Vol. 172, No. 2

Expression of the Escherichia coli K5 Capsular Antigen:Immunoelectron Microscopic and Biochemical Studies with

Recombinant E. coliKLAUS-DIETRICH KRONCKE,' GRAHAM BOULNOIS,2 IAN ROBERTS,2 DIETER BITTER-SUERMANN,3

JOCHEN R. GOLECKI,4 BARBARA JANN,' AND KLAUS JANN1*

Max-Planck-Institut fur Immunbiologie, Freiburg, Federal Republic of Germany'; Department of Microbiology,University of Leicester, Leicester, United Kingdom2; Institut fur Medizinische Mikrobiologie, Medizinische HochschuleHannover, Hanover, Federal Republic of Germany3; and Institut fur Biologie 2, Mikrobiologie, Universitat Freiburg,

Freiburg, Federal Republic of Germany4

Received 5 June 1989/Accepted 13 November 1989

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has beencloned previously, and three gene regions responsible for polymerization and surface expression have beendefined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310,1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressingthe K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemicalanalysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a

well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparationsand ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutantrevealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsularpolysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide,the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccha-rides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharidefrom the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and bothpolysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was

smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membraneand that gene region 1 directs the transport of the lipid-substituted capsular polysaccharide through theperiplasm and across the outer membrane.

The K5-specific capsular polysaccharide of Escherichiacoli has the structure 4)-,B-glucuronyl-(1,4)-ox-N-acetylglu-cosaminyl-(1, (34). As a consequence of its structural iden-tity with desulfoheparin, the first polymeric intermediate inthe biosynthesis of heparin (18, 21), humans and animals are

practically unable to raise antibodies against the K5 polysac-charide. As a result, E. coli expressing this nonimmunogeniccapsular polysaccharide is extremely virulent. The isolationof a K5-specific coliphage (12) and the construction of a

hybridoma cell line producing K5-specific monoclonal anti-body (5, 27) made the K5 capsule amenable not only todiagnostic and epidemiological studies but also to a molec-ular and cellular analysis of its expression.

It was shown that the K5 capsular polysaccharide belongsto a group of polysaccharides (group II) which also includesthe Kl, K7, K12, K92, and K100 polysaccharides (13a, 16).Genetic analyses (Sa, 7, 26, 27, 30-32, 35) revealed that thecapsule genes are organized in the same way in all strainstested and that they consist of three regions. Region 2 (7kilobases [kb] in E. coli K5) contains the polymerizationgenes. This region is flanked by region 1 and by region 3,both of which are responsible for the translocation of the K5polysaccharide and its surface expression as a capsule.Whereas region 2 is characteristic of the capsule type andvaries in E. coli with different capsular polysaccharides (Kantigens), regions 1 and 3 serve functions which are the same

* Corresponding author.

in all E. coli expressing group II capsules and are exchange-able within these E. coli strains (Sa, 26, 27). It was shownthat region 1 and region 3 mutants are acapsular but seem toproduce the capsular polysaccharide (Sa, 6, 27). Specificextraction procedures, by either saline extraction, osmoticshock, or cell desintegration, revealed that whereas no

polysaccharide could be extracted from region 2 mutants,both region 1 and 3 mutants contained intracellular capsularpolysaccharide. The location of the intracellular product wasnot defined unambiguously. To analyze the cellular locationand the physical nature of the capsular material in wild-typeE. coli K5, a K5 recombinant, and mutants defective inregions 1, 2, and 3, we studied the bacteria from thesesources by immunoelectron microscopy and compared theresults with those obtained from biochemical analyses ofcapsular material which was isolated from the differentbacterial strains.

