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0021-9193/84/091047-06$02.00/0

Copyright © 1984,American SocietyforMicrobiology

Effect of

Bacteriophage P1

Lysogeny on

Lipopolysaccharide

Composition

and the Lambda

Receptor of Escherichia coli

JUAN M. TOMASAND WILLIAM W. KAY*

Departmentof Biochemistry andMicrobiology, University ofVictoria, Victoria, British Columbia V8W 2Y2, Canada Received12 April 1984/Accepted13 June 1984

Theouter membraneofEscherichia coliwasalteredas a consequenceoflysogeny by bacteriophages P1 and P1cmts. The predominantchangewas a reduction in the sizeof lipopolysaccharideto aheptose-deficient form. P1cmtslysogenswerestillsensitiveto severalbacteriophagesbut wereresistantto Avir. Neither wholecells nor solubilized outer membranesfrom P1 cmtslysogens were ableto inactivate X vir, and 32P-labeled X vir was unabletoadsorb toP1 cmtslysogens. P1 cmts lysogenswere alsoaffected inmaltose transport. Thelevel of periplasmic maltose-binding proteinwasreducedsomewhat,buttherewasnosignificant reductionin the level of theoutermembraneXreceptor(LamB).These membrane abnormalities were all corrected in strains cured of P1 cmts. It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequentlythe function ofthe A receptor.

P1 cmts is a recombinant bacteriophage derived fromP1

andan Rplasmid(R14; 20). It iswidelyused becauseofits

efficiency in establishing lysogeny, its temperature-sensitive controlof the lytic cycle,anditsconvenienceas a transduc-tion vehicle, since it carries the easily recognizable Tn9

element encoding chloramphenicolacetyltransferase (CAT). As aprophage, P1 proviral DNA is maintained as a

single-copy plasmid (34). It is unusual among temperature viral genomes in that P1 functions are less conservatively

clus-teredthanotherviralgenomes (9, 26,35). Mutants in theP1 immunity system, such as P1 vir, are lytic and unable to

establish lysogeny (34). The P1 plasmid also causes exoge-nous DNA lacking the appropriate modifications to be

inactivatedandlost (4).

The bacterialreceptorfor P1 is lipopolysaccharide(LPS) core oligosaccharide (3), a relatively invariant structure amongenterobacteria.This may explain thewidehost range

of P1, which is unlike those ofsomebacteriophages (suchas

X) whichare thought to have morespecific receptors, such as outer membrane (OM) proteins, LPS 0 antigen, or

combinations of both (14, 24, 39, 42). The well-known low

efficiency ofplating (EOP) of A virions onP1 lysogens has

beenassumed to be due to restriction. However, because of the unusual nature ofP1 and because we had previously

observed that A vir was not inactivated by whole cells of

Escherichia coliP1 cmts lysogens, wedecided to reinvesti-gatethisassumption.The results hereinclearly demonstrate

thatA phage do not adsorb toP1 cmts lysogens even in the presenceof copiousquantities of the LamB OM protein and

thatthe major OM modification is in LPS structure. MATERIALS AND METHODS

Bacterial strains. The strains, theirproperties, and origins are listed in Table 1. E. coli C600 and its P1 cmts

lysogen

(GY2780) were used predominantly. A lysogens of E. coli wereconstructed with A Tn2 (10), butat a

10-3-fold-reduced

efficiency in P1 cmts lysogens. P1 cmts lysogens ofE. coli strains were established by the method ofTyler and

Gold-berg (38). P1 cmts was cured at a frequency of

_10-4

by selecting forchloramphenicol sensitivity afterpenicillin en-richment(8). P1 cmtscould not be induced in cured strains,

*Correspondingauthor.

P1 cmtssensitivitywasregained,and noCATactivitycould be measured.

Bacteriophages and colicins. P1 cmts(20),P1 vir(34),A vir

(2), andXTn2 (10) aredescribed elsewhere. Phages T4, T6,

4)80,andcolicinVwere labstock;TulAandTP1 weregifts ofM. Schwartz, Paris, France. P1 cmts, P1 vir, X vir, and A Tn2 were always grown on C600, which is nonlysogenic.

