JOURNAL OF BACTERIOLOGY, July 1980, p. 328-337 Vol. 143, No. 10021-9193/80/07-0328/10$02.00/0

Cross-Reactivity of Major Outer Membrane Proteins ofEnterobacteriaceae, Studied by Crossed

ImmunoelectrophoresisHARMEN HOFSTRA,* MAARTEN J. D. VAN TOL, AND JACOB DANKERT

Laboratory for Medical Microbiology, State University Groningen, and Department ofHospital Infections,University Hospital A.Z.G., Groningen, Oostersingel 59, The Netherlands

Outer membrane fractions were prepared from 11 bacteria in the familyEnterobacteriaceae: Escherichia coli serotypes 01K-, 04K2, 026K60, 075K-,and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumo-niae, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providenciastuartii. All strains studied were found to contain one non-peptidoglycan-bound,heat-modifiable outer membrane protein, and one or two peptidoglycan-associ-ated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossedimmunoelectrophoresis using sodium dodecyl sulfate-polyacrylamide gel electro-phoresis for separation of the antigens in the first dimension of the procedure wasshown to provide a useful model system for studying the antigenic relationshipsof the major outer membrane proteins in Enterobacteriaceae species. Peptido-glycan-bound major outer membrane proteins of all bacteria studied reacted withantiserum against the purified peptidoglycan-bound matrix protein I of E. coli026K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II* of E. coli 026K60 in both their unmodified and in theirheat-modified forms. These results indicate that the genes coding for the majorouter membrane proteins in the family Enterobacteriaceae have been well enoughconserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.

The cell envelope of Enterobacteriaceae con-tains a complex mosaic of antigens. The 0-anti-genic part of the lipopolysaccharide and thecapsular K antigen are used to classify them intonumerous serotypes, which show only weak an-tigenic cross-reactivity (22). Apart from theseserotype-antigens, a number of common anti-genic surface structures have been described inthe Enterobacteriaceae. The R-core oligosac-charide (18) and the lipid-A part (8) of thelipopolysaccharide, as well as Kunin commonantigen (15), are probably shared by most enter-obacterial strains. The free form lipoprotein ofthe Escherichia coli outer membrane wasshown to be immunochemically cross-reactivewith the corresponding lipoprotein in many en-terobacterial strains (21).

Little attention has been paid to the immu-nology of the 27,000- to 40,000-dalton majorproteins of the enterobacterial outer membrane.These proteins have been very well character-ized in E. coli K-12. Protein Ia and Ib (29) or band c (19) are tightly but not covalently boundto the peptidoglycan layer. The heat-modifiableprotein II* (9) or d (19) is not associated withthe peptidoglycan. The presence of peptidogly-

can-bound and non-peptidoglycan-bound majorouter membrane proteins has been detected invarious Enterobacteriaceae species (20). Apply-ing an interfacial immunoprecipitation test, wewere able to detect antigenic cross-reactivity ofthese proteins in several E. coli serotypes, Sal-monella typhimurium, Klebsiella pneumoniae,and Proteus vulgaris (12). Using the sodiumdodecyl sulfate (SDS)-polyacrylamide gel im-munoperoxidase technique (34) it was possibleto demonstrate the antigenic relationship be-tween the major outer membrane proteins of E.coli and four Proteus species (13), although inCNBr cleavage experiments no similarities werefound between the peptidoglycan-bound pro-teins of E. coli and P. mirabilis (6).Crossed immunoelectrophoresis has been

widely used for the immunochemical analysis ofbacterial envelope structures (23, 28, 31). How-ever, attempts to identify the major outer mem-brane proteins of E. coli by this technique havebeen unsuccessful (32). Using specific antiseraagainst proteins I and II* of E. coli 026K60 ina crossed immunoelectrophoresis technique,modified according to Converse and Papermas-ter (4), we identified the peptidoglycan-bound

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CROSS-REACTIVITY OF OUTER MEMBRANE PROTEINS 329

and both the heat-modified and the unmodifiedform of the non-peptidoglycan-bound majorouter membrane protein of the homologousstrain (35).

In the present study we report the investiga-tion of outer membrane preparations derivedfrom several E. coli serotypes and related En-terobacteriaceae species by crossed immunoe-lectrophoresis. We have found that all strainsexamined by us possess peptidoglycan-boundand non-peptidoglycan-associated major outermembrane proteins which are immunologicallyrelated to the corresponding proteins in E. coli.

MATERIALS AND METHODSBacterial strains and growth conditions. Bac-

terial strains used in this study are described in Table1. Cells were grown in mineral medium supplementedwith yeast extract and glucose (5) on a rotary shaker(New Brunswick Scientific Co., New Brunswick, N.J.)at 200 rpm, 37°C. Overnight cultures were diluted 1:50with fresh medium and grown for 4 h (late exponentialphase of growth). Cells were harvested by centrifuga-tion at 6,000 x g for 10 min at 4°C.

Isolation of outer membrane. Cells of E. colistrains (about 20 g, wet weight) were suspended in 150ml of 50 mM Tris-hydrochloride (pH 7.8) containing1 mM EDTA and sheared in a homogenizer (MSE,London) for 1 min to remove extracellular and flagellarmaterials. Cells were resedimented and subsequentlydisrupted by three passages through an X-press (LKB,Bromma, Sweden) at a working pressure of 100,000 to120,000 lb/in2. The crude envelope fraction was sedi-mented by ultracentrifugation (Heraeus, Christ, Os-terode, Federal Republic of Germany) at 100,000 x g

for 45 min at 4°C. Outer membranes were obtained byTriton X-100 (Sigma Chemical Co., St. Louis, Mo.)extraction of the crude membrane fraction essentiallyby the method of Schnaitman (30) as described indetail for E. coli 026K60 (5).