MATERIALS AND METHODS

Chemicals. Alcian Blue 8GX, dithiothreitol, and Agar VIIfor electron microscopy were from Sigma, Deisenhofe,Federal Republic of Germany; acrylamide, N,N-methyl-enebisacrylamide, N,N,N',N'-tetramethylethylenediamine(TEMED), Epon 812, dodecyl-succinic anhydride, meth-ylphthalic anhydride, 2,4-6-tri(dimethylaminoethyl)phenol,lysozyme, osmium tetroxide, and uranyl acetate were allfrom Serva, Heidelberg, Federal Republic of Germany;cetyltrimethylammonium bromide (Cetavlon) as well as all

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TABLE 1. Bacteria used

Relevant SourceStrain number properties (reference)

20026 [(Bi8337-41)] Encapsulated wild-type (26, 34)010:K5:H4

21241 [LE392] Acapsular K-12, used (26)as host

21484 [LE392(pGB118)] E. coli K-12 recombi- (26)nant expressing theKS capsule

21487 [LE392(pGB118iA1)] Region 1 deletion This papermutant

21496 [LE392(pGB118: Region 2 insertion (6)TnlO00-16)] mutant

21500 [LE392(pGB118: Region 3 insertion (6)Tn1000-24)] mutant

media for cultivation of bacteria were from E. Merck AG,Darmstadt, Federal Republic of Germany; the componentsof the Lowicryl K4M resin: triethylene glycol dimethylacrylate, hydroxypropyl methacrylate, n-hexacyl methacry-late, and benzoin methylester were from Chemische WerkeLowi, Waldkrainburg, Federal Republic of Germany; TSKwas from LKB, Grafelfing, Federal Republic of Germany;silver stain kit was from Bio-Rad, Munich, Federal Republicof Germany; and gold conjugated anti-mouse immunoglobu-lin antibody was from Janssen, Kaldenkirchen, FederalRepublic of Germany.

Buffers. Kellenberger buffer (17) consisted of a stocksolution containing, per liter, sodium acetate 3H20 (1.94g), sodium barbital (2.94 g), and NaCI (3.4 g). To 10 ml of thissolution, 0.1 M HCl (14 ml), CaCl2 (30 mg), and deionizedwater (26 ml) was added, and the pH was adjusted to 7.1 withsodium hydroxide. Barbital buffer contained, per liter, 5,5-diethylbarbituric acid (1.84 g) and its sodium salt (10.3 g); thepH was adjusted to 8.6.

Bacteria and cultivation. The bacteria used in this studyare listed in Table 1. For the electron microscopic analysis,the bacteria were grown in Merck Standard I mediumcontaining, per liter, peptone (15.6 g), yeast extract (2.8 g),NaCl (5.6 g), and glucose (1.0 g). For isolation of the K5polysaccharide, the bacteria were grown on agar (2%) con-taining Merck Standard I medium. For the cultivation ofplasmid containing bacteria, the medium was supplementedwith ampicillin (100 ,ug/ml).DNA analysis. Plasmid DNA was isolated, digested with

restriction enzymes, and analyzed by agarose gel electro-phoresis as described previously (26, 27).Monoclonal anti-K5 antibody. Hybridoma cells (clone

1091) producing the immunoglobulin M-type monoclonalantibody (5, 25) were cultivated in minimal essential Eaglemedium containing streptomycin (100 ,.g/ml), penicillin (100,ug/ml), tylosin (10 jig/ml), and fetal calf serum (10%). Thecell culture supernatants were centrifuged in a Christ centri-fuge at 3,000 rpm twice for 30 min, and ammonium sulfatewas added to the supernatant to 50% saturation. The precip-itate was dissolved in 20 mM Tris hydrochloride (pH 7.0)containing glycine (10 mM) and NaCl (125 mM). This solu-tion was centrifuged (10,000 x g for 5 min) and subjected tohigh-pressure liquid chromatography on a TSK 3000 column.Elution was carried out with the same buffer at a flow rate of

0.5 ml/min. Fractions (0.5 ml) were monitored at 276 nm, andpeak fractions were tested with the enzyme-linked immuno-sorbent assay (8, 28) with the purified K5 polysaccharide asantigen. The active fraction contained 2.5 mg of protein perml and had an enzyme-linked immunosorbent assay titer of1:25,000.

Analytical methods. Glucuronic acid was determined di-rectly in the polysaccharide with the carbazole reagent, andglucosamine was determined after hydrolysis (4 M HCl at100°C for 18 h) with the Elson Morgan reagent. Immunoelec-trophoresis was performed according to I. 0rskov and F.0rskov (22) with the monoclonal anti-K5 antibody. Poly-acrylamide gel electrophoresis was run by the method ofPelkonen et al. (24) in 20-cm gels of 15 or 25% (SO V for 1 hor 350 V for 3.5 h). The gels were stained by the AlcianBlue-periodate-silver method (20, 23).