X vir was labeled with

32Pi

as follows. E. coli C600 was grown in minimal medium (phosphate-free) containing 33 ,uM32p;(26.4,uCiml-

1)

and10 mMmaltose.A vir wasplated onthesecells intryptonebrothsoftagar andincubated for8 h. 32P_labeled A vir was recovered from the lysates after

centrifugation to remove cells (106 PFU/3,740 cpm).

Media. Davis minimal medium containing 0.2% proteose peptone no. 3 (Difco Laboratories) was used. Maltose (10 mM)was added toinduce maltose transportand the LamB

protein. Luria broth (LB) and tryptone brothwere used as

rich media and weresupplemented with5 x

10-3

CaCl2 and

10 mM maltosefor phage assays.

Phage inactivation experiments. Bacteriophages

(103

PFU) were incubated with

107

cells, 200

p.g

of deoxycholate

(DOC)-solubilized OM protein,or 200 ,ugofpurified LPS for

20 min at 37°C. Chloroform (3 to 4 drops) was added and

mixed for60s, and themixturewascentrifugedat12,000 xg

for 10 min. The supernatants were diluted and assayed directly onE. coli C600. Control experiments were carried outwithout cells. For these inactivation studies, cells were growninLBsupplemented with 5 x

10-3

MCaCl2for P1 vir and tryptone brothsupplemented with 10 mM maltosefor A vir.

32P-labeled X vir at 2 x 106 PFU was incubated at 37°C

with 2 x

107

E. coli cells which hadbeenpreviously grown on Davis minimal medium plus 10 mM maltose to induce LamB protein. At various time intervals, 100-,ul samples

were either centrifuged at 12,000 x gfor 15min orfiltered through

0.45-p.m

filters and washed twice with 5 ml of minimal medium. The supernatants or filtered cells were then assayedforradioactivity.

Cell surface isolation and analyses.

Periplasmic proteins

were released by osmotic shock (41). Cell

envelopes

were preparedbyFrenchpressure cell lysis at 16,000lb/in2of whole

cells, followed by removal of unbroken cells at

10,000

x g for 10 min and

finally by

sedimentation of the membrane fraction at 100,000 x g for 2 h.

Cytoplasmic

membranes 1047

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1048 TOMAS AND KAY

TABLE 1. Bacterial strains

Strain Properties Source

Escherichia coli

C600 Wild-type, rough LPS Lab stock (3)

GY2780 P1 cmts lysogen of E. coli C600 J. Blanco, Valencia, Venezuela

KT60 XTn2lysogen of E. coli C600 This study

KT61 A Tn2lysogen of E. coli GY2780 This study

CSH51 araA(lac-pro) rpsL thi J. H. Miller (25)

CSH57 ara leulacY purE gal trp his argG malA rpsL xylmtl ilu metA J. H. Miller (25) or B thi

KT64 P1 cmts lysogen of E. coli CSH51 This study

KT62 P1 cmts lysogen of E. coli CSH57 This study

KT82 E. coli GY2780 cured ofP1cmts This study

KT201 E.coli CSH51 cured ofP1 cmts This study

KT202 E. coli CSH57 cured ofP1 cmts This study

JB135 HfrG6mal7W-I; zah-735::TnJO;A(argF-lac)U169; zjb-729::TnIO; J. M. Brass, Constance, Federal

zja-742::TnlO; AmalE444 Republic ofGermany

JB135 HfrG6mal7T-I; zah-735::TnlO;A(argF-lac)U169;zib-729::TnIO; J. M.Brass,Constance, Federal

zja-742::TnlO; malE259 Republic of Germany

KT98 E. coli C600pBR325::Tn9

Salmonella typhimurium

SU453 hisF trpB metA rpsLxyl K. E.Sanderson, Calgary,

Can-ada

SA33 proA tre+clb+ galErfa K. E.Sanderson, Calgary,

Can-ada

were solubilized twice with sodium N-lauryl sarcosinate

(12), and the OM fraction was sedimented as before. OM

proteinsweresolubilized in 1% DOC and2mMEDTA(30).

Membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)by a modification (1) of the Laemmli procedure (23). Protein gels were routinely stained with Coomassie blue. Protein concentrations were determined by the Lowry procedure,

using bovineserumalbumin as astandard.