Outer membranes from the other enterobacterialspecies were isolated by a rapid method developed byB. Witholt and P. Sloots, Laboratory for Biochemistry,State University, Groningen, The Netherlands (un-published procedure).

Briefly, late-exponential-phase cells from a 3-literculture (about 10 g, wet weight) were washed with0.9% (wt/vol) NaCl and suspended in 50 ml 100 mMTris-hydrochloride buffer (pH 8.0) containing 2.5 mMEDTA. Subsequently, cells were subjected to a seriesof short sonic oscillations in an ultrasonic power unit(MSE, London), each sonication (20 to 30 s) beingfollowed by cooling on ice for 2 min. Sonic treatmentwas continued until the optical density (OD) of thesuspension, measured at 450 nm (Beckman Instru-ments, Inc., Fullerton, Calif.) had decreased to ap-proximately 10% of the original OD. Unbroken cells

were removed by centrifugation (6000 x g, 10 min,4°C). The supernatant fraction was centrifuged at30,000 x g for 20 min at 4°C. The sediment was washedtwice with deionized water and lyophilized (Edwards,Crawley, U.K.). The resulting preparations appearedto consist of practically pure outer membrane material

TABLE 1. Bacterial strains used in this studyStrain or species Source/reference Origin

E. coli serotype 01K- DHIG" (12) NHFhE. coli serotype 04K2 DHIG (12) Wound"E. coli serotype 026K60 DHIG (5) NHFE. coli serotype 075K- DHIG (12) NHFE. coli serotype 0111K58 E. HeathdS. flexneri DHIG (this study) FPDeS. typhimurium phage DHIG (12) NHF

type VI 260K. pneumoniae DHIG (12) NHFS. marcescens DHIG (this study) NHFP. vulgaris DHIG (12) NHFP. mirabilis DHIG (13) NHFP. stuartii DHIG (this study) NHF

a Department of Hospital Infections, University Hospital,Groningen, The Netherlands.hNHF, Normal human feces.' Isolated from an infected wound in the oral cavity of a seal

in Pieterburen seal nursery, Pieterburen, The Netherlands.d Generously donated by B. Witholt, Laboratory for Bio-

chemistry, Groningen, The Netherlands.e Feces of a patient with diarrhea.

when analyzed by SDS-polyacrylamide gel electropho-resis (SDS-PAGE). A Triton X-100 extraction of thesefractions according to Schnaitman (30), to removecontaminating cytoplasmic membrane material, didnot significantly change the SDS-PAGE profiles ofthese preparations. Therefore, this extraction step wasomitted.

Isolation of the outer membrane proteins of E.coli 026K60. The non-peptidoglycan-bound, heat-modifiable outer membrane protein, designated II*(9), d (19), or B (26), was extracted from 200 mg ofouter membranes with 50 mM Tris-hydrochloridebuffer (pH 7.8) containing 0.5% (wt/vol) SDS for 1 hat 370C. After ultracentrifugation (100,000 x g, 45 min,15°C), the insoluble material was reextracted twice toremove any protein II* left. The peptidoglycan-boundprotein I (29), Rosenbusch matrix protein (27), wasextracted from the remaining insoluble material with50 mM Tris-hydrochloride (pH 7.8) containing 2%(wt/vol) SDS by heating for 5 min at 1000C, followedby ultracentrifugation. The insoluble material fromthis extraction, mainly consisting of the murein-lipo-protein complex (3), was reextracted under the sameconditions to remove any protein I left, washed twicewith distilled water, and lyophilized. Protein I andprotein II* preparations were purified by repeatedcolumn chromatography on Bio-Gel P-150 (BioRadLaboratories, Richmond, Calif.) as described previ-ously in detail (14).

Isolation of lipopolysaccharide. Lipopolysac-charide of E. coli 026K60 was isolated by the methodof Westphal et al. (37), as previously described (12).

Preparation of OM protein antisera. Rabbitswere immunized with 5 mg of protein I or protein II*of E. coli 026K60 suspended in 0.5 ml of isotonicsaline and the same volume of Freund complete ad-juvant. The proteins were administered intramuscu-larly in the upper hind leg region at days 0 and 28.Blood was collected 3 weeks after the second injection.Serum was obtained by centrifugation of the clottedblood, and stored in 1-ml portions at -70°C. The

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330 HOFSTRA, VAN TOL, AND DANKERT

specificity of the antisera was investigated by theenzyme-linked immunosorbent assay (ELISA) as de-scribed elsewhere in detail (14). The antiserum againstprotein I turned out to contain a considerable numberof antibodies directed against the murein-lipoproteincomplex. Both antisera contained a low titer of anti-lipopolysaccharide. Antibodies directed against lipo-

polysaccharide or murein-lipoprotein were absorbedfrom the sera by stepwise addition of these antigensfrom 20-mg/ml suspensions in phosphate-buffered sa-

line (pH 7.2). Each addition was followed by incuba-tion for 30 min at 37°C and centrifugation (10,000 x g,10 min) to remove immune complexes. The absence ofantibodies against murein-lipoprotein and lipopolysac-charide in the absorbed antisera was confirmed byELISA.ELISA. ELISA was carried out in microtiter trays