Isolation of the K5 polysaccharide. The capsular polysac-charide was isolated from agar-grown E. coli 21484 byextraction with saline and precipitation with cetyltrimethyl-ammonium bromide (Cetavlon) and was purified as de-scribed previously (11, 34).For the isolation of polysaccharide from E. coli 21487 and

21500, the bacteria were disintegrated with a French press(77 kg/cm2) and the homogenate was then centrifuged(100,000 x g for 1 h). The bacteria were also spheroplastedwith lysozyme-EDTA in 20% sucrose, and the spheroplasts,with or without fixation with glutaraldehyde, were removedby centrifugation (1,500 x g for 20 min). From all superna-tants, the polysaccharide preparations were obtained byprecipitation with Cetavlon and were purified (11, 34).Removal of the phosphatidic acid from the K5 polysaccha-

ride. For the chromatographic detection of the phosphatidicacid, loosely attached and noncovalently bound lipid wasremoved by extraction with chloroform:methanol (1:1 [vol/vol]). A sample (5 mg) of the polysaccharide obtained fromthe aqueous phase by freeze drying was heated in 1 ml ofcitrate buffer (10 mM; pH 4.0) for 20 min at 100°C. After thesolution was cooled, it was extracted twice with chloroform:methanol (1:1 [vol/vol]). The concentrated organic phasewas chromatographed on silica gel 60 with chloroform:methanol:water (65:25:4, by volume) as solvent. The lipidwas visualized with the molybdate-hydrazine reagent (13).The aqueous phase was lyophilized, and the residue, whichconsisted of the polysaccharide, was used for immunoelec-trophoresis.

Plasmolysis of the bacteria for electron microscopy. Plas-molysis of the bacteria was performed by the method of Smitand Nikaido (33). Briefly, the bacterial suspension (2 ml) wascentrifuged, and the sediment was carefully suspended in 0.5ml of phosphate-buffered saline (PBS)-0.1% glutaraldehyde-20% sucrose. After 5 min at 37°C, 0.5 ml of PBS-8%glutaraldehyde-28.6% sucrose was added, and the mixturewas kept at 37°C for 1 h. The plasmolyzed bacteria werecentrifuged, and the pellet was used for electron microscopicanalysis.Sample preparations for electron microscopy. The bacteria

were grown in Merck Standard I broth to a cell density ofabout 1 x 108 to 2 x 108 per ml. For embedding, the bacterialsuspensions (2 ml) were incubated with sodium azide (finalconcentrations, 5 mM). For stabilization of extracellularpolysaccharide, SO ,ul of the diluted anticapsular monoclonalantibody was added. After 30 to 60 min at 37°C, the bacteriawere fixed with glutaraldehyde (final concentration, 2%) inKellenberger buffer (17), centrifuged, and transferred tolow-temperature gelling agar (Agar VII; Sigma) at 37°C.After the agar was solidified, it was cut into small cubes.

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EXPRESSION OF THE E. COLI KS CAPSULAR ANTIGEN

Sample embedding in Epon. The agar cubes containing thebacteria were fixed with glutaraldehyde (2% [vol/vol]) for 1 hafter they were fixed with osmium tetroxide (1% [vol/vol]),dehydrated, embedded, and polymerized in Epon 812 (19).Sample embedding in Lowicryl K4M. The progressive

lowering of temperature embedding technique (2, 3) wasused with minor modification. The agar cubes were fixedwith glutaraldehyde (2% [vol/vol]) in Kellenberger buffer for1 h at 1°C and then dehydrated with ethanol (EtOH) asfollows: 30% EtOH (-10°C for 1 h), 50% EtOH (-20°C for1 h), 70% EtOH (-35°C for 1 h), 90% EtOH (-35°C for 1 h),100% EtOH (-35°; twice for 1 h each time). To preventdetachment of the bacteria from the surrounding resin in theultrathin sections, uranyl acetate (0.5% [vol/vol]) was addedat the 70% EtOH step (1, 9).

