinct inhibition but a slight temporary in-crease of the permeation rate. Addition of alarger amount of colicin E3 (18 pLg/ml) showedalmost the same profile.Dose effect of colicin E2 on the inhibition

of the ONPG permeation. Dose effect of colicinE2 on the killing and the inhibition of ONPGpermeation was measured under the two condi-tions, that is, relatively anaerobic and aerobicones. Under the former condition, a culture ofCP78 grown aerobically in nutrient broth (8x 108 cells/ml) and treated with chloram-phenicol was divided into several 0.5-ml por-tions in small test tubes (13-mm diameter)and then incubated with various amounts ofcolicin E2 without shaking. The aerobic condi-tion was achieved by shaking 5.0 ml of the

similar cultures in L-shaped tubes. As shownin Fig. 3, the killing effect of colicin E2 wasremarkably stimulated by aeration. The singlehit dose of the colicin calculated from theamount to give 36% survival was 9.1 moleculesof colicin E2 per bacterium under the aerobiccondition and 158 molecules per bacterium

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TIME (min)FIG. 2. Inhibition of ONPG permeation rate of

CP78 by colicins El, E2, and E3. The initial rate ofpermeation without the colicins was that producing67.5 nmol of o-nitrophenol for 3 min by 4 x 108cells. Symbols: 0, E1 (0.7 pg/ml, 35 killing unitslcell); 0, E2 (0.2 pg/ml, 275 killing unitslcell); A,E3 (0.12 pg/ml).

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FIG. 3. Dose effect of colicin K2 on killing andinhibition of ONPG permeation rate of CP78. Sym-bols: surviving cells (0) and ONPG permeation rate(0) under the aerobic condition; surviving cells(A) and ONPG permeation rate (A) under the rela-tively anaerobic condition.

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under the anaerobic condition. The dose ofcolicin E2 necessary to obtain the half-maxi-mum inhibition was 9 and 338 molecules percell, respectively, which corresponded to about1 and 2 killing units of the colicin per bacteriumunder each condition. The maximum inhibitionon ONPG permeation was about 67% in bothconditions, and the addition of the largeramounts of the colicin did not increase theextent of the inhibition. The residual activityto transport ONPG at the higher colicin con-centrations was effectively inhibited by 5 mMthiodigalactoside with 2 mM KCN. This in-dicates that the residual transport is stillmediated by the specific carrier, M protein (8),and not a nonspecific diffusion due to destruc-tion of the membrane structure.

Confirmation of colicin E2 protein as theinhibitory effector to the ONPG permeationsystem. When colicin E2-resistant mutants ofCP78 selected for resistance to BF23 phage wereexamined, their ONPG permeation systemswere found to be unsusceptible to the colicin.This suggested that the inhibition of permea-tion by our colicin preparation was due to thecolicin itself and not to some other con-taminants. To confirm this further, the colicinpreparation was fractionated by Ampholineelectrophoresis and fractions were assayed forthe killing activity and the permeation in-hibitory activity. As shown in Fig. 4, bothactivities coincided completely with proteinpeaks. The main two peaks had isoelectricpoints of pI 7.63 and 7.43, which showed goodcoincidence with the data of Herschman andHelinski (13). The third small peak of pl7.25 also showed both activities. This com-ponent seemed to be a third conformer ofcolicin E2 hitherto unknown.

Effect of colicin E2 on transport of TMG.The inhibitory effect of colicin E2 on the(3-galactoside permease system of CP78 wasfurther analyzed by measuring transport of[14C]TMG. 3-Galactosidase and- its permeaseof CP78 cells were induced by the growth inC medium containing TMG instead of IPTGso as to avoid competitive interference of IPTGwith accumulation of radioactive TMG. Theaddition of colicin E2 caused a rapid decreasein the influx rate starting within a few minutes(Fig. 5). About 20% of the transport activityremained at the maximum inhibition with thelarger amounts of the colicin.To analyze the effect of colicin E2 on the

transport system of TMG further, the changein amounts of intracellular [14C]TMG due tothe colicin action was measured. The cultureof CP78 grown in C medium induced withTMGwas added with [14CITMG in the presence

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FIG. 4. Ampholine electrophoresis of colicin E2preparation. Permease inhibiting activity wasassayed by incubating 2 !d of the each 100-folddiluted fraction with 0.4 ml of the chloramphenicol-treated culture of CP78 for 20 min at 37 C underthe relatively anaerobic condition and measuringthe residual activity to hydrolyze ONPG. Symbols:(A) *, optical density at 280 nm; 0, pH. (B) *, per-centage of inhibition of ONPG permeation rate; 0,killing activity.