(M24AR; Greiner, Niirtingen, Federal Republic ofGermany) coated with protein I, protein II*, lipopoly-saccharide or murein-lipoprotein. The assay was per-formed as described previously in detail (14).SDS-PAGE. SDS-PAGE was performed by using

the discontinuous buffer system, described by Laem-mli (16), on slab gels prepared by the method of Ames(1). The separation gel contained 11% (wt/vol) acryl-amide and 0.44% (wt/vol) bisacrylamide; the stackinggel contained 3% acrylamide and 0.12% bisacrylamide.Both gels contained 0.1% (wt/vol) SDS.To detect both the peptidoglycan-bound and the

non-peptidoglycan-associated proteins, we heatedouter membrane samples at 100°C for 20 min in 62.5mM Tris-hydrochloride buffer (pH 6.7) containing 2%(wt/vol) SDS, 10% (vol/vol) glycerol, and 0.001% (wt/vol) bromophenol blue (sample buffer). Approxi-mately 20 pl of sample, containing 10 to 15 jtg of outermembrane, was applied per gel slot.To investigate the presence of non-peptidoglycan-

bound major outer membrane proteins in the strainsstudied, we dispersed outer membrane preparations toa concentration of 1 mg/ml in 62.5 mM Tris-hydro-chloride (pH 6.7) containing 2% (wt/vol) SDS andincubated them at 37°C for 30 min. The insolublematerial (peptidoglycan and peptidoglycan-boundproteins) was sedimented by centrifugation at 110,000x g for 30 min at 15°C. Possible heat modifiability ofthe non-peptidoglycan-bound proteins was investi-gated by heating one half of the supernatant fractionat 100°C for 10 min, while the other half was kept atroom temperature. Subsequently both samples were

supplemented with glycerol (10% vol/vol) and bro-mophenol blue (0.001% wt/vol). From each sample, 30p1, containing about 5 ,ug of protein, was applied pergel slot. The sedimented material was dispersed insample buffer (ca. 2 mg/ml) and heated at 100°C for20 min. From these samples 20 tl was applied per gel

slot to detect outer membrane proteins that had re-

mained associated with the peptidoglycan layer duringthe extraction at 370C.

Electrophoresis was carried out at 20 mA for 5 to 6h. Staining the slab gels with Coomassie brilliant blue,destaining, and photography, as well as the applicationof standard proteins for molecular weight estimation,were the same as previously described (5).Crossed immunoelectrophoresis. Crossed im-

munoelectrophoresis was performed essentially by the

method of Converse and Papermaster (4), as previ-ously applied for the analysis of the major outer mem-brane proteins of E. coli 026K60 (35). In the firstdimension of this procedure, the outer membrane pro-teins were separated by SDS-PAGE. Outer membranepreparations were dispersed to a concentration of 3mg/ml in 6.25 mM Tris-hydrochloride buffer (pH 6.7)containing 0.2% (wt/vol) SDS, 10%o (vol/vol) glycerol,and 0.01% (wt/vol) bromophenol blue. Subsequentlysamples were heated at 100°C for long enough toobtain about half of the non-peptidoglycan-bound pro-tein in its heat-modified form, while the other halfremained unmodified. Outer membrane preparationsof E. coli serotypes, S. flexneri, and K. pneumoniaewere heated for 1 to 2 min to achieve this purpose.Samples of S. typhimurium, P. vulgaris, P. mirabilis,and P. stuartii were boiled for 20 min, while S. mar-cescens outer membranes had to be boiled for 30 to 40min. From each sample 20 to 30 pl, corresponding to60 to 90 jig of outer membranes, was applied per gelslot. SDS-PAGE was carried out at 20 mA for 7 h, toachieve a proper separation of the major outer mem-brane proteins in the 27,000-to-40,000 molecularweight range. After electrophoresis each polyacryl-amide gel was cut longitudinally into 5-mm wide strips,each containing one electrophoretic outer membraneprotein profile. About 20 strips were obtained fromeach gel. Three of these strips were stained withCoomassie brilliant blue to detect the localization ofthe major outer membrane proteins in the unstainedstrips. For electrophoresis in the second direction, anunstained gel strip, containing the electrophoretic pro-tein profile of one outer membrane preparation, wasincorporated into an antibody containing agarose gelsystem, with an intermediate gel, containing 1.5% (vol/vol) Triton X-100 (Sigma), as described previously indetail (35). The dimensions of the glass plates were 70by 70 by 0.8 mm. The dimensions of the Triton X-100-containing intermediate gel and the antibody-contain-ing gel were 5 by 70 by 1.2 mm and 45 by 70 by 1.2mm, respectively. For all agarose parts of the agarose-polyacrylamide gel system, 1% (wt/vol) agarose(Hoechst, Federal Republic of Germany) in 80 mMTris-hydrochloride, 40mM sodium-acetate, and 1 mMEDTA (pH 7.4) was used. The concentration of theantisera in the antibody-containing agarose gel partswas related to the reciprocal ELISA titer against theirappropriate antigen. Consequently, 6% (vol/vol) ofantiserum against protein I of E. coli 026K60 (titer,20,000 in ELISA) or 1.5 to 2% (vol/vol) of antiserumagainst protein II* of E. coli 026K60 (titer, 80,000 inELISA) or a combination of both was used for theanalysis of outer membrane preparations from E. coliserotypes, S. flexneri, S. typhimurium, K. pneumo-niae, and S. marcescens, whereas 16% (vol/vol) ofanti-I or 4% (vol/vol) of anti-II* was used in crossedimmunoelectrophoresis of P. vulgaris, P. mirabilis,and P. stuartii outer membranes. Electrophoresis inthe second direction was performed at 2 V/cm for 18to 20 h in a water-cooled electrophoresis cell (Shan-don, London, U.K.). After electrophoresis, gels werecovered with several sheets of filter paper and pressed.Subsequently gels were washed with several changesof fresh 0.1 M NaCl and deionized water. Finally, gelswere air dried, stained with 0.25% (wt/vol) Coomassie

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CROSS-REACTIVITY OF OUTER MEMBRANE PROTEINS 331

brilliant blue R 250 in methanol-acetic acid-water (45:10:45 by vol), and destained in the same solvent mix-ture. The resultant pattems were photographed onAgfa Ortho 25 professional document copying film.