Infiltration of the samples with increasing concentrationsof Lowicryl K4M resin in 100% EtOH at -35°C was done asfollows: Lowicryl:EtOH (1:3) for 2 h, Lowicryl:EtOH (1:1)overnight, Lowicryl:EtOH (3:1) for 2 h, and Lowicryl for 2h, then for 4 h, and then overnight. The sample waspolymerized in gelatin beem capsules by indirect UV irradi-ation (360 nm) for 12 h at -45°C. During the next 12 h ofpolymerization, the temperature was slowly raised to 4°Cand further hardening was achieved by direct UV irradiationat 4°C for 3 h.Specimen preparations and electron microscopy. Epon-

embedded samples were cut into ultrathin sections (50 to 60nm). The sections were placed on Formvar-coated nickelgrids and stained with uranyl acetate and lead citrate.Ultrathin sections from Lowicryl-embedded samples werelabeled by the immunogold technique as follows. To reduceunspecific background labeling, the grids were floated on asolution of milk powder (1% in PBS) for 30 min and werewashed twice with PBS. They were transferred to a solutionof the anticapsular monoclonal antibody (diluted 1:500 inPBS) and incubated at room temperature for 2 h. After beingwashed five times with PBS, the grids were transferred to asolution of gold (5 or 15 nm) conjugated anti-mouse immu-noglobulin M antibody (diluted 1:100) and incubated for 1 h.After the sections were washed once with PBS and fourtimes with distilled water, they were stained for 7 min withuranyl acetate (2% [wt/wt]), washed, and air dried. Allsamples were examined in a Philips 400 T/ST microscope at80 kV.

RESULTS

Characterization of bacteria. The cloning and characteri-zation of the K5 genes have been described previously (26,27), as have the insertion mutants pGB118::TnJO00-16 andpGB118::TnlO00-24 (strains 21496 and 21500). The formerinsertion was in region 2, and the latter was in region 3 of theK5 genes. pGB118A1 (strain 21487) is a spontaneous deletionof pGB118 which removes about 3 kb of region 1 of the K5genes. The deletion in pGB118A1 gives rise to the samephenotype as other deletions and insertion mutations whichlie in region 1 of the Kl, K7, and K5 genes (7). The extent ofthe deletion was mapped by performing single and doublerestriction enzyme digests of pGB118 and pGB118A1.

Electron microscopic demonstrations of the K5 capsule. Theelectron microscopic demonstration of bacterial capsulescannot be achieved by conventional methods without priorstabilization of these delicate and highly hydrated structures(3a). A previously described KS-specific monoclonal anti-body (5, 25) was used to stabilize the K5 capsule expressedby E. coli 21484, the recombinant expressing the K5 capsule.

j-

FIG. 1. Electron microscopic demonstration of the KS-specificcapsule of wild-type 20026 (010:K5:H4) from which the capsulegenes have been cloned (26). The sample was prepared by ultrathinsectioning of antibody-treated and Epon-embedded cells. Bar, 0.5,Lm.

The K5 capsule could also be demonstrated well in ultrathinsections of bacteria which had been stabilized with themonoclonal antibody and then embedded in Epon (3a, 4, 19).Whereas in wild-type E. coli K5 strains all bacteria werecovered with a capsule which appeared thin (50 to 100 nm)(Fig. 1), in E. coli 21484, only about 25% of the bacteria hada capsule, which was up to 850 nm thick and often envelopedseveral cells (Fig. 2). The reason for the expression pattern

FIG. 2. Electron microscopic demonstration of the KS-specificcapsule of the KS capsule-expressing recombinant E. coli 21484.The samples were prepared by ultrathin sectioning of antibody-treated and Epon-embedded cells. Bar, 0.5 p.m.

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1088 KRONCKE ET AL.

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FIG. 3. Electron micrographs of ultrathin sections from Lowicryl K4M-embedded region 1 mutant E. coli 21487 (A), region 3 mutant E.coli 21500 (B), and region 2 mutant E. coli 21496 (C). The capsular material was labeled by the immunogold technique. Bar, 0.5 ,um; diameterof the gold particles is 15 nm in panel A and 5 nm in panel B.

of the capsule in recombinant E. coli 21484 is unclear. Weare currently analyzing this phenomenon.