Whole-cellLPSwas

analyzed

by

SDS-PAGE after

protein

aran-ha

-S

_TiUA

1

2

3

4

56

7

8

FIG. 1. SDS-PAGE of whole-cell LPS from E. coli and S.

typhimurium. Cells(3x 107)wereincubated with proteinaseKfor4

hat55°Cinthepresenceof 1%SDS. Afterboilingfor5min,10-,ul

samples wereapplied toeach lane, and the gelwas silver stained

(37). Lanes: 1,E.coli C600;2,GY2780(P1cmtslysogen); 3, KT82 (P1cmtscured);4,CSH57;5, KT62 (P1cmtslysogen);6,KT202(P1

cmtscured);7, S. typhimurium SU453 (smooth); and8, S.

typhimur-iumSA33 (heptose-deficient LPSmutant).

digestion(37) (Fig. 1). LPSwasalsopurified by the method of Westphal and Jann (40) as modified by Osborn (13).

PurifiedLPSwashydrolyzed with 1 N HCl for 2 hat100°C.

Colorimetric analysis of deoxy-D-manno-octulosonic acid

(KDO) andL-glycero-D-manno-heptOse(heptose)contentof LPS was by the method of Osborn (13, 28). Organic

phos-phatewas assayed by the method of Bartlett(5). Monosac-charides were also analyzed as theiralditol acetate

deriva-tivesby gas-liquidchromatographyon a3% SP-3840column (Supelco) at 225°C. Alditol acetate

carbohydrate

standards

wereeitherpurchased fromSupelcoorprepared

by

standard methods.

Maltose transport. Cells

growing

under

inducing

condi-tionswereharvestedat

mid-log phase

and washed twice with fresh medium(without

maltose);

35 ,ug

(dry

weight)

of cells

wasincubated with 1,uM

[14C]maltose

at

37°C,

andsamples

were removed at 30-s intervals for 10 min,

filtered,

and

washedwith minimal medium. Thefilterswere dissolved in PCS (AmershamCorp.) and

assayed

for

radioactivity.

CAT activity.

Exponential-phase

cells

(40

ml)

growing

in

LB were

harvested,

washed twice with an

equal

volume of

0.1 M

Tris-hydrochloride (pH 7.8),

and

resuspended

in 4 ml

ofthesame buffer.

Resuspended

cellswere sonicated for 1

min, and unbrokencellswere removedat30,000 x gfor 15 min. CAT wasassayed at 37°C (33).

RESULTS

Establishment of lysogeny. P1 cmts

lysogeny

was easily

established in three different E. coli strains: C600, CSH51,

and CSH57. These lysogens occurred at high frequency

(-10-3).

Lysogenywasconfirmedinthesestrainsbytesting

for chloramphenicol, CAT activity in cell extracts, and

resistance to P1 cmts and by the recovery of P1 cmts particlesfrom

lysed

cells 3 hafterinductionat 42°C (30min).

All these strains were still sensitive to bacteriophages T4,

T6,

480,

TU1A, and TP1 aswellascolicin Vbut showeda pronounced reduction in EOP (0.001%) for X vir (7). For

comparison, we establishedX lysogenyin E. coliC600 and GY2780 by using X Tn2 and selecting for ampicillin resist-J. BACTERIOL.

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TABLE 2. Chemical compositionofLPS fromE.coli, P1cmts

lysogens,andcured lysogens

Strain KDOO ~~Heptose" Organic Strain

KDo"

(pmol/mg

ofLPS)

phosphate'

C600 0.35 0.18 0.66

GY2780 0.32 0.01 0.44

KT82 0.34 0.18 0.70

aKDO and heptose were assayed by gas-liquid chromatography and colorimetrically.

bOrganicphosphatewasassayed bythemethodof Bartlett(5).

ance(Apr). Theselysogenswerechecked for resistance to X Tn2andforthepresence ofkTn2particlesaftertemperature

induction.

Effect ofP1 cmtslysogenyonLPS. SinceP1 cmtslysogens

were apparently resistant to A vir, we examined the OM

composition. When whole-cell LPS was analyzed on SDS

gels bythemethod of Tsai and Frasch(37),it was apparent that there was a majorreduction in LPS size of the E. coli

CSH57 P1 cmts lysogen KT62 (Fig. 1). The LPS size

difference between E. coli C600 and its P1 cmts lysogen GY2780 is too small to be significantly differentiated by these gels since E. coli C600 is already a "rough" LPS mutant. However, a clear difference in chemical composi-tion was apparent (Table 2). When cured ofP1 cmts, these strains regained theirnormal

higher-molecular-weight

LPS

and were also shown to regaintheir normal EOP for A vir. Nochanges in LPS profilewere detected in KTn2

lysogens

ofE. coli strains (data notshown), suggesting this effect is phage specific.