RESULTSMajor outer membrane proteins in the

30,000- to 40,000-dalton range. Outer mem-brane preparations of the bacterial strains usedin this study were analyzed by SDS-PAGE afterheating at 1000C for 20 min in SDS-containingsample buffer. The electrophoretic profiles ob-tained after this treatment showed two or threemajor protein bands in the 33,000- to 40,000-dalton range (Fig. 1). When outer membranepreparations were extracted with the samebuffer at 370C for 30 min, followed by centrifu-gation to sediment the peptidoglycan and pep-tidoglycan-associated proteins, SDS-PAGE pro-files of the resultant supernatants showed onlyone major protein band. The apparent molecularweights of these proteins are given in Table 2.Heat treatment ofthe extracted proteins (1000C,10 min) before SDS-PAGE analysis resulted ina significant increase oftheir apparent molecularweights in SDS-PAGE (Table 2). Molecularweight estimations of the outer membrane pro-teins that remained associated with the pepti-doglycan layer during the extraction in SDS-containing buffer at 370C are given in Table 2.Comparison of the data listed in Table 2 with

the SDS-PAGE profiles shown in Fig. 1 revealsthat all strains tested by us possess one non-peptidoglycan-bound, heat-modifiable majorouter membrane protein and one or two pepti-doglycan-associated outer membrane proteins.In E. coli serotypes, S. flexneri, S. typhimurium,K. pneumoniae, and S. marcescens, the highermolecular weight outer membrane proteins inthe 33,000- to 40,000-dalton range turned out tobe peptidoglycan bound while the lower molec-ular weight proteins were heat modifiable andnot peptidoglycan bound. In P. vulgaris, P. mi-rabilis, and P. stuartii, the situation of theseproteins in SDS-PAGE profiles is apparentlyreversed with regard to each other.Crossed immunoelectrophoresis. The re-

sults of crossed immiunoelectrophoresis experi-ments carried out with outer membranes of dif-ferent E. coli strains separated in the first di-mension and antisera against outer membraneprotein I or II* of E. coli 026K60, or a combi-nation of both sera applied in the second dimen-sion, are shown in Fig. 2. When outer membranesof the homologous strain E. coli 026K60 wereseparated in the first dimension, the applicationof anti-protein I in the second direction resultedin the formation of a single immunoprecipitaterepresenting the 36,000-dalton peptidoglycan-

40 K.

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_,,.......30 K.

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A S C D E F G H I J K L

FIG. 1. SDS-PAGE profiles of the major outermembrane proteins in the 30,000- to 40,000-daltonrange. Outer membrane preparations were heated at100°C for 20 min in SDS-containing sample bufferbefore application to the gel slots. (A) E. coli 01K7,(B) E. coli 04K2, (C) E. coli 026K60, (D) E. coli075K7, (E) E. coli O111K58, (F) S. flexneri, (G) S.typhimurium, (H) K. pneumoniae, (I) S. marcescens,(J) P. vulgaris, (K) P. mirabilis, and (L) P. stuartii.

TABLE 2. Apparent molecular weights of the majorouter membrane proteins of the bacterial strainsused in this study and their relationship to the

peptidoglycan layerMol wt

Non-peptidogly-can-bound heat- Major outer

Strain modifiable major membrane pro-outer membrane teins, peptidogly-

proteins can bound at37°C

370C 100°CE. coli 01K- 28" 35 37E. coli 04K2 27 33 36.5, 38E. coli 026K60 27 33 36, (37)bE. coli 075K- 27 33.5 (36.5), 38E. coli OlllK58 27 33 (36), 36.5S. flexneri 26.5 34 37S. typhimurium 28 35 36.5, 38K. pneumoniae 28 34 36.5S. marcescens 28 34.5 (36) 40.5P. vulgaris 29 40 37P. mirabilis 30.5 40 38P. stuartii 31 39 35.5

Molecular weights are given in thousands.Minor proteins are given in parentheses.

bound major outer membrane protein (Fig. 2A).The minor 37,000-dalton protein did not giverise to a separate peak. Application of antiserumagainst protein II* gave a double peak withfusion (Fig. 2B). The left-hand peak correspondsto the heat-modified form of protein II* (33,000daltons), and the right-hand one represents theunmodified form of this protein (27,000 daltons).As outer membranes were boiled shortly (lessthan 2 min) before electrophoresis in the firstdimension, both the heat-modified and the un-modified forms of the non-peptidoglycan-boundprotein II* were present. Application of a com-bination of both antisera resulted in a combinedpattern (Fig. 2C). This pattern shows clearly