Electron microscopic analysis of capsular material inexpression mutants. We had previously found that mutationsin gene regions 1 and 3, which both determine the translo-cation of the capsular polysaccharide to the cell surface,result in acapsular bacteria. However, these mutants are stillable to produce capsular polysaccharide (5a, 6, 26). Toobtain information on the location of the capsular material inthese mutants, we analyzed ultrathin sections of Lowicryl-embedded bacteria by the immunogold method. E. coli 21496(region 2 mutant) showed no label at all, in keeping with amutated polymerization system (Fig. 3C); E. coli 21487(region 1 mutant) was heavily labeled with gold spheres atthe cell membrane (Fig. 3A). E. coli 21500 (region 3 mutant)had little label associated with the membrane, the major partof the label being in the cytoplasm (Fig. 3B). The labelingwas observed with about 25% of the cells in region 1 and 3mutants, which is in keeping with the fact that in E. coli21484, only about 25% of the cells expressed the K5 capsule.In wild-type E. coli KS, no intracellular gold label could bedetected (data not shown).We wanted to know whether the region 1 mutant had the

capsular material at the cytoplasmic membrane and how

intimately it was associated with this membrane. Therefore,we spheroplasted the bacteria with EDTA-lysozyme in 20%sucrose prior to embedding and immunogold labeling ofultrathin sections. Most of the gold spheres were found inthe periplasm, and only little label was seen in associationwith the cytoplasmic membrane (Fig. 4).

Isolation of the K5 polysaccharide from the bacteria. Theextracellular K5 polysaccharide was isolated from the agar-grown clone E. coli 21484 by saline extraction. Polysaccha-rides from the region 1 and 3 mutants were obtained eitherfrom the supernatants of a cell homogenate (French press) orfrom the supernatants of spheroplasts with or without glu-taraldehyde stabilization. Cetavlon precipitation was used inall cases for the precipitation of the polysaccharides (11, 34).The yields of the polysaccharide preparations are shown inTable 2. The data show that the K5 polysaccharide can beobtained from region 1 mutants but not from region 3mutants by spheroplasting after fixation of the spheroplastswith glutaraldehyde. Unfixed spheroplasts of region 3 mu-tants released most of the K5 polysaccharide, probablybecause of their instability under the conditions used. Com-plete recovery of polysaccharide was obtained from cellhomogenates (ultracentrifuged French pressure cell homoge-nates) of both mutants. All polysaccharide preparations

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EXPRESSION OF THE E. COLI KS CAPSULAR ANTIGEN

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FIG. 5. Immunoelectrophoretic patterns of capsular materialfrom the K5-expressing recombinant E. coli 21484 (A), region 1mutant E. coli 21487 (B), and region 3 mutant E. coli 21500 (C). Thesamples were applied to the wells before (1) or after (2) heating at pH4 (100°C for 20 min). Monoclonal anti-K5 antibody was used forprecipitation.

FIG. 4. Electron micrograph of an ultrathin section from region 1mutant E. coli 21487 after the bacteria were spheroplasted andembedded in Lowicryl K4M. The capsular material was labeled bythe immunogold technique. Bar, 0.5 pm.

contained equimolar amounts of glucuronic acid and N-acetylglucosamine, and all reacted with the monoclonalanti-K5 antibody in immunoelectrophoresis. These resultsindicate that these preparations contained the capsular K5polysaccharide.

Analysis of lipid substitution. It was reported previouslythat the capsular K5 polysaccharide is substituted at thereducing end with a phosphatidic acid (29; B. Jann and K.Jann, unpublished results; see also reference 10). This lipidsubstitution may be of importance for polysaccharide syn-thesis and translocation. We therefore analyzed the extra-cellular capsular polysaccharide and polysaccharides ob-tained from region 1 and region 3 mutants for the presence ofcovalently bound lipid. After extraction of contaminatinglipid, the polysaccharide preparations were subjected to mildacid hydrolysis and extracted with chloroform-methanol.The concentrated extract was subjected to thin-layer chro-matography on silica gel. Whereas the extracts from hydro-lyzates of the polysaccharide from E. coli 21484 and E. coli21487 (region 1 mutant) exhibited a spot which had the samechromatographic mobility and staining properties as 1,2-

TABLE 2. Yields of the capsular material obtained by Cetavlonprecipitation from various preparations of region 1 mutant

E. coli 21487 and region 3 mutant E. coli 21500

Capsular material (mg)Cetavlon precipitate from from E. coli"

supernatant of:21487 21500

French press homogenate 2.2 1.2Fixed spheroplasts 1.2 0.05Unfixed spheroplasts 2.2 0.7

a Yields are expressed as milligrams per gram of bacteria (dry weight).

di-palmitoyl-sn-glycerol-3-phosphate, no spot at all could beobserved with the extract from the hydrolyzate of the region3 mutant (results not shown).