Todescribe this effectonLPS more

thoroughly,

LPSwere purified fromthesevarious strainsand chemically

analyzed

(Table 2).TheP1 cmtslysogen GY2780 contained a heptose-deficient form ofLPS, and thecured

lysogen

regained this

carbohydrate. No other normal LPS core

carbohydrates

weredetected,indicatingthatE.coliC600 isan

rfaE,

D,orC mutant,chemotypeRd, and that thecured

lysogen

is essen-tially equivalent.

Effect of lysogeny on phage inactivation. In theory, the

alteration ofcore oligosaccharide should have affected P1

phage absorption (3). Either whole cells or purified LPS

could be used to inactivateP1 vir(Table 3). Whole cellsor

LPS from C600 inactivated P1 vir as expected, but neither

the P1 cmts

lysogen

GY2780 nor LPS from this strain

inactivated P1 vir. A KTn2lysogen of C600 (KT60) and its

corresponding LPS: readily inactivatedP1 vir;however,aP1 cmtsKTn2double

lysogen

anditscorrespondingLPS had no

effect.TheP1 cmts-curedstrain KT82 recovered the normal property ofinactivating P1 vir.

TABLE 3. Inactivation ofP1 virandX virbywholecellsand OM components

% Inactivation ofP1 vir %Inactivation ofXvirby:

Strain by:

Wholecells' LPSb Wholecells' DOC-OMd

C600 90.0 31.1 90.0 54.3

GY2780 <0.1 <0.1 0 0

KT82 87.8 31.2 89.2 52.7

KT60 89.9 33.2 91.6 62.2

KT61 <0.1 <0.1 0 0

a CellsweregrowninLB.

bPurified LPS(1 mg

ml-1).

cCellsweregrown in LBplus0.2%maltose.

dOM solubilized in

1%

DOC(30).

When asimilar seriesofKvirinactivationexperimentswas

conducted with eitherwhole cells or DOC-solubilized OM from maltose-induced cells (which have previously been shown to contain theLamB protein [30]) good K vir inactiva-tion was found. Neither theP1cmtslysogenGY2780 nor the

P1 cmts X Tn2 double lysogen was able to inactivate A vir. The K Tn2single lysogen KT60 was similar inthis regard to its C600 parent. As before, curing of the P1 cmts lysogen resulted in the ability to inactivate K vir. Therefore, P1 cmts

lysogenyrenders E. coli unable to inactivate K vir even in the presence of the LamB protein (see Fig. 3). As aconfirmation

of this observation,weexamined the adsorption

characteris-tics of

32P-labeled

A vir to maltose-induced cells of E. coli

C600, the P1 cmts lysogen GY2780, and the cured strain KT82(Fig.2A). Onlythe parent and cured strains caused the

lossof

32P-labeled

K vir fromthe incubation mixture when a

phage-inactivation protocol was used; in a filter assay (Fig. 2B), only these same cells bound

32P-labeled

A vir. Under

these conditions, theP1 cmtslysogenhad only0.03%of the

32P-labeled

A vir binding activity of the nonlysogens.

Effect ofP1cmtslysogenyon maltosetransport and maltose-inducible proteins. In view of the observation that the A-receptoractivity wasnonfunctionalin P1 cmtslysogens, we

examined the activity of the maltose transport system in

these strains under inducing conditions (Table 4). The P1

cmts lysogens were partially defective (-35% of wild-type

activity) in the transport of maltose, and this defect was

corrected upon curing of the P1 cmts lysogen. No maltose transport defect was apparent in the K Tn2 single lysogen. The periplasmic and OM fractions of these cells were also

examined by SDS-PAGE(Fig. 3) to try tolocalize the defect. Intheperiplasmicfraction there were few changes. Howev-er, expressionof thelevel of maltose-binding protein (MBP)

(molecular mass, 37 kilodaltons

[Kda])

(19) was somewhat

reduced in the maltose-induced P1 cmts single and double

CE,)