of chloramphenicol. It was incorporated rapidlyinto the cells and reached complete equilibriumabout 15 min after the addition at 37 C.Colicin E2 was added to this equilibratedcultures, and amounts of intracellular TMGwere sequentially measured by the rapid filtra-tion technique. A rapid decrease in the levelof intracellular TMG occurred within a fewminutes after the colicin addition. The exitrate of intracellular TMG was acceleratedabout 5 min after the colicin addition andfinally reached less than 17% of the initiallevel (Fig. 6). Considerably good coincidenceof this profile with that of the influx rateobtained by measuring ONPG hydrolysis de-scribed above suggested that colicin E2 inhibitedthe influx of TMG without significant ac-celeration or inhibition of the exit velocity.Although these experiments were performed

in C medium to compare with the followingexperiments about potassium leakage, similar

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FIG. 5. Inhibition of the influx rate of [4(by colicin E2. Rates were indicated by the azof radioactivity incorporated for 30 s of incuunder the condition described in the text. IE2 was added at time 0. Symbols: 0, contiE2 (0.02 pg/ml, 29 killing units/cell).

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FIG. 6. Leakage of the intracellular [14CJTMG bycolicin E2. The colicin was added at time 0. Sym-bols: 0, control; A, E2 (0.02 pg/ml, 29 killing unitslcell); 0, E2 (0.066 pg/ml, 95.7 killing units/cell).

effects were also observed with cells grownin nutrient broth.Leakage of potassium ion from cells by

colicin E2. Experiments using radioactive4KCQl showed that almost complete loss of theintracellular potassium ion was caused bycolicin E2. Cells preloaded with 42KCI andresuspended in fresh potassium free C mediumwere challenged with colicin E2 in the presenceof chloramphenicol. Extensive loss of the in-tracellular potassium ion began within 2 or 3min after the addition of the colicin, whichwas almost the same time as the initiation ofthe inhibition of the /3-galactoside permease(Fig. 7). The mode of the leakage by colicinE2 was different from that by colicin E1 whichshowed no lag period; however, almost 99%of the intracellular potassium ion was releasedafter a prolonged incubation with 27.5 killingunits of colicin E2 per bacterium. No sig-nificant lysis of cells was observed during theexperimental periods by turbidimetry or totalcell counting.

Effect of colicin E2 on transport of L-proline. The inhibitory effect of colicin E2 on

7TMG the transport of -proline was also observed.nounts The colicin challenge was performed with cells,bation of CP78 which were grown in M medium andColicin treated with chloramphenicol. Radioactive L-rol , proline was added 1 min after the addition of

the colicin, and the radioactivity incorporatedinto the cells was measured sequentially. Theinhibition of uptake velocity appeared 2 min

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FIG. 7. Leakage ofthe intracellularpotassium ionby colicins E2 and El. Symbols: 0, control; A, E,(0.008 pg/ml, 11 killing units/cell); *, E2 (0.02pg/ml, 27.5 kiling units/cell); x, El (0.1 pg/ml,about 5 killing units/cell).

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after the colicin challenge, and distinct leakageof the incorporated radioactivity started 5 to 6min after the addition of colicin E2 (Fig. 8).Degradation of DNA by colicin E2. De-

gradation of DNA into acid-soluble materialsby colicin E2 was measured with cells of CP78in C medium. Simultaneous measurements ofthe inhibition of the ONPG permeation ratewere also carried out. As shown in Fig. 9, theextensive degradation of DNA began 10 minafter the colicin challenge. The initiation ofDNA degradation was apparently delayed com-pared to the initiation of the permease in-hibition; however, it seems possible that limitedendonucleolytic cleavage of DNA occurs at theearlier stage.

Sensitization of the transport systems tocolicin E2 by X-lysogenization. Among variousstrains of E. coli treated, all the strains whichshowed marked susceptibility to the permeationinhibitory activity of colicin E2 were lysogensfor X phage. In contrast, transport systems ofthe nonlysogenic strains, W2252T- and B, wereconsiderably less susceptible. These results sug-gested the possibility that the presence of Xprophage affected the susceptibility of themembrane transport systems to the colicin.To investigate this possibility, the prophagewas cured to derive the isogenic nonlysogenicstrains, and susceptibilities of their ONPGpermeation systems to colicin E2 were comparedwith that of the parent. The permeation sys-tems of all the nonlysogenic derivatives, e.g.,

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FIG. 8. Inhibition of L-[14C]proline uptake bycolicin E2. Colicin E2 was added at the arrow Aand (14C]proline at the arrow B. Symbols: 0, con-

trol; *, E2 (0.125 pg/ml, about 100 killing units/cell).