Immunoelectrophoresis of the K5 polysaccharide prepara-tions. Because of micelle formation, lipid-substituted capsu-lar polysaccharides have a lower electrophoretic mobilitythan does the unsubstituted form. This difference has beendemonstrated by immunoelectrophoresis (29). We thereforesubjected the various K5 polysaccharide preparations to an

Al1 2 3

B

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FIG. 6. Polyacrylamide gel electrophoresis in 25% gel (A) and in1S% gel (B) of the capsular polysaccharides obtained from thecapsule-expressing recombinant E. coli 21484 (lanes 1), region 1mutant E. coli 21487 (lanes 2), and region 3 mutant E. coli 21500(lane 3). In 15% gels, the preparation used in lane 3 ran out of thegel.

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1090 KRONCKE ET AL. J. BACTERIOL.

immunoelectrophoretic analysis with the monoclonal anti-K5 antibody for detection. A diagram representing theresults is shown in Fig. 5. The polysaccharide from theregion 1 mutant exhibited the same immunoelectrophoreticprecipitation pattern as the polysaccharide from E. coli K-12recombinant expressing the K5 polysaccharide. The pres-ence of the two precipitation arcs is in agreement with theprevious finding (29; Jann and Jann, in press) on wild-type E.coli that only part of a preparation of group II capsularpolysaccharides is lipid substituted. The polysaccharidefrom the region 3 mutant showed only the fast-moving band,which is characteristic of unsubstituted polysaccharides.Mild acid hydrolysis of the polysaccharide preparations (pH4; 100°C for 20 min) removed the lipid, and the productsshowed only the fast-moving arc in immunoelectrophoresis.It is noteworthy that the polysaccharide from the region 3mutant did not alter its electrophoretic mobility upon treat-ment with dilute acid.

Polyacrylamide gel electrophoresis. It was recently re-ported (24) that acidic capsular polysaccharides can beanalyzed by polyacrylamide gel electrophoresis, resulting inladder-like patterns similar to those shown by lipopolysac-charides in sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (14, 23). Such an analysis was performed withthe polysaccharide preparations from the capsule-expressingclone and from the region 1 and 3 mutants. The polysaccha-ride from the region 1 mutant has about the same migrationpattern as the extracellular polysaccharide from the K5expressing clone, and the polysaccharide from the region 3mutant contains molecules which are smaller, as indicatedby the ladder-like pattern (Fig. 6).

DISCUSSION

The capsular polysaccharides (K antigens) of E. coli aredivided, on the basis of chemical, biochemical, and serolog-ical properties, into two groups (13a, 16). Group II com-prises the capsular polysaccharides of extraintestinal E. coli,which are in several respects comparable to those of Neis-seria meningitidis and Haemophilus influenzae. The genes ofseveral representatives of group II capsules, such as Kl, K5,K7, K12, and K92, have been cloned and analyzed (Sa, 7, 26,30, 32, 35). These studies have revealed a gene organizationwhich is common in E. coli expressing the above-mentionedcapsules and probably also in other E. coli with group IIcapsular polysaccharides (Sa, 6, 26 ). The capsule genes areorganized in three adjacent regions to which specific func-tions were ascribed. Central region 2 directs the polymeriza-tion of the respective polysaccharides and was termed thesynthesis region. Its structure and size (7 kb in E. coli K5)differ according to the structure and specificity of the dif-ferent E. coli K antigens. Region 1, which is to one side ofregion 2, was reported to direct the translocation of thepolysaccharide to the cell surface and was termed thetransport region. Whereas this region has been intensivelystudied (Sa, 35), not much is known about region 3, which islocated to the other side of region 2. The data obtained so farindicate that region 3 determines some ill-defined modifica-tion of the polysaccharide, possibly involving lipid substitu-tion (6, 29). It also seems to be involved in translocation ofthe polysaccharide to the cell surface. Region 1 (8 to 9 kb)and region 3 (1.8 to 2 kb) have the same structure andfunction in all E. coli with group II capsular polysaccharideshitherto studied.The studies on the capsular K5 polysaccharide of E. coli

