Vol. 161, No. 2

Membrane Proteins Correlated with Expression of the PolysialicAcid Capsule in Escherichia coli Ki

CHRISTOPHER WHITFIELD,1t* ERIC R. VIMR,2 t J. WILLIAM COSTERTON,1 AND FREDERIC A. TROY2

Department of Biology, University of Calgary, Calgary, Alberta, Canada T2N 1N4,1 and Department of BiologicalChemistry, University of California School of Medicine, Davis, California 956162

Received 30 July 1984/Accepted 31 October 1984

Growth of Escherichia coli Kl strains at 15°C results in a defect in the synthesis or assembly of the Klpolysialic acid capsule. Synthesis is reactivated in cells grown at 15°C after upshift to 37°C, and activationrequires protein synthesis (Whitfield et al., J. Bacteriol. 159:321-328, 1984). Using this temperature-induceddefect, we determined the molecular weights and locations of membrane proteins correlated with the expressionof Kl (polysialosyl) capsular antigen. Pulse-labeling experiments demonstrated the presence of 11 proteinswhose synthesis was correlated with capsule appearance at the cell surface. Using the differential solubility ofinner and outer membranes in the detergent Sarkosyl, we localized five of the proteins in the outer membraneand four in the inner membrane. The subcellular location of two of the proteins was not determined. Fiveproteins appeared in the membrane simultaneously with the initial expression of the Kl capsule at the cellsurface. One of these proteins, a 40,000-dalton protein localized in the outer membrane, was identified as porinprotein K, which previously has been shown to be present in the outer membrane of encapsulated E. coli. Thepossible role of these proteins in the synthesis of the polysialosyl capsule is discussed.

The Kl capsular polysaccharide (CPS) produced by strainsof Escherichia coli Kl is comprised of polysialic acid (poly-N-acetylneuraminic acid) joined by a-2-8 ketosidic linkages(2, 3, 5, 10, 11). The ability to produce Kl CPS is recognizedas an important correlate of invasiveness in E. coli strains(25).

Several aspects of the biosynthetic pathway for Kl CPShave been characterized, in particular the role of the sialyl-transferase complex in synthesis of the polysialosyl capsularpolymers (reviewed in reference 29). In contrast, relativelylittle is known concerning the mechanism(s) of Kl chaininitiation or translocation of the CPS from the site ofsynthesis at the inner membrane (IM) to the outer leaflet ofthe outer membrane (OM). Our recent studies directedtoward understanding these processes have been based onthe observation that the synthesis of polysialic acid istemperature sensitive in E. coli, with no Kl capsule beingsynthesized at temperatures below the phase transitiontemperature of the bulk membrane lipids (6, 30). Becausemembranes prepared from cells grown at 15°C containsialyltransferase that is capable of elongating exogenouslyadded oligosialyl acceptors, the defect induced by growth at15°C appears to reflect an inability to synthesize or assemblean endogenous acceptor into the sialyltransferase complex(30). Activation of polysialic acid synthesis has been shownto occur when membranes prepared from E. coli cells grownat 15°C are incubated in vitro at 33°C for 2 to 4 h (32a).Activation was obligatorily coupled to protein synthesis, andspecific polypeptides that are correlated with in vitro activa-tion have been identified (33a).We have recently shown that activation of polysialic acid

synthesis in vivo also requires protein synthesis (34) but thatthe elongation is apparently independent of protein synthesis

* Corresponding author.t Present address: Department of Microbiology, University of

Guelph, Guelph, Ontario, Canada NlG 2W1.t Present address: VMBSB, University of Illinois, Urbana, IL

61801.

both in vivo (34) and in vitro (Whitfield and Troy, in press).This suggests that the requirement for protein synthesis isrelated to either initiation of Kl CPS synthesis or transloca-tion of polymeric chains from the IM to the periphery of theOM. In this paper we identify and locate specific membraneproteins whose synthesis is correlated with Kl capsuleexpression in growing E. coli Kl cells.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The conditionsused for growth of E. coli K-235 (O1:K1:H-) have beendescribed previously (31). For 35S labeling, cells were grownin M63 minimal medium (16) supplemented with 2% D-glu-cose and L-[35S]methionine.

