Vol. 169, No. 12JOURNAL OF BACTERIOLOGY, Dec. 1987, p. 5546-55550021-9193/87/125546-10$02.00/0Copyright © 1987, American Society for Microbiology

Major Membrane Surface Proteins of Mycoplasma hyopneumoniaeSelectively Modified by Covalently Bound Lipid

KIM S. WISE* AND MARY F. KIMDepartment of Microbiology, School of Medicine, University of Missouri-Columbia, Columbia, Missouri 65212

Received 10 April 1987/Accepted 13 August 1987

Surface protein antigens of Mycoplasma hyopneumoniae were identified by direct antibody-surface bindingor by radioimmunoprecipitation of surface '25I-labeled proteins with a series of monoclonal antibodies (MAbs).Surface proteins p70, p65, p50, and p44 were shown to be integral membrane components by selectivepartitioning into the hydrophobic phase during Triton X-114 (TX-114)-phase fractionation, whereas p41 wasconcomitantly identified as a surface protein exclusively partitioning into the aqueous phase. Radioimmuno-precipitation of TX-114-phase proteins from cells labeled with [35S]methionine, "4C-amino acids, or [3H]palmitic acid showed that proteins p65, p50, and p44 were abundant and (with one other hydrophobic protein,p60) were selectively labeled with lipid. Covalent lipid attachment was established by high-performance liquidchromatography identffication of [3H]methyl palmitate after acid methanolysis of delipidated proteins. Anadditional, unidentified methanolysis product suggested conversion of palmitate to another form of lipid alsoattached to these proteins. Alkaline hydroxylamine treatment of labeled proteins indicated linkage of lipids byamide or stable 0-linked ester bonds. Proteins p65, p50, and p44 were highly immunogenic in the natural hostas measured by immunoblots of TX-114-phase proteins with antisera from swine inoculated with wholeorganisms. These proteins were antigenically and structurally unrelated, since hyperimmune mouse antibodiesto individual gel-purified proteins were monospecific and gave distinct proteolytic epitope maps. Intraspeciessize variants of one surface antigen of M. hyopneumoniae were revealed by a MAb to p70 (defined in strain J,ATCC 25934), which recognized a larger p73 component on strain VPP11 (ATCC 25617). In addition, MAbto internal, aqueous-phase protein p82 of strain J failed to bind an analogous antigen in strain VPP11. Thesestudies establish that a highly restricted set of distinct, lipid-modified hydrophobic membrane proteins aremajor surface antigens of M. hyopneumoniae and that structural variants of surface antigens occur within thisspecies.

The class Mollicutes represents a divergent and rapidlyevolving group of small procaryotes taxonomically unifiedby their common lack of a cell wall and the presence of asingle limiting membrane (10, 20, 36). This surface is clearlycritical to the interaction of these organisms with theirenvironment; in particular, membrane-associated proteinsare likely to mediate a variety of transport functions andmust contribute to the surface topology of Mollicutes, dic-tating their interactions with a variety of hosts, both asparasites of host cell surfaces and as potential targets of theimmune response.

Recently, a novel group of membrane proteins covalentlymodified by lipids has been demonstrated in four species ofMollicutes: Mycoplasma capricolum (5, 6), Mycoplasmahyorhinis (3), Acholeplasma laidlawii (7, 17), and Spiro-plasma citri (37a). The abundance and variety of lipid-modified proteins are indeed remarkable in these organismscompared with the relatively limited number found in othereubacteria (22, 23). Although surface antigenic variation inM. hyorhinis has been shown to involve lipid-modifiedmembrane proteins (3), the general significance of theseproteins, their exact orientation in the membrane, and theirfunctions have yet to be determined. Despite uncertainty asto whether these proteins will be universally found in abun-dance throughout the Mollicutes, their presence in orga-nisms with such limited genomic capacity in itself empha-sizes their probable importance.

* Corresponding author.

In recent studies of Mycoplasma hyopneumoniae, a majorpulmonary pathogen of swine (21, 32), we used a panel ofmonoclonal antibodies (MAbs) to analyze the surface anti-genic structure and variation expressed within this species ofMollicutes. We report here (i) the identification of majorintrinsic surface membrane proteins covalently modified bylipids, (ii) the limited presence of only four such proteins inthis species, (iii) the immunogenic properties and antigenicuniqueness of these proteins, and (iv) the nature and com-plexity of covalent lipid attachment. In addition, two MAbswere used to determine antigenic variation among typestrains of this species, one of which involves expression ofalternative forms of an integral membrane surface protein.Finally, an abundant, unmodified surface protein partition-ing into the aqueous phase during Triton X-114 (TX-114)fractionation was identified, which may be extrinsic to themembrane of the organism.