0

M-.5

1.0

35:

cL

0.5

cm

CE

0

6

4

2

x -x

I

B

-)

10t

5

L

5

10

5

10

15

20

MINUTES

FIG. 2. Binding of32P-labeledK virtodifferent E.coli strains. X

virwaslabeledbylysisof

32Pi-labeled

cellsofE.coliC600.Phage bindingwasassayed bydisappearanceof32P-labeledphage(A)and by directbindingtowhole cells(B) (see text). Background adsorp-tion was determined with S. typhimurium SA33 as a control. Symbols: A,E.coliC600; x,P1cmtslysogen GY2780;*,P1

cmts-cured strainKT82.

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1050 TOMAS AND KAY

TABLE 4. Maltosetransportactivity inE.coli strains

Maltose

Strain Description

transport'

(p.mol min-mg-1[drywt]) C600 Parent 6.96 ± 0.3b GY2780 P1 cmtslysogen 2.46 t 0.1 KT60 XTn2lysogen 8.64 t0.4 KT61 P1cmtsX Tn2 doublelysogen 2.82 t0.2 KT82 GY2780cured of P1 cmts 6.78 t0.4

aCellswereinduced for transportby growth in LBplus10mMmaltose. Assays were done with[U-14CJmaltoseat5 x 10-7Mand with 0.035 mg of (dry weight) cellsml-'.

bAverageof three separatedeterminations. Standarderrors areindicated.

lysogens, which correlated withthe reduction in transport.

Ascontrols, theseproteinprofileswerecompared with those from strains JB135 and JB136 (malE mutants) which were

specifically defective in the expression of this 37-Kda

pro-tein. Also, no changes in the uptake of D-glucose or

D-galactose in P1 cmts lysogens were detected under these

conditions (datanotshown). From OM SDS-PAGE

profiles

(Fig. 3B), it was clear that no significant defects in the

expression of the LamB protein (48 Kda) occurred in these strains. However, an 18-Kdaprotein, inducible by maltose,

was significantly reduced, but notabsent, in P1 cmts

lyso-gens. The role of this protein is unknown. Nevertheless, theseresults indicate that

expression

and

disposition

ofthe

LamBprotein (X receptor) is apparently normal inP1 cmts lysogens, butthey are still unabletoabsorb X

phage (Table

3).

DISCUSSION

The most striking effect of P1 cmts

lysogeny

is that it

clearly

affects LPS core

biosynthesis, presumably

at the

level ofheptose or

nucleotide-heptose

synthesis

or at the

enzymewhich transfers

nucleotide-heptose

tothe

KDO-lipid

Areceptor,since the LPS

synthesized

inP1cmts

lysogens

is

heptose deficient. Since both the establishment of

lysogeny

andthesubsequent

curing

of the P1 cmts

plasmid

occur at a

high

frequency,

these effects must be due to a

phage

conversion effect and do not occur

by

mutation reversion. LPSconversionsby

lysogenic

phages suchas 15havebeen described before (31, 32) but

normally

donotaffect theLPS coreregion. However, the F'lac episome has recently been reportedtoaffect theglycosylation of LPScore oligosaccha-ride, precluding infectivity ofbacteriophage

4X174

(27).

Plasmid-mediated modificationshavealsobeenpreviously described (16,

21). However,

similar results with respect to OM LPS

composition,

maltosetransport, andXvir

inactiva-tion were also obtained with

wild-type

P1

phage

(data

not shown). The introduction of the Tn9 element into P1 does notappear to beresponsiblefor thelysogeniceffect onLPS,

since strain KT98(C600containingpBR325carryingtheTn9

element) had a normal EOP for A vir and P1 vir (datanot

shown).Itwould makesense,teleologically,forP1toinvoke

thiseffect to prevent furtherlysogenyorsuperinfection. It is well known that P1 bacteriophages use LPS core as a receptor, and the data here indicate that theheptose moiety isnormally required.

The effect of this LPS conversion on OM membrane systems is of interest. Surprisingly, it appears to affect the

functioningof the LamBprotein (48 Kda).Itis apparent that even in the presence of ample LamB protein, there is

virtuallynoA vir receptoractivityinP1cmts

lysogens.