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FIG. 2. Crossed immunoelectrophoretic analysis ofouter membrane fractions from several E. coli serotypes.Outer membrane preparations were heated at 100°C for 1 to 2 min in sample buffer containing 0.2% (wt/vol)SDS and were subsequently subjected to SDS-PAGE on slab gels. Unstainedpolyacrylamide gel strips, eachcontaining one electrophoretic outer membrane protein profile, were incorporated into antibody-containingagarose gel systems, prepared as described in the text. (A to C) E. coli 026K60; (D to F) E. coli O1K; (G toI) E. coli 04K2; (J to L) E. coli 075K7; (M to 0) E. coli 0111K58. Antibody gels contained 6% (vol/vol) ofspecific antiserum against the peptidoglycan-bound matrix protein I of E. coli 026K60 (left row, A to M), 1.5to 2.0% (vol/vol) of specific antiserum against the non-peptidoglycan-bound protein II* of E. coli 026K60 (Bto N), or a combination of both antisera (C to 0). The anode was to the right in the first dimension of theprocedure (SDS-PAGE) and on top in second-dimension electrophoresis. Coomassie brilliant blue-stainedreference strips, corresponding to the ones used for second-dimension electrophoresis, were photographedbelow each pattern to show the positions of the outer membrane protein antigens after first-dimensionelectrophoresis.

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CROSS-REACTIVITY OF OUTER MEMBRANE PROTEINS 333

that proteins I and II* reacted as nonidenticalantigens.When outer membranes of heterologous E.

coli strains were used, comparable patterns wereobtained in crossed immunoelectrophoresis (Fig.2D to 0). In all strains the higher molecularweight peptidoglycan-bound proteins (36,000 to39,000 daltons) reacted with antiserum againstprotein I of E. coli 026K60 (Fig. 2D, G, J, andM). In the pattern obtained with outer mem-branes of serotype 04K2, both peptidoglycan-bound proteins reacted with anti-protein I (Fig.2G). Minor proteins, present in the outer mem-branes of E. coli 075K- and 0111K58 (Table 2),did not give rise to separate immunoprecipitates(Fig. 2J and M). In all strains both the unmodi-fied and the heat-modified form of the non-pep-tidoglycan-bound protein reacted with antise-rum against protein II* of E. coli 026K60 (Fig.2E, H, K, and N). This was also the case with E.coli 01K-, the non-peptidoglycan-bound proteinof which shows a higher molecular weight, bothin its unmodified and in its heat-modified form(Fig. 2E). When a combination of antiseraagainst proteins I and II* of E. coli 026K60 wasused in the second dimension, a combined pat-tern was obtained with outer membrane prepa-rations of all heterologous E. coli strains, com-parable to the one obtained with the homologousE. coli 026K60 (Fig. 2F, I, L, and 0).The results of crossed immunoelectrophoretic

analysis of outer membrane preparations of S.flexneri, S. typhimurium, K. pneumoniae, andS. marcescens (Fig. 3) exhibited a great similar-ity to those obtained with outer membranes ofE. coli serotypes. All peptidoglycan-bound pro-teins reacted with the antiserum against proteinI of E. coli (Fig. 3A, D, G, and J). This was alsothe case with S. typhimurium, which possessestwo peptidoglycan-associated major outer mem-brane proteins (Fig. 3D). All heat-modifiable,non-peptidoglycan-bound proteins reacted withanti-protein II* of E. coli 026K60 in both theirunmodified and their heat-modified form (Fig.3B, E, H, and K). The use of a combination ofantisera against proteins I and II* of E. coli026K60 resulted in combined patterns, compa-rable to those obtained with E. coli serotypes(Fig. 3C, F, I, and L).The application of outer membrane prepara-

tions of P. vulgaris, P. mirabilis, and P. stuartiiin crossed immunoelectrophoresis experimentsresulted in the patterns shown in Fig. 4. Theouter membrane proteins of these species re-acted considerably less strongly with antiseraagainst proteins I and II* of E. coli 026K60 thandid the corresponding proteins in outer mem-brane preparations of the other strains tested.The concentrations of the antisera in the second

dimension of crossed immunoelectrophoresis ofProteus and Providencia outer membranes hadto be raised substantially to obtain visible peaksin the antibody-containing gel parts. However,in spite of increased antiserum concentrations,the immunoprecipitates obtained remained veryfaint and ill-formed in comparison with thoseobtained after application of outer membranepreparations of the other bacteria studied. In allthree species the peptidoglycan-bound majorprotein reacted with anti-protein I of E. coli026K60 (Fig. 4A, D, and G), whereas both theheat-modified and the unmodified form of thenon-peptidoglycan-bound protein reacted withanti-protein II* (Fig. 4B, E, and H). The appar-ent molecular weights of the heat-modifiedforms of the non-peptidoglycan-bound proteinsof these strains are higher than the molecularweights of their peptidoglycan-bound proteins(c.f. Table 2). This implies that the peak of thepeptidoglycan-bound protein is situated be-tween the two peaks of both forms of the non-peptidoglycan-bound protein in crossed immu-noelectrophoretic profiles (Fig. 4C, F, and I).