described here were undertaken in an attempt to understand

the process of capsule expression and to correlate thefindings with genetic data. The following interpretation ispresented as a working hypothesis for further investigation.The electron microscopic results on region 3 mutant E.

coli 21500 seem to depict the earliest block in the expressionof the capsular K5 polysaccharide, probably during ordirectly after polymerization. Analysis of ultrathin sectionsby the immunogold technique revealed that the majority ofthe K5 polysaccharides was located in the cytoplasm, andonly very little material was associated with the cytoplasmicmembrane. Since polysaccharide synthesis is known tooccur at the cytoplasmic membrane (15, 16), the findingsuggests that the polymerization takes place at the inner faceof the cytoplasmic membrane. The nature of the mutation inregion 3 is not known, but two possibilities exist. First, themutation actually blocks the substitution of the polysaccha-ride with the phospholipid which may be essential for itstranslocation across the cytoplasmic membrane; as a result,the material accumulates in the cytoplasm. Second, thedirect effect of the mutation is a loss of the ability totranslocate the polysaccharide across the cytoplasmic mem-brane (for example, due to lack of a transporter or energiz-er), and the absence of the phospholipid is a secondaryphenomenon. The finding that the K5 polysaccharide iso-lated from the cytosol of the region 3 mutant has no lipid andthat it is smaller than the extracellular polysaccharide doesnot favor one or the other interpretation.The transport of the K5 polysaccharide from the (outer

face of the) cytoplasmic membrane through the periplasmand the outer membrane is blocked in region 1 mutant E. coli21487. Immunogold electron microscopy showed that in thismutant, the K5 polysaccharide was almost entirely associ-ated with the membrane. After spheroplasting of the cells,the material was found in a periplasmic location, only part ofit being attached to the outer face of the cytoplasmicmembrane. The polysaccharide isolated from supernatantsof glutaraldehyde-fixed spheroplasts of the region 1 mutanthad the same size as the extracellular K5 polysaccharideand, like the latter, it was substituted with a phosphatidicacid. This again shows that the site where phosphatidic acidis attached must be the cytoplasmic membrane. Gene region1 determines five proteins which seem to be necessary forpolysaccharide transport and surface expression (Sa, 32). Itwill be interesting to know whether the mutational loss ofdistinct transport proteins results in distinct types of trans-port blocks.

ACKNOWLEDGMENTThis work was supported by the Deutsche Forschungsgemein-

schaft (DFG grant Ja 115/28).

LITERATURE CITED1. Acetarin, J.-D., E. Carlemaim, and W. Villiger. 1986. Develop-

ments of new LowicryIR resins for embedding biological speci-mens at even lower temperature. J. Microsc. (Paris) 143:81-88.

2. Acker, G., D. Bitter-Suermann, U. Meier-Dieter, H. Peters, andH. Mayer. 1986. Immunocytochemical localization of entero-bacterial common antigen in Escherichia coli and Yersiniaenterocolitica cells. J. Bacteriol. 168:348-356.

3. Armbruster, B. L., E. Carlemalm, R. Chiovetti, R. M. Garavito,J. A. Hobot, E. Kellenberger, and W. Villiger. 1982. Specimenpreparation for electron microscopy using low temperatureembedding resins. J. Microsc. (Paris) 126:77-85.

3a.Bayer, M. E. 1990. Visualization of the bacterial polysaccharidecapsule. Curr. Top. Microbiol. Immunol. 150:129-158.

4. Bayer, M. E., and H. Thurow. 1977. Polysaccharide capsule ofEscherichia coli: microscope study of its size, structure, and

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sites of synthesis. J. Bacteriol. 130:911-936.5. Bitter-Suermann, D., I. Goergen, and M. Frosch. 1986. Mono-

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