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 a10-3-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 aftercentrifugation 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 and10 mM maltosefor phage assays.
Phage inactivation experiments. Bacteriophages
(103
PFU) were incubated with107
cells, 200p.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 sampleswere 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 wholecells, followed by removal of unbroken cells at
10,000
x g for 10 min andfinally by
sedimentation of the membrane fraction at 100,000 x g for 2 h.Cytoplasmic
membranes 1047on March 14, 2021 by guest
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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 afterprotein
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
standardswereeitherpurchased fromSupelcoorprepared
by
standard methods.Maltose transport. Cells
growing
underinducing
condi-tionswereharvestedat
mid-log phase
and washed twice with fresh medium(withoutmaltose);
35 ,ug(dry
weight)
of cellswasincubated with 1,uM
[14C]maltose
at37°C,
andsampleswere removed at 30-s intervals for 10 min,
filtered,
andwashedwith minimal medium. Thefilterswere dissolved in PCS (AmershamCorp.) and
assayed
forradioactivity.
CAT activity.
Exponential-phase
cells(40
ml)
growing
inLB were
harvested,
washed twice with anequal
volume of0.1 M
Tris-hydrochloride (pH 7.8),
andresuspended
in 4 mlofthesame buffer.
Resuspended
cellswere sonicated for 1min, and unbrokencellswere removedat30,000 x gfor 15 min. CAT wasassayed at 37°C (33).
RESULTS
Establishment of lysogeny. P1 cmts
lysogeny
was easilyestablished in three different E. coli strains: C600, CSH51,
and CSH57. These lysogens occurred at high frequency
(-10-3).
Lysogenywasconfirmedinthesestrainsbytestingfor 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). Forcomparison, 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
LPSand 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 chemicallyanalyzed
(Table 2).TheP1 cmtslysogen GY2780 contained a heptose-deficient form ofLPS, and thecuredlysogen
regained thiscarbohydrate. No other normal LPS core
carbohydrates
weredetected,indicatingthatE.coliC600 isan
rfaE,
D,orC mutant,chemotypeRd, and that thecuredlysogen
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 straininactivated P1 vir. A KTn2lysogen of C600 (KT60) and its
corresponding LPS: readily inactivatedP1 vir;however,aP1 cmtsKTn2double
lysogen
anditscorrespondingLPS had noeffect.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. coliC600, 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 aphage-inactivation protocol was used; in a filter assay (Fig. 2B), only these same cells bound
32P-labeled
A vir. Underthese 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 somewhatreduced in the maltose-induced P1 cmts single and double
CE,)
0
M-.5
1.0
35:
cL
0.5
cm
CE0
6
4
2
x -xI
B
-)
10t
5
L5
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;*,P1cmts-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 theexpression 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
anddisposition
oftheLamBprotein (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 itclearly
affects LPS corebiosynthesis, presumably
at thelevel ofheptose or
nucleotide-heptose
synthesis
or at theenzymewhich transfers
nucleotide-heptose
totheKDO-lipid
Areceptor,since the LPSsynthesized
inP1cmtslysogens
isheptose deficient. Since both the establishment of
lysogeny
andthesubsequentcuring
of the P1 cmtsplasmid
occur at ahigh
frequency,
these effects must be due to aphage
conversion effect and do not occur
by
mutation reversion. LPSconversionsbylysogenic
phages suchas 15havebeen described before (31, 32) butnormally
donotaffect theLPS coreregion. However, the F'lac episome has recently been reportedtoaffect theglycosylation of LPScore oligosaccha-ride, precluding infectivity ofbacteriophage4X174
(27).Plasmid-mediated modificationshavealsobeenpreviously described (16,
21). However,
similar results with respect to OM LPScomposition,
maltosetransport, andXvirinactiva-tion were also obtained with
wild-type
P1phage
(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.
Someheptose-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 oB
4 6 n_.1 _ _ .~~~~~~~1FIG. 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.J. BACTERIOL.
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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
J.M.T. from theUniversity ofVictoria. LITERATURE CITED
1. Ames, G.F.-L., E. N. Spudich, and H. Nikaido. 1973. Protein composition oftheoutermembrane of Salmonellatyphimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117:406-416.