Bacteriophage adsorption experiments. Temporal expres-sion of the Kl capsule was monitored by adsorption ofbacteriophage K1F, which is specific for Kl-encapsulated E.coli. The experimental procedures used have been describedin detail previously (34).

L-[35S]methionine labeling of membrane proteins. Radioac-tive labeling was carried out by using mid-exponential phasecells. Routinely, 100 ,uCi of L-[35S]methionine (1,100Ci/mmol; Amersham Corp.) was added to 10 ml of cells,followed by incubation for 2 min. The bacteria were har-vested by centrifugation at 3,000 x g for 5 min and washedonce in 10 mM Tris-hydrochloride (pH 7.6). The washedcells were suspended in 3 ml of 10 mM Tris-hydrochloride -1mM dithiothreitol (pH 7.6) and disrupted by ultrasonication.Unbroken cells were removed by centrifugation at 3,000 x gfor 10 min. Total cell membranes (TM) containing both IMand OM were pelleted from the cell-free lysate by ultracen-trifugation at 200,000 x g for 60 min, washed once, andsuspended in 0.2 ml of 10 mM Tris-hydrochloride-1 mMdithiothreitol (pH 7.6).

Preparation of inner and outer enriched membrane frac-tions. Membrane fractions enriched for either IM or OMwere prepared by differential solubilization of TM by usingthe detergent Sarkosyl (sodium lauroyl sarcosinate; SigmaChemical Co.). The method of Filip et al. (14) was adapted

743

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744 WHITFIELD ET AL.

ipmiw OPIo iiisidmwm..'

.;,jl,-i.:;. -:.e .; .: I"

.4

-0

-a

-9

-4

~A* Omee

dob 4**

20.1K-

X--18Kii 5 b l^ ' j;

144K--

FIG. 1. Autoradiogram of SDS-PAGE of TM isolated from E.coli K-235 after temperature upshift. The methods used for pulse-labeling cells with L-[35S]methionine, preparation of TM, and SDS-PAGE are described in the text. Lane A contained zero-time controlcells; E. coli cells grown to mid-log phase at 15°C were harvestedand suspended in fresh M63 medium equilibrated at 15°C; this wasfollowed by pulse-labeling. Lanes B through F contained TMpreparations from identical cells that were shifted to 37°C at zerotime. Samples were removed and pulse-labeled at 5 min (lane B), 10min (lane C), 15 min (lane D), 20 min (lane E), and 30 min (lane F)after the temperature upshift. Lane G contained control cells whichwere grown and pulse-labeled at 37°C. The appearance of polysialicacid capsule at the cell surface was monitored by bacteriophageadsorption experiments. In this and subsequent figures, the onset ofbacteriophage binding is indicated by an arrow above the appropri-ate time point. The standard proteins used are indicated on the leftof the gel, and the newly synthesized proteins are indicated on theright.

for use with small volumes; 50-,ul portions of 35S-labeled TMwere diluted to 0.5 ml with 100 mM Tris-hydrochloride (pH7.6) containing 0.5% (wt/vol) Sarkosyl. The membraneswere mixed vigorously and extracted at room temperaturefor 30 min. Soluble and particulate fractions were separatedby centrifugation in a Beckman Airfuge at 140,000 x g for 5min; the resulting supernatant contained partially purifiedIM proteins. The Sarkosyl-insoluble pellet contained OMand small amounts of contaminating IM. The residual IMwas removed by a second Sarkosyl extraction, and subse-

quent extractions had no further effect on the resulting OMprotein profile. The OM fraction and the initial IM extractwere solubilized in electrophoresis sample buffer (18). Therelative purity of the membrane fractions was identical to thepurity obtained by the original method, as assessed bysodium dodecyl sulfate (SDS)-polyacrylamide gel electropho-resis (PAGE).