MATERIALS AND METHODS

Mycoplasma strains and culture conditions. The sources ofmycoplasma strains M. hyorhinis GDL, M. hyopneumoniae(suipneumoniae) J ATCC 25934, M. fermentans PG-18M713-002-084), M. pulmonis Ash M717-002-084, and M.bovis ATCC 25523 have been previously described (27, 28).M. hyopneumoniae VPP11 ATCC 25617 and M. flocculareATCC 27399 were obtained from the American Type CultureCollection, Rockville, Md.Mycoplasma stocks were obtained as filter-cloned cultures

(30) or were purified as described previously (27). All myco-plasma species were grown in broth culture at 37°C in

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TABLE 1. Properties of MAbs to M. hyopneumoniaea

Clone Protein Epitope ondesignation recognized surface"

F187C55A IG2a(K) p82F187C77A IG2a(K) p70F177C21A IG1(K) p66F188C42A IG1(K) p65 +F187C19A IG1(K) p50 +F187C84A IG2a(K) p44 +F187C70A IG1(K) p41 +

a MAbs have been characterized elsewhere (32a) except those to p70 andp82, which are fully described in the current report.

b Presence (+) or absence (-) determined by indirect immunofluorescentstaining of immobilized, intact mycoplasmas (32a).

spinner flasks (Bellco Glass, Inc., Vineland, N.J.). A modi-fied Hayflick broth medium containing 20% fetal bovineserum (35) was used to grow all species except M.hyopneumoniae and M. flocculare, which were grown in FFmedium (9) containing 20% porcine serum. Mycoplasmaswere harvested from logarithmic-phase broth cultures bycentrifugation and stored at -70°C in phosphate-bufferedsaline (PBS) as described previously (27).MAbs. Construction and characteristics of MAbs used in

this study have been described elsewhere (32a), with theexception of MAb to the p70 protein of M. hyopneumoniaeJ (clone F187C77A), which was derived from the same initiallibrary of hybridomas. The properties of MAbs are alsooutlined in Table 1. Indirect immunofluorescence assays forthe binding of MAbs to the surfaces of immobilized intactorganisms are also described elsewhere in detail (32a).

Radiolabeling of mycoplasmas. To metabolically label my-coplasmas, 20 ml of a logarithmic-phase broth culture wasremoved and centrifuged for 15 min at 10,000 x g. The pelletwas suspended in 2.0 ml of medium containing the appropri-ate label (see below) and incubated in static culture for 18 hat 370C. After the labeling period, cells were harvested bycentrifugation. The medium was removed, and cells werewashed once with ice-cold PBS and prepared for detergent-phase fractionation.For biosynthetic labeling with palmitic acid, a toluene

solution containing [9, 10-3H(N)]palmitic acid (specific ac-tivity, 30 Ci/mmol; New England Nuclear Corp., Boston,Mass.) was dried under nitrogen and redissolved in ethanol.Then, 50 RI containing 1 mCi of [3H]palmitic acid was addedto 2.0 ml of mycoplasma growth medium. The final ethanolconcentration in the labeling medium did not exceed 2.5%(vol/vol).For biosynthetic labeling with methionine, methionine-

free medium was prepared by dialyzing complete myco-plasma medium against several changes of methionine-freeRPMI 1640 (Hazelton Dutchland, Inc., Denver, Pa.). Dia-lyzed medium was supplemented with 100 ,uCi of L-[35S]methionine (specific activity, 1,127 Ci/mmol; New En-gland Nuclear) per ml and used for labeling.For biosynthetic labeling with amino acids, mycoplasma

growth medium was supplemented to 20 pCi/ml with a14C-amino acid mixture (55 mCi/matom carbon; New En-gland Nuclear).