2. Appleyard, R. K. 1954. Segregation of new lysogenic types
duringgrowth ofadoublylysogenic strain derivedfrom
Esche-richiacoli K12. Genetics39:440.
3. Archibald,A.R.1980.Virusreceptors,p.261-276.InBacterial
viruses.Chapman & Hall, Ltd., London.
4. Barksdale, L., and S. B. Arden. 1979. Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Mi-crobiol. 28:265-299.
5. Bartlett, G. R. 1959.Phosphorusassayincolumn chromatogra-phy.J. Biol. Chem.234A:466-468.
6. Beher, M. G., and C. A. Schnaitman. 1981. Regulation ofthe OmpAoutermembraneproteinof Escherichiacoli.J.Bacteriol. 147:972-985.
7. Birge, E.A.1981.Bacterial andbacteriophagegenetics,p.
176-177. Springer-Verlag,NewYork.
8. Carlton, B. C., and B. J. Brown. 1981. Gene mutation, p. 222-242. In P.Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N.R. Kreig, and G. B. Phillips (ed.),
Manualof methods forgeneral bacteriology. AmericanSociety forMicrobiology, Washington, D.C.
9. Chattoraj, D. K. 1977. Genetic andphysical map of bacterio-phage P1,p.733-736. In A.Bukhari,J.Shapiro,and S.Addyer (ed.), DNA insertion elements, plasmids and episomes. Cold
Spring HarborLaboratory, ColdSpring Harbor,N.Y. 10. Davis, R. W., D. Botstein, and J. R. Roth. 1980. Advanced
bacterial genetics.ColdSpring Harbor Laboratory, ColdSpring Harbor, N.Y.
11. Endermann, R., J. Hindennach, and V. Henning. 1978. Major proteins of the Escherichia coli outer cell envelope membrane. Preliminary characterization of the phage A receptor protein. FEBS Lett. 88:71-74.
12. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergentsodium-lauryl sarcosinate. J. Bacteriol. 115:717-722.
13. Hanson, R. S., and J. A. Phillips. 1981. Chemicalcomposition, p. 328-364. In P.Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Kreig, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C.
14. Hantke, K. 1978. Major outer membrane proteins of E. coli K12 serve asreceptors for the phages T2 (proteinIa)&434(protein 16). Mol. Gen. Genet. 164:131-135.
15. Havekes, L. M., B. J. J.Lugtenberg, and W. P. M. Hoekstra. 1976. Conjugationdeficient E. coli K12 F- mutants with hep-tose-lesslipopolysaccharide. Mol. Gen. Genet. 146:43-50. 16. Hoffman, J., B. Lindberg, M. Glowacka, M. Derylo, and Z.
Lorkiewicz. 1980. Structural studies of the lipopolysaccharide from Salmonella typhimurium 902 (Col II drd 2). Eur. J. Biochem. 105:103-107.
17. Hofnung, M., D.Hatfield, and M. Schwartz. 1974. malB region in Escherichia coli K12: characterization of new mutations. J. Bacteriol. 117:40-47.
18. Kadner, R. J., and P. J. Bassford, Jr. 1978. The role of the outer membrane in active transport, p. 426. In Bacterial transport. Marcell Dekker, Inc., New York.
19. Kellermann, O., and S. Szmelcman. 1976. Active transport of maltose in Escherichia coli K12. Involvement of a "periplas-mic" maltose binding protein. Eur. J. Biochem. 47:139-149. 20. Kondo, E., and S. Mitsuhashi. 1964. Drugresistance of enteric
bacteria. IV. Active transducing bacteriophage P1 CM pro-duced by thecombination ofRfactor withbacteriophageP1.J. Bacteriol. 88:1266-1276.
21. Kopecko, D. J.,0.Washington, and S. B. Formal. 1980. Genetic and physical evidence for plasmid control of Shigella sonnei formIcellsurface antigen. Infect. Immun. 19:207-214. 22. Koplow, J., and H. Goldfine. 1974. Alterations in the outer
membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. J. Bacteriol. 117:527-543.