Preparation of peptidoglycan-associated proteins. Peptido-glycan-associated proteins were prepared by using a modi-fication of the method of Rosenbusch (24). Samples (50 ,ul) of35S-labeled TM were diluted with 0.45 ml of 10 mM Tris-hy-drochloride (pH 8.0) containing 2% (wt/vol) SDS. Extractionwas performed at 32°C for 30 min. Particulate fractionscontaining peptidoglycan and associated proteins were ob-tained as a pellet following centrifugation at 140,000 x g for5 min in a Beckman Airfuge. Optimal purity was achievedfollowing a second extraction. The residual pellet was sus-pended in 0.1 ml of electrophoresis sample buffer at 100°Cfor 10 min.

Trypsin treatment of TM. A 0.02-ml portion of radiola-beled TM was diluted with 0.18 ml of 50 mM Tris-hydro-chloride (pH 7.0) containing trypsin at a final concentrationof 50 ,ug/ml. Trypsin treatment was performed at 37°C for 60min, and digested TM was harvested by centrifugation at140,000 x g for 5 min in a Beckman Airfuge. The pellet waswashed twice in 10 mM Tris-hydrochloride (pH 7.6) andfinally dissolved in electrophoresis sample buffer (18). Con-trol samples were processed in the same way except thattrypsin was omitted.SDS-PAGE and autoradiography. SDS-PAGE was per-

formed by the method of Laemmli (18), using 12% (wt/vol)acrylamide in the resolving gel and 4% acrylamide in thestacking gel. Samples were dissolved in Laemmli electropho-resis sample buffer (18) prior to SDS-PAGE. Solubilizationwas performed at 100°C for 10 min unless otherwise indi-cated. Equivalent amounts of radioactivity (routinely 250,000cpm) were applied to each sample well. Gels were stainedwith Coomassie brilliant blue R-250. Destained gels weredried under a vacuum and subjected to autoradiography byexposure against Cronex 6-Plus X-ray film (Du Pont Co.) at-70°C. Films were developed according to the instructionsof the manufacturer.

Radioactive counting procedures. Radioactivity was quan-tified by scintillation counting, using a Beckman modelLS8000 liquid scintillation counter. Samples (0.01 ml) weredried onto squares ofWhatman filter paper and counted in 10ml of Omnifluor scintillant (New England Nuclear Corp.).

RESULTS

Membrane proteins correlated with expression of Kl CPS.The range of membrane proteins whose presence was cor-related with expression of Kl CPS was established intemperature upshift experiments. E. coli K-235 cells weregrown to mid-exponential phase at 15°C, conditions underwhich no Kl CPS is synthesized (6, 30). The cells wereharvested by centrifugation and suspended in fresh M63growth medium equilibrated at 37°C. At appropriate timeintervals samples were withdrawn for bacteriophage adsorp-tion studies, an indicator of Kl CPS expression, as de-scribed previously (34). An additional 10-ml sample wastransferred to a separate flask and incubated for 2 min at37°C in M63 medium containing L-[35S]methionine. Cellsgrown and radiolabeled at 15 and 37°C were used as con-trols. Cells were immediately used as a source of TM andsolubilized in SDS in preparation for SDS-PAGE.

A B C D E F G

94K-

67K-

43K

30K-

.. ... ...... __ __^ * * ,tS ::;.,|., ^,lE, ... ...i_ _.______ -___ __: _- __

-----_F_f = sS ------ --

x.e -t twMs>e S. ..... .. xw:-t -t-_t_

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BIOSYNTHESIS OF E. COLI K1 CAPSULE 745

CD E FI.. X..;.:f

A B

67K

K .... ..

.....