Some

heptose-deficient LPSmutantsof E. coli have been shown to be deficient in OM protein content (22, 29). The P1 cmts

lysogens described here, however, were not measurably deficient in OM proteins; the reason forthisdiscrepancy is unknown,butotherheptose-deficientmutantsdo notexhibit

large reductions in OM proteins either (6, 15). Maltose-induced wild-type cells or lysogens displayed equivalent amounts of LamB protein according to SDS-PAGE of OM

preparations (Fig. 3), but this proteinclearly does not act as an effective receptorfor K vir, since this phage will simply not bind to P1 cmts lysogens. Since the LamB protein is clearly required forK-receptor activity (30, 36), we suggest the true receptor to be an LPS-LamB protein complex. Heptose-deficient LPS mutantshave already been reported tobedefective in K-phage inactivation butweresuggestedto be dueto areduced level of LamB protein (29). There isno currentevidence available to necessarily preclude the exis-tenceof more than one receptor for K. In this regard, neither protease norheat treatment of crudeOMextractscontaining LamBprotein destroys K inactivation (30).

Further evidence for a disfunction in this system is the apparent reduced rate of maltose transport in P1 cmts lysogens. It seems likely that as an inducer, maltose is entering theP1 cmtslysogens less efficiently and results ina reducedexpression of the MBP. Other periplasmicproteins

MALTOSE

+ - + - + - + -A -. ....-

-0o

0 0 STRAINS W o 4 o

B

4 6 n_.1 _ _ .~~~~~~~1

FIG. 3. SDS-PAGE of theperiplasmicandGMproteinsfrom E. colistrains. (A) Periplasmic proteinswereobtainedbythe method of Willis et al. (41). (B) OM were obtained as sodium N-lauryl sarcosinate-insoluble material (12). Cells were grown in Davis minimal medium plusproteose peptone no. 3 (Difco)either in the presence (+) orabsence (-) of 10 mM maltose (induced versus

uninduced).Bio-Radstandards(14.4, 21.5, 31.0, 45.0, 66.2,and 92.5 Kda)wereusedtocalculate molecularmass.Eachlane contained 40 (A)or50

jig

(B)ofprotein. The 48-Kda LamBprotein isincluded.

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are relatively unaffected. The LamB protein and the MBP

are the end products of divergent expression of the malB

region (17). Maltose diffuses through the OM via porins

other than LamB (18), thus thestrainsarenottotally devoid of maltose transportactivity. Themaltose-inducible 18-Kda OMproteinmayplayaroleinthisprocess.Theinduction by

maltose of OM proteins other than LamB has previously

been observed (11, 30), and all the functions of the mal

genes, such asmalF, areas yetunknown (17).

Itisusuallyexplained that Xvirions normally havealow

EOPonP1lysogensbecausethe P1plasmid restricts exoge-nous DNA (4, 7). However, the data here indicate that before this there existsamechanism invoked by the P1cmts plasmid which prevents A virions from recognizing their receptor. X virions, suchas Tn2used here,canstillinfect

P1 cmts lysogens but at a vastly reduced EOP. Having

survived the restriction system, subsequently recovered A phages will reinfect P1 lysogens withanEOPof -1, but this

ismostlikely the result ofmutation in thesecond-generation

A phages. X phages grown on E. coli GY2780 (P1 cmts

lysogen) are able to reinfect E. coli GY2780 and C600

(nonlysogen)atanEOPof 1.The A phages obtained

original-ly from E. coli GY2780 and recultured on E. coli C600 are

stillabletoreinfect both strains atan EOP of1.

It is becoming increasingly popular to view OM protein bacteriophagereceptors asLPS-protein complexes (14, 24, 39, 42). Ifasimilar LPS effectonthe X-phage OMreceptoris correct, then presumably other, even more specific, OM

proteins might also be affected. Indeed, we have recently

observedan even moredrastic effect of P1cmtslysogenyon

phosphatetransport in E.coli P1 cmtslysogens (manuscript in preparation).

ACKNOWLEDGMENTS

We thank J. M. Somers and K. Widenhorn for critical discus-sions, and J. M. Brass, M. Schwartz, and K. E. Sanderson for bacterialorphagestrains.

Thisworkwassupported by the Natural Sciences and

Engineer-ingResearch Council of Canada andbyapostdoctoralfellowshipto

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References

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