DISCUSSIONInvestigations reported here indicate that the

application ofSDS-PAGE in the first dimensionof crossed immunoelectrophoresis provides auseful model system for the immunological anal-ysis of gram-negative outer membrane proteins.It facilitated the characterization of the majorouter membrane proteins of several Enterobac-teriaceae species as common envelope antigens.Previous studies using crossed immunoelectro-phoresis resulted in a detailed immunochemicalanalysis of membrane material derived fromMicrococcus lysodeikticus (23), Neisseria gon-orrhoeae (28, 31), and N. meningitidis (11). Re-cently, crossed immunoelectrophoresis was usedfor the resolution of cytoplasmic and outer mem-branes of Escherichia coli (32). However, onlyBraun's lipoprotein (3) and lipopolysaccharidewere detected as major antigens of the E. coliouter membrane in their system. Attempts toidentify the major proteins in the 33,000- to40,000-dalton range, e.g., the peptidoglycan-bound matrix protein, were unsuccessful, despitetheir abundance in the outer membrane. Severalpossible explanations can be given for the in-ability of Smyth et al. (32) to detect the matrixprotein in crossed immunoelectrophoresis pat-terns. First, the anti-envelope serum used bythem probably contained antibodies against thematrix protein I in its native configuration, sit-uated as a trimer in the outer membrane struc-ture (24). We found that such antibodies reactonly weakly with denatured protein I, isolatedby the method of Rosenbusch (27) by boiling in

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334 HOFSTRA, VAN TOL, AND DANKERT

A

DC

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J KFIG. 3. Crossed immunoelectrophoretic analysis of outer membrane preparations derived from (A to C) S.

flexneri, (D to F) S. typhimurium, (G to I) K. pneumoniae, and (J to L) Serratia marcescens. Outer membranefractions of S. flexneri and K. pneumoniae were heated at 100°C for 1 to 2 min in sample buffer containing0.2% (wt/vol) SDS, whereas outer membranes of S. typhimurium and S. marcescens were boiled in the samebuffer for 20 and 40 min, respectively. First- and second-dimension electrophoreses were performed asdescribed in the test and in the legend to Fig. 1. Antibody gels contained 6% (vol/vol) of antiserum againstprotein I of E. coli 026K60 (A to J), 1.5 to 2% (vol/vol) of anti-protein II* of E. coli 026K60 (B to K), or acombination of both antisera (C to L). Coomassie brilliant blue-stained polyacrylamide reference strips werephotographed below each pattern, to show the positions of the major outer membrane proteins after first-dimension electrophoresis. The anode is to the right and on top in each pattern.

SDS (H. Hofstra and J. Dankert, unpublisheddata). Second, the use of agarose gels not con-taining SDS for separation of the antigens in thefirst dimension may have prevented the matrixprotein from moving like a discrete antigen dur-ing this part of their procedure. In a recent studywe reported the use of SDS-PAGE in the firstdimension of crossed immunoelectrophoresis(35), essentially by the method of Converse and

Papermaster (4). This technique, combined withthe application of specific outer membrane pro-tein antisera in the second dimension of theprocedure, gave rise to clearly visible immuno-precipitates accounting for the peptidoglycan-bound matrix protein I and both the heat-mod-ified and the unmodified form of the non-pepti-doglycan-bound major outer membrane proteinII*. The main benefits of the application ofSDS-

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FIG. 4. Crossed immunoelectrophoretic characterization of antigenic cross-reactivity of the major outermembrane proteins in P. vulgaris, P. mirabilis, and Providencia stuartii with the corresponding proteins ofE. coli 026K60. Outer membrane preparations of (A to C) P. vulgaris, (D to F) P. mirabilis, and (G to I) P.stuartii were heated at 100°C for 20 min in sample buffer containing 0.2% (wt/vol) SDS before electrophoresisin the first dimension (SDS-PAGE). Second-dimension electrophoresis was performed as described in the textand in the legend to Fig. 1. Antibody gels contained 16% (vol/vol) of antiserum against protein I of E. coli026K60 (A, D, and G), 4% (vol/vol) of antiserum against protein II* of E. coli 026K60 (B, E, and H), or acombination of both sera (C, F, and I). Coomassie brilliant blue-stained polyacrylamide reference strips,photographed below each pattern, show the position of the major outer membrane proteins after first-dimension electrophoresis. The anode is to right and top in each pattern.

PAGE in the first dimension of crossed immu-noelectrophoresis are the high resolution at-tained by this method and the possibility ofstaining reference strips to detect the exact lo-calization of the antigens separated. A disadvan-tage of our method is the necessity to solubilizethe membrane material by boiling in SDS-con-taining buffer before electrophoresis in the firstdimension. This implies that antigens are ana-lyzed in a denatured form, which may seriouslyimpair the formation of immunoprecipitates inthe antibody-containing agarose gel when anti-sera which have been elicited against the anti-gens in their native configuration are applied.

Results presented in this study indicate thatthe antigenic cross-reactivity of the major outermembrane proteins is a widespread phenome-non in the family Enterobacteriaceae. However,crossed immunoelectrophoresis as applied inthis study is not a suitable method for the quan-tification of the degree of immunological rela-tionship between corresponding proteins in dif-ferent enterobacterial strains, as it is not possible

to standardize all factors ultimately contributingto the area and shape of the precipitin arcsdeveloped in the antibody-containing gel.Notwithstanding this restriction it was possibleto divide the strains tested into three groups,pertaining to the antigenic cross-reactivity oftheir major outer membrane proteins with thecorresponding proteins of E. coli 026K60. Onegroup, containing the E. coli serotypes, washighly cross-reactive with E. coli 026K60. Thereactions of their outer membrane proteins withthe antisera against proteins I and II* of E. coli026K60 reflected those of the outer membraneproteins of the homologous strain itself. Themajor outer membrane proteins of S. flexneri, S.typhimurium, K. pneumoniae, and S. marces-cens reacted slightly less strongly with antiseraagainst proteins I and II* of E. coli 026K60. Weused the same concentrations of specific antiserain the second dimension agarose gels for thesespecies as we had applied for E. coli serotypes.However, immunoprecipitation peaks obtainedwith outer membrane preparations of these four

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336 HOFSTRA, VAN TOL, AND DANKERT

species were always fainter and mostly lowerthan those formed with outer membraixe pro-teins of E. coli serotypes. The third group, com-posed of Proteus species and P. stuartii, showedmuch weaker antigenic cross-reactivity of theirmajor outer membrane proteins with those of E.coli 026K60. In this group 4% (vol/vol) of anti-protein II* and 16% (vol/vol) of antiserumagainst protein I had to be used to obtain visibleprecipitin arcs.