...3K

.I30K-

43K-

30K_

94K

67K-

:nsswrws,jagrX. i 8

59K: _ _~~~56K: _ _:777..rvF -77-

polypeptides with Mr ranging from 78,000 (78K) to 18K weresynthesized after the temperature shift. Four proteins (59K,56K, 40K, and 27K) were clearly identified. The remainingtwo (78K and 18K) were present in trace amounts; althoughthese proteins were reproducibly detected as faint bands onoriginal autoradiograms, their presence could not be demon-strated after photography. All six polypeptides were presentjn the membranes of bacteria grown at 37°C. Of theseproteins, four (78K, 59K, 56K, and 27K) were detectable inthe membrane 5 min after the temperature shift. Synthesis ofthe 40K and 18K proteins was temporally correlated with

A B C

{..

t;

.;.1::

,

D E F

I

2OAK QL * *

||E _~~~18K

1440-K

FIG. 2. Autoradiogram of SDS-PAGE of OM isolated from E.coli K-235 after temperature upshift. Sarkosyl-insoluble OM wereprepared as described in the text. Lane A contained control (zero-time) OM prepared from E. coli cells grown at 15°C, and lane Fcontained OM prepared from cells grown and labeled at 37°C. LanesB through E contained OM extracted from cells pulse-labeled at 5,10, 15, and 20 min after upshift, respectively. Molecular weightmarkers are indicated on the left side of the gel, and newlysynthesized proteins are indicated on the right.

The time at which Kl CPS appeared at the cell surfacewas indicated by an increase in bacteriophage binding, andthe kinetics of this process have been reported in detailelsewhere (34). In all experiments described below, a 10- to12-min lag occurred between temperature upshift and theappearance of Ki CPS at the cell surface. The time at whichbacteriophage binding increased is indicated in each figureby an arrow.The protein profiles ofTM prepared from cells in a typical

temperature shift experiment are shown in Fig. 1. Six

20.1 K--4-18 K

14.4K-

FIG. 3. Autoradiogram of SDS-PAGE of IM isolated from E. coliK-235 after temperature upshift. Sarkosyl-soluble IM were isolatedas described in the text. Lane A contained control (zero-time) IMprepared from cells grown at 15°C. Lane F contained IM from cellsgrown and pulse-labeled at 37°C. Lanes B through E contained IMisolated from cells pulse-labeled at 5, 10, 15, and 20 min after thetemperature shift, respectively. Molecular weight markers are indi-cated on the left side of the gel, and newly synthesized proteins areindicated on the right.

VOL. 161, 1985

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746 WHITFIELD ET AL.

TABLE 1. Molecular weights and probable locations ofmembrane proteins whose synthesis was correlated with

expression of polysialic acid at the cell surface of E. coli K-235'Presence in: Probable TemporalProtein

TM OM IM location correlationb

78K Tr + Tr OM74K - + Tr OM59K + + Tr OM56K + + Tr OM'40K + + Tr OM Yes36K - - + IM Yes28K - - + IM27K + + +26K - - + TM Yes25K - - + IM Yes18K Tr Tr Tr Yes

a Data compiled from five independent labeling experiments. Typical gelsare shown in Fig. 1 through 3.

b Proteins appearing in membrane profiles at the same time that Kl CPSwas initially expressed at the cell surface.

c Protein present in trace quantities at 15°C but increasing in concentrationafter temperature upshift.

d Protein found in similar concentrations in both IM and OM.

expression of the Kl capsule since these proteins were notpresent at 5 min but were visible in the membrane concom-itant with expression of the capsule after 10 min (Fig. 1, laneC).

Localization of membrane protein changes to IM or OM.IM and OM were enriched by their differential solubilities inthe detergent Sarkosyl. These experiments were carried outin order to localize the proteins described above to either theIM or the OM. Further enrichment of each membrane typealso allowed us to detect proteins which were synthesizedonly in trace quantities and which were not visible in TMpreparations without overloading the gel.