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FIG. 9. DNA degradation and inhibition ofONPG permeation rate by colicin E2. Colicin E2 (0.02pg/ml, 25 killing units/cell) was added at time 0.Symbols: 0, residual DNA; *, ONPG permeationrate.

strain 353 (Table 1), showed distinct decreasesin the susceptibility to the colicin.

Further analyses of the effect of X prophagewere made by comparing susceptibilities of thereconstructed series of strains lysogenic forvarious X mutants. As a starting nonlysogenicstrain, the X-susceptible derivative of strains353 and W2252T- was used. Table 1 showspotassium leakage by colicin E2 in these strains.Slight leakage of the intracellular potassiumion was induced in the nonlysogenic strains bythe colicin; however, lysogenization for wildX phage and Xind- caused marked sensitiza-tion to the colicin. Since expression of almostall X genes except for the cI and rex areprevented completely in cells carrying Xind-,it may be concluded that the sensitization isdue either to the cI or to the rex gene of Xprophage. On the other hand, cells carryingXimm434 in which the X-specific immunityregion, including the cI and rex genes, wasdisplaced by the comparable region of prophage434 showed no increased susceptibility of thepotassium transport system. Then, it seemedhighly probable that the rex gene of X pro-phage, responsible for the specific exclusion ofrII mutants of T4 phage of the X-lysogeniccells, was involved in the observed sensitizationto colicin E2. This was really confirmed withthe transport systems for TMG and potassiumion in cells carrying Xrex-. As shown inTable 2, the sensitization of both transport

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TABLE: 1. Effect of X-lysogenization on leakage ofpotassium ion from cells by colicin E2

Colicin E2 added (14)a

Strains 0 5 10

Counts/min 9 Counts/min Counts/min %

CP78(A) 8,270 100 219 2.38 113 1.36353Xs 9,641 100 7,885 81.6 7,281 75.5353Xs(X) 9,131 100 749 8.20 811 8.89353XM(Xind-) 7,886 100 405 5.13 398 5.05353X8(Ximm434) 1,994 100 1,749 87.6W2252T- 6,497 100 5,901 90.9 5,633 86.8W2252T-(X) 7,618 100 585 7.68 519 6.82

a Colicin E2 solution (20 jug/ml) was added to 5-ml cultures of each strain (5 x 108 to 8 x 108 cells/ml)preloaded with 42KCl and incubated for 20 min at 37 C aerobically in the presence of chloramphenicol. Theresidual intracellular radioactivity was measured.

TABLE 2. Effect of the rex gene on leakages of intracellular TMG and potassium ion

Es added (1d)

Strains EOP a of T4rII Intracellular TMG8 intracellular K+ b

0 2 10 0 5 20

353XA 100 16,161 14,902 11,892(100) (92.3) (73.5)

353Xs(X) 0 15,586 4,207 4,017 9,035 287 260(100) (27.1) (25.8) (100) (3.18) (2.88)

353Xs(Xrex-5a) 100 15,325 14,432 12,876 7,940 6,658 6,672(100) (94.2) (83.8) (100) (84.0) (84.1)

353Xs(Xrex-Qam) <5c 9,357 153 144(100) (1.63) (1.54)

EEOP, Efficiency of plating.b Radioactivities (counts per minute) of [14C]TMG or 42K+ remaining in cells after the incubation with

colicin E2 (20 ,ug/ml) for 20 min at 37 C were indicated. Incubation condition was the same as that in Table 1.Values in parentheses are percentages of the amounts in the absence of the colicin.

c Only extremely faint plaques are visible.

systems by A prophage failed to occur withxrex-5a, which caused no effective exclusionor T4rII, whereas Arex-Qam, whose Rex-property was suppressed in cells of 353X8,behaved like wild X concerning the sensitiza-tion of the transport systems. It is reasonablyconcluded that the sensitization to colicin E2by X prophage depends solely on the rex geneproduct.