In E. coli 04K2 and S. typhimurium bothpeptidoglycan-bound major proteins reactedwith the antiserum against protein I of E. coli026K60. This indicates that these strains pos-sess more than one immunologically identicalpeptidoglycan-bound major outer membraneprotein. However, it should be noted that thepeptidoglycan-bound protein of E. coli 026K60is also composed of more than one polypeptidespecies. We do not know how far the subcom-ponents of the peptidoglycan-bound protein inour strains correspond to peptidoglycan-associ-ated proteins of E. coli K-12, designated b andc (19), Ia and Ib (29), or la and lb (2). Besidesthese, other peptidoglycan-bound proteins havebeen described in E. coli K-12, e.g., protein 2(25), e (33), E (7), and Ic (10). There is no reasonto expect that the situation in the outer mem-brane of our clinically isolated strains of E. coliwill be less complicated. Proteins b and c of E.coli K-12 were shown to differ considerably withrespect to the electrophoretic mobility of frag-ments obtained in cyanogen bromide or proteo-lytic cleavage experiments (17, 36). We cannotexclude the possibility that both components ofprotein I of E. coli 026K60 acted as differentimmunogens, after administration to rabbits. Inthat case it would be plausible that in E. coli04K2 and S. typhimurium the two peaks formedwith anti-protein I were induced by differentantibody populations present in this antiserum.

In conclusion, crossed immunoelectrophoreticanalysis of enterobacterial outer membranepreparations, using the SDS-PAGE for separa-tion of the antigens in the first dimension of theprocedure, potentiated the definition of the ma-jor outer membrane proteins as common surfaceantigens in Enterobacteriaceae. This indicatesthat the antigenic structure of these proteins hasbeen very well conserved during evolution. Theirpotential as cross-protective antigens in experi-mental infections with heterologous Enterobac-teriaceae is presently under investigation.

ACKNOWLEDGMENTS

We are grateful to B. Witholt for instructing us in hismethod for rapid isolation of gram-negative outer membranesand for his donation of E. coli 0111K58. We thank R. W.Rozeboom for his skillful technical assistance and M. TenKate for typing the manuscript.

LlTERATURE CITED1. Ames, G. F.-L. 1974. Resolution of bacterial proteins by

polyacrylamide gel electrophoresis on slabs. Membrane,soluble, and periplasmic fractions. J. Biol. Chem. 249:634-644.

2. Bassford, P. J., D. L Diedrich, C. A. Schnaitman,and P. Reeves. 1977. Outer membrane proteins ofEscherichia coli. VI. Protein alteration in bacterio-phage-resistant mutants. J. Bacteriol. 131:608-622.

3. Braun, V., and K. Rehn. 1969. Chemical characteriza-tion, spatial distribution and function of a lipoprotein(murein-lipoprotein) of the E. coli cell wall. Eur. J.Biochem. 10:426-438.

4. Converse, C. A., and D. S. Papermaster. 1975. Mem-brane protein analysis by two-dimensional immunoelec-trophoresis. Science 189:469-472.

5. Dankert, J., and H. Hofstra. 1978. Antibodies againstouter membrane proteins in rabbit antisera preparedagainst Escherichia coli 026K60. J. Gen. Microbiol.104:311-320.

6. Datta, D. B., C. Kramer, and U. Henning. 1976. Dipo-loidy for a structural gene specifying a major protein ofthe outer cell envelope membrane from Escherichiacoli K-12. J. Bacteriol. 128:834-841.

7. Foulds, J., and T.-J. Chai. 1978. New major outer mem-brane protein found in an Escherichia coli tolF mutantresistant to bacteriophage TuIb. J. Bacteriol. 133:1478-1483.

8. Galanos, C., 0. Luderitz, and 0. Westphal. 1971. Prep-aration and properties of antisera against the lipid-Acomponent of bacterial lipopolysaccharides. Eur. J. Bio-chem. 24:116-122.

9. Garten, W., I. Hindennach, and U. Henning. 1975.The major proteins of the Escherichia coli outer cellenvelope membrane: Characterization of proteins II'and III, comparison of all proteins. Eur. J. Biochem. 59:215-221.

10. Henning, U., W. Schmidmayr, and I. Hindennach.1977. Major proteins of the outer cell envelope mem-brane of Escherichia coli K12: multiple species of pro-tein I. Mol. Gen. Genet. 154:293-298.

11. Hoff, G. E., and C. E. Frasch. 1979. Outer membraneantigens of Neisseria meningitidis group B serotype 2studied by crossed immunoelectrophoresis. Infect. Im-mun. 25:849-856.

12. Hofstra, H., and J. Dankert. 1979. Antigenic cross-reac-tivity of major outer membrane proteins in Enterobac-teriaceae species. J. Gen. Microbiol. 111:293-302.