All six of the newly synthesized proteins detected in TM(Fig. 1) were also present in'Sarkosyl-insoluble OM (Fig. 2),and an additional protein (74K) was also detected. In thispartially purified OM preparation, 56K and 27K polypep-tides appeared to be present in trace amounts in membranesfrom cells grown at 15°C and were considered by us sincethey increased in concentration after the temperature shift.The two polypeptides present as trace components of TM(the 78K and 18K proteins) were clearly evident followingenrichment for OM. The 18K protein was detectable as adiscrete band, and the 78K protein was detectable as thelower component of a closely migrating doublet. Only tracequantities of five proteins (78K, 74K, 59K, 56K, and 40K)were also detectable in the Sarkosyl-soluble IM fraction(Fig. 3). These results indicated either contamination of IMwith small amounts of OM or the trapping of trace quantitiesof OM proteins during synthesis at the IM. From the relativeenrichments, these five proteins (78K, 74K, 59K, 56K, and40K) were localized in the OM. Two proteins (27K and 18K)were present in similar quantities in both IM and OM, andtheir precise location was not resolved. The possibility thatthe 27K and 18K proteins reflect contamination by peri-plasmic components was considered, but is unlikely giventhe extensive washing and detergent solubilization tech-niques employed.Four additional proteins, which were not visible in either

TM (Fig. 1) or OM (Fig. 2), were detected in Sarkosyl-sol-uble IM fractions (Fig. 3). We presumed that one protein(36K) was masked by the major 36.5K OM protein. Signifi-cant amounts of a 36.5K polypeptide were present in the IM,

possibly either resulting from contamination with residualOM or reflecting synthesis of an OM protein at the IM.However, the reduction in the amount of 36.5K protein wassufficient to reveal the presence of the closely migrating 36Kcomponent. The 28K, 26K, and 25K proteins may be syn-thesized at levels too low to be detectable in routine TMgels. Synthesis of three of these IM localized proteins(namely, 36K, 26K, and 25K) was temporally correlatedwith Kl capsule expression.The results obtained from these SDS-PAGE studies are

summarized in Table 1.Characterization of the 40K protein as porin protein K. The

40K protein change was selected for further study becauseof previous reports that a 40K major OM protein, subse-quently shown to be a porin (28, 33), was correlated withencapsulation of E. coli strains having different capsularserotypes (1, 23, 32).The basic properties of the 40K protein are shown in Fig.

4. Samples were prepared from cells radiolabeled 30 minafter temperature upshift. The 40K protein was present inlarge quantities in TM (Fig. 4, lane A) and, in confirmation ofthe results shown in Fig. 2, was markedly enriched in OM(Fig. 4, lane B). Smaller amounts of a 40K protein in IM

A B C D E F Gvm_

94K~ 7M

43K- _

40K

38-5K

- 3 7K-;36-5K

--- 30K--- -

20-1K

14.4K

FIG. 4. Properties of the 40K protein cotrelated with expressionof Kl CPS in E. coli K-235. TM (lane A) were prepared frombacteria labeled 30 min after temperature upshift and were used as asource of OM (lane B) and IM (lane C). Lane D shows the effect oftrypsin treatment on the profile of TM. Lane E contained peptido-glycan-associated proteins extracted from TM. Lanes F and G showthe effect of solubilization at 100°C (lane F) or 37°C (lane G) on theprotein profile of OM. The positions of molecular weight markers,the 40K protein, and other major OM proteins are indicated.

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BIOSYNTHESIS OF E. COLI Kl CAPSULE 747

(Fig. 4, lane C) may reflect either synthesis of an OM proteinat the IM or contamination with residual OM, as discussedabove. Alternatively, a 40K IM protein may also be present.Treatment of TM with trypsin did not degrade three of thefour major OM proteins (40K, 38.5K, and 37K); the fourth(36.5K) showed extensive proteolysis (Fig. 4, lane D).Extraction of peptidoglycan-associated proteins revealedthree major bands (40K, 38.5K, and 37K) (Fig. 4, lane E).