23. Laemmli, U. K. 1970.Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.
24. Lugtenberg, B., and L. Van Alphen. 1983. Molecular architec-tureand functioning of the outer membrane of Escherichiacoli and other gram-negative bacteria. Biochim. Biophys. Acta 737:51-115.
25. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 26. Murialdo, H., and A. Becker. 1978. Head morphogenesis of
complex double-stranded deoxyribonucleic acid bacterio-phages. Microbiol. Rev. 42:529-576.
27. Ohkawa,T.1982.The coreoligosaccharide in LPS oftheTer-15 mutant after the transformation ofF'-lac episome. Biochem. Biophys. Res.Commun. 108:1413-1417.
28. Osborn, M. J. 1966. Preparation oflipopolysaccharide from mutantstrains ofSalmonella. Methods Enzymol. 8:161-164. 29. Randall, L. L. 1975. Quantitation of the loss of the
bacterio-phage lambda receptor protein from the outer membrane of lipopolysaccharide-deficient strains of Escherichiacoli. J. Bac-teriol. 123:41-46.
30. Randall-Hazelbauer,L.,and M. Schwartz.1973.Isolation ofthe bacteriophage lambda receptor from Escherichiacoli.J. Bacte-riol. 116:1436-1446.
31. Robbins,P. W.,I. M.Keller, A. W.Wright,and R.Bernstein. 1965. Enzymatic and kinetic studies on the mechanism of0 antigen conversion by bacteriophage 15. J. Biol. Chem.
on March 14, 2021 by guest
http://jb.asm.org/
1052 TOMAS AND KAY
240:384-390.
32. Robbins, P. W., aid T. Uchida. 1962. Studiesonthe chemical
basis of the phage conversion of 0 antigens in the E group
Salmonella. Biochemistry 1:323-335.
33. Shaw, W. V., and R. F. B,rodsky. 1968. Characterization of
chloramphenicol acetyltransferasefrom
chloramphenicol-resis-tantStaphylococcusaureus.J. Bacteriol. 95:28-36.
34. Sternberg, N., and R. 1-oess. 1983. The moleculargeneticsof bacteriophageP1. Annu. Rev. denet. 17:123-154.
35. Susskind, M.M., andD. Botstein. 1978. Moleculargeneticsof bacteriophage P22. Microbiol. Rev.42:385-413.
36. Szmelcman, S.,M.Scpwartz,T.J. Silhavy,and W. Boos.1976. MAltose transport in Escherjchia coli K12. A comparison of
transportkineticsin
wild-type
and X-resistantmut,antswith the dissociation constants of the maltose-binding protein asmea-sured byfluorescencequenching. Eur. J.Biochem. 65:13-19.
37. Tsai,C.M.,and C.E.Frasch. 1982. A sensitivesilver stain for
detecting lipopolysaccharides in polyqcrylamide gels. Anal.
Biochem. 119:115-119.
38. Tyler, B. M., and R. B. Goldberg. 1976. Transductiop of chromosomal genes betweenenteric bacteria by bacteriophage P1. J. Bacteriol. 125:1105-1111.
39. Van Alphen, L., B. Lugtenberg, E. T. Rietschel, and C. Mombers.1979. Architecture of the outer membrane of Esche-richia coli K12. Phase transitions of the bacteriophage K3 receptorcomplex. Eur. J. Biochem. 101:571-579.
40. Westphal,O., and K. Jann. 1965. Bacteriallipopolysaccharides. Extraction with phenol-water and further applications of the procedure.Methods Carbohydr. Chem. 5:83-91.
41. Willis, 1]. C., R. G.Morris, C. Cirakoglu, G. D. Schellenberg, N. H. Gerber, and; C. E. Furlong. 1974. Preparation of the periplasmic binding proteins from Salmonella typ/pimurium and Escherichia coli. Arch. Biochem. Biophys. 161:64-75. 42. Yu, F., H.Yamada, and S. Mizushima. 1981. Role of
lipopoly-saccharide in the receptor function for bacteriophage TuIb in Escherichia coli. J. Bacteriol. 148:712-715.
J. 13ACTERIOL.