DISCUSSIONThe transport inhibitory effect of colicin E2

on the X-lysogenic cells described here wassimilar to that of colicin E1; however, thepresence of an induction period in the action ofcolicin E2 discriminates these two types ofcolicins. The inhibition of membrane transportsystems by colicin E2 takes place in two stagessequentially. The first slowly proceeding inhibi-tion stage begins immediately after the colicinadsorption, and after 4 or 5 min the second

stage starts to cause a marked inhibition of theinflux rate and rapid leakage of the metabo-lites. Since chloramphenicol was added to thesystem to prevent induction of X and thelysogen for the induction-negative mutant ofX phage (Xind-) showed distinct susceptibilityto the colicin, the possibility is negated thatthe progressive inhibition of the transportsysems is an indirect result of the prophageinduction triggered by the colicin action. Luskand Nelson (17) reported that colicin K had aninduction period depending upon the tempera-ture and the multiplicity, whereas colicin Eldid not. Experiments at a different temperatureand multiplicity of E2 would establish howsimilar its induction period is to that ofcolicin K.The remarkable sensitization of the mem-

brane transport systems to colicin E2 by X-lysogenization is caused by the rex gene func-tion of the prophage. The addition of colicin

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E2 to cells carrying Xrex+ phage seems toinhibit the oxidative phosphorylation accom-panying with the inhibition of the active trans-port systems ofthe cell membrane (unpublisheddata). Inhibition of 32P incorporation by colicinE2 has also been reported in a strain notstated to be X lysogenic (28). Since initiationand maintenance of the DNA degradation di-rected by colicin E2 require energy (14), therex gene-dependent reduction of the DNAdegradation appearing in later periods of thecolicin challenge (23) may be explained by thismembrane attacking effect of the colicin stim-ulated by the rex gene.The mechanism of the apparent sensitization

of the cell membrane to the colicin action bythe rex gene product cannot be decided fromthe results presented here. Although theprimary function of the rex gene product hasnot yet been identified, several observationsseem to suggest its close relationship with thecell membrane. It has been reported that rIIexclusion by the Rex function is suppressed bythe presence of higher concentrations of mag-nesium ion (11) or sucrose with calcium ion(7), and abnormalities of cation transport andleakage ofpyridine nucleotides occur during therII infection of a lysogen for Arex+ (27).Premature lysis after infection by Ti phagewas also observed to be due to the rex gene(9). These experiments imply that the Rex func-tion may affect cell membrane stability andpermeability in the cases of the several in-fectious events. It may be assumed that colicinE2 itself has an intrinsic activity to affect thestructure of the cell membrane, and the rexgene product has modified the cell membraneto make it susceptible to the colicin action insome way. However, several alternative possi-bilities should also be considered. For instance,it is possible that colicin E2 is activated bythe rex gene product to produce the membraneattaking activity in a similar manner to thecase of colicin E3, whose intrinsic ribonucleolyticactivity is unmasked by endolytic cleavage toproduce an active fragment (21). On the otherhand, it seems also possible that the rex geneproduct is activated by the colicin to revealits latent membrane attacking activity. In thiscontext, it should be noted that activation ofperiplasmic endonuclease I by colicin E2 hasbeen postulated (1, 2). Further experimentsare needed to settle these problems; however,this system may be useful to analyze not onlythe mode of action of colicin E2 but also therex gene function which has hitherto been un-known.The strong inhibition of transport systems by

colicin E2 may be a principal cause for the

killing of X-lysogenic cells. In contrast, non-lysogenic cells such as E. coli B are suscepti-ble to the lethal effect of the colicin, whereastheir transport systems show considerable resis-tance (1 killing unit of colicin E2 for E. coliB is about 18 molecules per bacterium under theaerobic condition). This fact poses a questionwhether the membrane attacking activity ofthecolicin plays a primary role for the killingof Rex- cells. However, the partial sensitivityof the transport systems, especially for (8-galactosides, occurs in nonlysogenic cells. Thisindicates that a similar reaction of the colicinagainst the membrane occurs to a smallerextent even in the absence of the rex geneproduct. It may be assumed that a minoralteration in the membrane of nonlysogeniccells induced by colicin E2 is sufficient tocause the effective killing. Although the demon-stration of an intrinsic nuclease activity incolicin E2 protein (25) suggests direct actionof the colicin molecules in the DNA degrada-tion, possible role of the membrane alterationinduced by colicin E2 for the killing and theDNA degradation should be carefully examinedfurther.

LITERATURE CITED1. Almendinger, R., and L. P. Hager. 1972. Role for

endonuclease I in the transmission process of colicinE2. Nature (London) New Biol. 235:199-203.

2. Almendinger, R., and L. P. Hager. 1973. Recon-stitution of colicin E2-induced deoxyribonucleic aciddegradation in spheroplast preparations. Antimicrob.Agents Chemother. 4:167-177.

3. Beppu, T., K. Kawabata, and K. Arima. 1972.Specific inhibition of cell division by colicin E2without degradation of deoxyribonucleic acid in anew colicin sensitivity mutant of Escherichia coli.J. Bacteriol. 110:485-493.