Solubilization of OM at 37°C resulted in a loss of the fourmajor OM protein bands from the profile (Fig. 4, lane G).Because of the formation of SDS-resistant oligomers (tri-mers) which form tight noncovalent associations with thepeptidoglycan (21), pore-forming proteins are characteristi-cally not solubilized below 60°C. Formation of such oligo-mers of protein K have been demonstrated previously (33).Using the two-dimensional technique of Hui and Hurlbert(17), which was designed to study heat-modifiable mem-brane proteins, we demonstrated that the 40K protein stud-ied here indeed produced higher-molecular-weight aggre-gates when it was solubilized at 37°C (data not shown).

Since the porins protein K (23, 28, 33) and PhoE (22) bothhave apparent monomeric molecular weights of 40,000,chymotryptic peptide maps of the 40K protein describedhere were produced by using the method of Cleveland et al.(7) and were compared with similar maps for authenticprotein K, OmpF, and PhoE. Our results (data not shown)indicated that the 40K protein was identical to authenticprotein K but differed in 5 of 10 peptides from both OmpFand PhoE. These results were predicted from detailed stud-ies reported previously by Sutcliffe et al. (28).These general properties suggest that the 36.5K protein is

probably OmpA and that the 37K, 38.5K, and 40K proteinsare OmpC, OmpF, and protein K, respectively.Assembly of protein K in pulse-chase experiments. Pulse-

chase experiments were used to investigate the possibilitythat growth at 15°C resulted in the accumulation of aprecursor form of protein K which could then be chased intothe mature form after temperature shift. E. coli K-235 cellswere pulse-labeled for 10 min in mid-exponential phase at150C with L-[35S]methionine. The labeled cells were washedin M63 medium containing 1 mM unlabeled methionine toremove any residual radioactive substrate. The cells weresuspended in the original volume of M63 medium containing1 mM methionine equilibrated at 37°C. Samples were re-moved at appropriate intervals over a 20-min period, andOM was prepared as described above. As shown in Fig. 5,the protein profile of the OM did not change over theexperimental period. No radioactive protein K appeared inthe OM. The time at which Kl CPS was expressed at the cellsurface, measured by bacteriophage absorption (Fig. 5,arrow), was identical to the time observed in previousexperiments. Coomassie blue staining (data not shown) ofthe polyacrylamide gel shown in Fig. 5 revealed the presenceof trace quantities of protein K in the OM at later times. Toensure that protein K synthesis did occur in these latersamples, sample of cells was removed 20 min after thetemperature shift and washed in M63 medium to removeresidual unlabeled methionine, and the cells were pulse-la-beled with L-[35S]methionine for 5 min at 370C. These cellswere then used to prepare OM. As shown in Fig. 5, lane F,radioactive protein K was present in the OM of these controlcells.

DISCUSSIONIn this study we identified 11 membrane proteins whose

synthesis is correlated with expression of the Kl polysialic

acid capsule at the cell surface. Of these 11 proteins, 5appeared in the membrane simultaneously with KI CPSexpression. Previous studies have indicated that the low-temperature-induced defect in synthesis of the polysialicacid capsule does not occur because of an inability tosynthesize the sialyltransferase, but rather occurs due to theinability of membranes from cells grown at 15°C to synthe-size or assemble an endogenous acceptor (30). Furtherconfirmation of this suggestion was obtained from studies inwhich we used a sialyltransferase-defective mutant of E. coliK-235, since identical protein changes occurred in upshiftexperiments with both wild-type and mutant bacteria (Whit-field, unpublished data). Therefore, we believe that the

A B C D E F

94K-

67K-

43K

I..'..I30K-

20.1K-

144K-

FIG. 5. Assembly of the 40K protein in pulse-chase experiments.The experimental protocol used is described in the text. E. coliK-235 cells grown at 15°C were pulse-labeled with L-[35S]methionineat 15°C for 10 min and chased with unlabeled methionine at 37°C.Lanes A through E contained OM prepared from cells taken at 0, 5,10, 15, and 20 min after the chase, respectively. Lane F containedcontrol cells which were removed 20 min after the chase, washed,and pulsed again at 37°C with L-[35S]methionine. Molecular weightmarkers are indicated on the left side of the gel.