13. Hofstra, H., and J. Dankert. 1980. Antigenic cross-reac-tivity of outer membrane proteins ofE. coli and Proteusspecies. FEMS Microbiol. Lett. 7:171-174.

14. Hofstra, H., and J. Dankert. 1980. Preparation andquantitative determination of antibodies against majorouter membrane proteins of Escherichia coli 026K60.J. Gen. Microbiol. 117:437-447.

15. Kunin, C. M., M. V. Beard, and N. E. Halmagyi. 1962.Evidence for a common hapten associated with endo-toxin fractions of Escherichia coli and other Entero-bacteriaceae. Proc. Soc. Exp. Biol. Med. 111:160-166.

16. Laemmli, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

17. Lee, D. R., C. A. Schnaitman, and A. P. Pugsley. 1979.Chemical heterogeneity of major outer membrane poreproteins of Escherichia coli. J. Bacteriol. 138:861-870.

18. Luderitz, O., A. M. Staub, and 0. Westphal. 1966.Immunochemistry of 0 and R antigens of Salmonellaand related Enterobacteriaceae. Bacteriol. Rev. 30:192-255.

19. Lugtenberg, B., J. Meyers, R. Peters, P. van derHoek, and L. van Alphen. 1975. Electrophoretic res-olution of the 'major outer membrane protein' of Esch-erichia coli K12 into four bands. FEBS Lett. 58:254-258.

J. BACTERIOL.

on May 8, 2020 by guest

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nloaded from

CROSS-REACTIVITY OF OUTER MEMBRANE PROTEINS 337

20. Lugtenberg, B., H. Bronstein, N. van Selm, and R.Peters. 1977. Peptidoglycan-associated outer mem-brane proteins in gram-negative bacteria. Biochim. Bio-phys. Acta 465:571-578.

21. Nakamura, K., R. M. Pirtle, and M. Inouye. 1979.Homology of the gene coding for outer membrane li-poprotein within various gram-negative bacteria. J.Bacteriol. 137:595-604.

22. Orskov, I., F. 0rskov, B. Jann, and K. Jann. 1977.Serology chemistry and genetics of 0 and K antigens ofEscherichia coli. Bacteriol. Rev. 41:667-710.

23. Owen, P., and M. J. R. Salton. 1975. Antigenic andenzymatic architecture of Micrococcus lysodeikticusmembranes established by crossed immunoelectropho-resis. Proc. Natd. Acad. Sci. U.S.A. 72:3711-3715.

24. Palva, E. T., and L L. Randall. 1978. Arrangement ofprotein I in Escherichia coli outer membrane: cross-linking study. J. Bacteriol. 133:279-286.

25. Pugsley, A. P., and C. A. Schnaitman. 1978. Outermembrane proteins of Escherichia coli. VI. Evidencethat bacteriophage-directed protein 2 functions as apore. J. Bacteriol. 133:1181-1189.

26. Reithmeier, R. A. F., and P. D. Bragg. 1974. Purifica-tion and characterization of a heat-modifiable proteinfrom the outer membrane of Escherichia coli. FEBSLett. 41:195-198.

27. Rosenbusch, J. P. 1974. Characterization of the majorenvelope protein from Escherichia coli: regular ar-rangement on the peptidoglycan and unusual dodecylsulfate binding. J. Biol. Chem. 249:8019-8029.

28. Salton, M. R. J., and C. Urban. 1978. Selective extrac-tion of gonococcal envelope antigens with thiocynates.FEMS Microbiol. Lett. 4:303-306.

29. Schmitges, C. J., and U. Henning. 1976. The major

proteins of the Escherichia coli outer cell-envelopemembrane. Heterogeneity of protein I. Eur. J. Biochem.63:47-52.

30. Schnaitman, C. A. 1971. Solubilization of the cytoplas-mic membrane of Escherichia coli by Triton X-100. J.Bacteriol. 108:545-552.

31. Smyth, C. J., A. E. Friedman-Kien, and M. R. J.Salton. 1976. Antigenic analysis of Neisseria gonor-rhoeae by crossed immunoelectrophoresis. Infect. Im-mun. 13:1273-1288.

32. Smyth, C. J., J. Siegel, M. R. J. Salton, and P. Owen.1978. Immunochemical analysis of inner and outermembranes ofEscherichia coli by crossed immunoelec-trophoresis. J. Bacteriol. 133:306-319.

33. van Alphen, W., N. van Selm, and B. Lugtenberg.1978. Pores in the outer membrane of Escherichia coliK12. Involvement of proteins b and e in the functioningof pores for nucleotides. Mol. Gen. Genet. 159:75-83.

34. van Raamsdonk, W., C. W. Pool, and C. Heyting.1977. Detection of antigens and antibodies by an im-muno-perozidase method applied on thin longitudinalsections of SDS-polyacrylamide gels. J. Immunol.Methods 17:337-348.

35. van Tol, M. J. D., H. Hofstra, and J. Dankert. 1979.Major outer membrane proteins of Escherichia colianalysed by crossed immuno-electrophoresis. FEMSMicrobiol. Lett. 5:349-352.

36. Verhoef, C., B. Lugtenberg, R. van Boxtel, P. deGraaff, and H. Verheij. 1979. Genetics and biochem-istry of the peptidoglycan-associated proteins b and cof Escherichia coli K12. Mol. Gen. Genet. 169:137-146.

37. Westphal, O., 0. Luderitz, and F. Bister. 1952. Uberdie Extraktion von Bakterien mit Phenol/Wasser. Z.Naturforsch. 7b:148-155.

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