4. Boon, T. 1971. Inactivation of ribosomes in vitroby colicin E3. Proc. Natl. Acad. Sci. U.S.A. 68:2421-2425.

5. Boon, T. 1972. Inactivation of ribosomes in vitroby colicin E3 and its mechanism of action. Proc.Natl. Acad. Sci. U.S.A. 69:549-552.

6. Bowman, C. K., J. Sidikaro, and M. Nomura. 1971.Specific inactivation of ribosomes by colicin E3in vitro and mechanism of immunity in colicino-genic cells. Nature (London) New Biol. 234:133-137.

7. Buller, C., and L. Astrachan. 1968. Replication ofT4rII bacteriophage in Escherichia coli K-12(A). J.Virol. 2:298-307.

8. Carter, J. R., Jr., C. Fox, and E. P. Kennedy.1968. Interaction of sugars with the membraneprotein component of the lactose transport systemof Escherichia coli. Proc. Natl. Acad. Sci. U.S.A.60:725-732.

9. Christensen, J. R., and J. M. Geiman. 1973. A neweffect of the rex gene of phage X: prematurelysis after infection by phage Ti. Virology 56:285-290.

10. Fields, K. L., and S. E. Luria. 1969. Effects ofcolicins El and K on transport systems. J. Bacteriol.97:67-63.

11. Garen, A. 1961. Physiological effects of rIl mutationsin bacteriophage T4. Virology 14:151-163.

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12. Gussin, G. N., and V. Peterson. 1972. Isolation andproperties of rex- mutants of bacteriophage lambda.J. Virol. 10:760-765.

13. Herschman, H. R., and D. R. Helinski. 1967. Purifi-cation and characterization of colicin E2 and colicinE3. J. Biol. Chem. 242:5360-5368.

14. Holland, E. M., and I. B. Holland. 1972. Kinetics ofcolicin Erinduced solubilization and fragmentationof Escherichia coli DNA in vivo. Biochim. Biophys.Acta 281:179-191.

15. Howard, B. 1967. Phage X mutants deficient in rIIexclusion. Science 158:1588-1589.

16. Hull, R. R., and P. Reeves. 1971. Sensitivity ofintracellular bacteriophage X to colicin CA42-E2.J. Virol. 8:355-362.

17. Lusk, J., and D. L. Nelson. 1972. Effects of colicinsEl and K on permeability to magnesium andcobaltous ions. J. Bacteriol. 112:148-160.

18. Nomura, M. 1964. Mechanism of action of colicins.Proc. Natl. Acad. Sci. U.S.A. 52:1514-1521.

19. Nomura, M., and A. Maeda. 1965. Mechanism of actionof colicins. Zentralbl. Bakteriol. Parasitenkd.Infektionakr. Hyg. Abt. I. Orig. 196:216-239.

20. Nose, K., and D. Mizuno. 1971. Retrictive effectof prophage A on DNA degradation in Escherichiacoli cells. Biochim. Biophys. Acta 246:20-28.

21. Ohsumi, Y., and K. Imahori. 1974. Studies on a factor

ANTIMICROB. AGENTS CHEMOTHER.

enhancing colicin E3 activity in vitro. Proc. Natl.Acad. Sci. U.S.A. 71:4062-4066.

22. Ringrose, P. 1972. Interaction between colicin E3 andDNA in vitro. FEBS Lett. 23:241-243.

23. Saxe, L. 5. 1974. Reduction of colicin Erinduced DNAbreakdown by the rex gene of X prophage. Virology60:288-292.

24. Saxe, L. 5. 1975. The action of colicin E2 on supercoiledA DNA. I. Experiments in vivo. Biochemistry 14:2051-2067.

25. Saxe, L. S. 1975. The action of colicin E2 on supercoiledA DNA. II. Experiments in vitro. Biochemistry14:2058-2063.

26. Sekiguchi, M. 1966. Studies on the physiologicaldefect in rI mutants of bacteriophage T4. J. Mol.Biol. 16:503-522.

27. Schwartz, S. A., and D. R. Helinski. 1971. Purifica-tion and characterization of colicin El. J. Biol. Chem.246:6318-6327.

28. Takagaki, Y., K. Kunugita, and M. Matsuhashi. 1973.Evidence for the direct action of colicin K on aerobic32P, uptake in Escherichia coli in vivo and in vitro.J. Bacteriol. 113:42-50.

29. Thirion, J. P., and M. Hofung. 1972. On some geneticaspects of phage A resistance in E. coli K-12.Genetics 71:207-216.

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