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748 WHITFIELD ET AL.

proteins identified in this study may be relevant for either thesynthesis or assembly of an endogenous acceptor, for bind-ing the CPS to the cell surface, or for processes involved intranslocation of CPS from the IM to the OM.Of the 11 proteins identified, the 40K protein was charac-

terized further. This protein was of particular interest be-cause of reports which indicated that the presence of a 40Kprotein, subsequently named protein K, was correlated withencapsulation in E. coli strains having different capsularserotypes (1, 23, 32). More recent studies have demonstratedthe OM pore-forming properties of protein K (28, 33). Ourresults show a temporal correlation between the insertion ofprotein K (and other proteins) into the membrane andsurface expression of polysialic acid CPS. Pulse-chase ex-periments showed no accumulation of protein K precursor at15°C, and thus de novo synthesis, rather than proteolyticprocessing of precursor pools, appears to occur after thetemperature upshift.The precise role of protein K in CPS expression remains

unclear. Current evidence supports a role for membranephospholipids in anchoring CPS to the cell surface (15, 26),and thus it is unlikely that protein K serves an attachmentrole. The possibility that protein K, by virtue of its pore-forming nature, may mediate the export of CPS has beenconsidered (33). Recent experiments in which the clonedgenes for Kl CPS biosynthesis were used (27) have demon-strated the requirement for a functional porin in the OM tofacilitate CPS expression at the cell surface (J. Foulds andW. Aaronson, Abstr. Annu. Meet. Am. Soc. Microbiol.1984, D21, p. 54). Any porin species could fulfill this role,but no information is available as to the relative efficiency ofeach porin species in facilitating expression. In this study,only protein K synthesis was temporally correlated withCPS expression; no expression of CPS occurred at 15°Cdespite the presence of OmpC and OmpF in the OM. Sincewe do not yet know how many independent aspects of Kibiosynthesis are defective at 15C, no conclusions can bedrawn from this observation alone.Recent studies by Di Rienzo and Inouye have provided

evidence for the cotranslocation of lipopolysaccharide fromthe IM to the OM with OM proteins (namely, matrix proteinand proteins 3 and 7) (13). It is possible that protein K mayfunction in an analogous manner for CPS translocationr, andthis may explain the requirement for continuous proteinsynthesis during the reactivation of Kl CPS synthesis (34).

It has been proposed that translocation of OM proteins,lipopolysaccharide, and surface appendages, such as pili,occurs at discrete sites (termed zones of adhesion or Bayerjunctions) at which the IM and OM come into contact (4).There are several models for the structure of these special-ized membrane domains (20), and it has been suggested byDe Leij et al. (8, 9) that these zones may be transientstructures generated by the synthesis and insertion of OMproteins. It has been proposed that export of CPS to the cellsurface occurs via Bayer junctions (4, 32a, 34). Therefore, itis possible that the insertion of porin proteins or perhapsother OM proteins also facilitates translocation of CPS in anonspecific fashion by stabilizing the Bayer junction atwhich export occurs. This would explain the observationthat any porin species is capable of facilitating Ki CPSexpression (Foulds et al., Abstr. Annu. Meet. Am. Soc.Microbiol. 1984).A further indication of the significance of protein K in this

particular system comes from experiments on in vitro acti-vation of Kl CPS biosynthesis (33a). Time-dependent acti-vation of the endogenous synthesis of sialyl polymers in

membranes from cells grown at 15°C required protein syn-thesis. Four proteins (40K, 21K, 17.5K, and 16.5K) werecorrelated with the activation process. No further character-ization of the 40K protein was possible in these in vitrostudies due to the small amounts of protein synthesized.Although we are not able to define precisely the function

of the proteins identified in this study, recent work withcloned Kl CPS biosynthetic genes tnay help clarify theproblem (27). Minicells have been used to identify enzymesinvolved in the synthesis of the CMP-NeuNac precursor,polysialic acid assembly, and CPS expression at the cellsurface. Interestingly, five proteins were involved in CPSexport (80K, 77K, 60K, 40K, and 37K). Proteins of similarsizes (78K, 74K, 59K, 40K, and 36K) were identified in theexperiments described here. Silver et al. (27) also detected19K and 18K proteins coded for by genes involved inpolysialic acid synthesis. An 18K protein is also describedhere. Whether these proteins are related to the 20K proteinshown previously to be missing in membranes from cellsgrown at 15°C and therefore perhaps related to the synthesisor assembly of the endogenous acceptor (30) remains to bedetermined.Both growth temperature (19) and membrane fluidity (12)

exert direct effects on membrane protein synthesis andassembly; therefore, precise definition of the range of pro-teins involved in CPS expression in E. coli Kl awaits furtherinvestigation. In the future studies will be carried out touncouple insertion of protein K and other proteins identifiedin this report in order to determine what direct effect theseproteins have on the in vivo activation of polysialic acidsynthesis.

ACKNOWLEDGMENTSThis study was supported by a fellowship from the Alberta Her-

itage Foundation for Medical Research (to C.W.) and by PublicHealth Service research grant AI-09352 from the National Institutesof Health (to F.A.T.).The excellent photographic work of Joyce Nelligan is gratefully

acknowledged.

LITERATURE CITED1. Achtman, M., A. Mercer, B. Kusecek, A. Pohl, M. Heuzenroe-

der, W. Aaronson, A. Sutton, and R. P. Silver. 1983. Sixwidespread bacterial clones among Escherichia coli Kl isolates.Infect. Immun. 39:315-335.

2. Barry, G. T. 1958. Colominic acid, a polymer of N-acetyl-neur-aminic acid. J. Exp. Med. 107:507-521.

3. Barry, G. T., and W. F. Goebel. 1957. Colominic acid, asubstance of bacterial origin related to sialic acid. Nature(London) 179:206-208.

4. Bayer, M. E. 1979. The fusion sites between outer and cytoplas-mic membranes of bacteria: their role in membrane assemblyand virus infection, p. 167-202. In M. Inouye (ed.), Bacterialouter membranes. John Wiley & Sons, Inc., New York.

5. Bolanos, R., and C. W. DeWitt. 1966. Isolation and characteri-zation of the Kl (L) antigen of Escherichia coli. J. Bacteriol.91:987-996.

6. Bortolussi, R., P. Ferrieri, and P. G. Quie. 1983. Influence ofgrowth temperature of Escherichia coli Kl on capsular antigenproduction and resistance to opsonization. Infect. Immun.39:1136-1141.

7. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K.Laemmli. 1977. Peptide mapping by limited proteolysis in so-dium dodecyl sulfate and analysis by gel electrophoresis. J.Biol. Chem. 252:1102-1106.

8. De Leij, L., J. Kingma, and B. Witholt. 1978. Insertion of newlysynthesized proteins into the outer membrane of Escherichiacoli. Biochim. Biophys. Acta 512:365-376.

9. De Leij, L., J. Kingma, and B. Witholt. 1979. Nature of the

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BIOSYNTHESIS OF E. COLI Kl CAPSULE 749

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25. Schiffer, M. S., E. Oliveira, M. P. Glode, G. H. McCracken, Jr.,M. Sarff, and J. B. Robbins. 1976. A review: relation betweeninvasiveness and the Kl capsular polysaccharide of Escherichiacoli. Pediatr. Res. 10:82-89.

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32a.Whitfield, C., D. A. Adams, and F. A. Troy. 1984. Biosynthesisand assembly of the polysialic acid capsule in Escherichia coliKl. Role of a low-density vesicle fraction in activation of theendogenous synthesis of sialyl polymers. J. Biol. Chem.259:12769-12775.

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34. Whitfield, C., E. R. Vimr, J. W. Costerton, and F. A. Troy.1984. Protein synthesis is required for in vivo activation ofpolysialic acid capsule synthesis in Escherichia coli Kl. J.Bacteriol. 159:321-328.

VOL. 161, 1985

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