Vectorial surface labeling with 1251 was carried out withintact, metrizamide-gradient-purified mycoplasmas (33).Surface iodination was performed by modification of theprocedure of Markwell (16). A 0.5-ml sample of metri-zamide-suspended mycoplasma was incubated for 1 h at 4°Cwith 500 PLCi of Na1251 (17mCi/mg, New England Nuclear)

and two lodobeads (an immobilized form of the catalystN-chlorobenzenesulfonamide; Pierce Chemical Co.,Rockford, Ill.). lodination was terminated by the removal ofthe lodobeads, and 0.5 ml of PBS was added to dilute themetrizamide. lodinated mycoplasmas were immediately har-vested by centrifugation at 12,000 x g for 2 min, washedtwice with PBS, and finally suspended in Tris-saline (10 mMTris, 150 mM NaCl, plI 7.4) before electrophoresis orTX-114 (Sigma Chemical Co., St. Louis, Mo.)-phase parti-tioning. Mycoplasmas incubated with Na125I in the absenceof lodobeads were not labeled. Organisms disrupted withdetergent (1% sodium dodecyl sulfate (SDS) or 1% TritonX-100) before iodination exhibited many more iodinatedpolypeptides than did mycoplasmas which had not beendetergent treated.TX-114-phase partitioning. Mycoplasmas from logarith-

mic-phase cultures were subjected to TX-114-phase parti-tioning as originally described by Bordier (2) and lateradapted to mycoplasmas (19). Briefly, mycoplasmas weresuspended in Tris-saline containing 1% (wt/vol) TX-114 and1 mM phenylmethylsulfonyl fluoride and were incubated at4°C for 30 min. This preparation was centrifuged at 49C for 3min at 10,000 x g to remove insoluble material. The super-natant was transferred to a new tube, incubated at 37°C for5 min to induce condensation of TX-114, and was thencentrifuged at 22°C for 5 min at 10,000 x g. The resultingheavy, detergent-enriched fraction (TX-114 phase) and alighter, detergent-depleted fraction (aqueous phase) wereplaced in separate tubes, readjusted to a concentration of 1%(wt/vol) TX-114, and repartitioned as described above. Afterthree cycles of phase fractionation, the condensed TX-114phase was adjusted to 1% (wt/vol) and used in parallel withthe aqueous phase for subsequent electrophoresis and im-munoblot analysis.

Electrophoresis and immunoblot techniques. SDS-poly-acrylamide gel electrophoresis (SDS-PAGE) Was performedby the method of Laemmli (14) with linear gradient gels aspreviously described (35). Samples were prepared by heatingat 100°C for 5 min in sample buffer (2% SDS-5% 2-mercaptoethanol-10% [vol/vol] glycerol-62.5 mM Tris, pH6.8). After electrophoresis, the gels were stained with anaqueous solution containing .025% (wt/vol) Coomassie blue(Bio-Rad Laboratories, Richmond, Calif.), 50% (vol/vol)methanol, and 10% (vol/vol) acetic acid. Gels were destainedin the same solution without Coomassie blue. Fluorographywas performed by a variation of the technique of Bonner andLaskey (1). Stained gels were treated after SDS-PAGE withglacial acetic acid for 1 h and then with 10% (wt/vol)2,5-diphenyloxazole (PPO) in glacial acetic acid for 1 h. Gelswere placed in water to precipitate the PPO, and afterextensive rinsing to remove acetic acid, were treated with40% (vol/vol) methanol and dried. Gels were then exposed topreflashed X-Omat-AR film (Eastman Kodak Co., Roches-ter, N.Y.) for 3 to 6 weeks at -70°C. Gels containing [355]methionine-labeled proteins were stained, dried, and autora-diographed by direct exposure to film. Molecular weightswere calculated from known standard markers (expressed inkilodaltons) including phosphorylase, 92; bovine serum albu-min, 60; ovalbumin, 45; carbonic anhydrase, 30; andcytochrome c, 12.5.

Nitrocellulose filter blots were prepared by the procedureof Towbin et al. (29) with slight modifications previouslyreported (35). Blots were incubated with hybridoma culturefluid containing MAb or with serum diluted in PBS contain-ing 10% fetal calf serum and then incubated with peroxidase-conjugated antibody against mouse or swine immunoglobu-

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lin G (IgG) (Cooper Biomedical, Inc., Malvern, Pa.); thebound antibody was visualized with the enzyme substrateO-dianisidine. Relative molecular weights were calculatedfrom blots of standards run simultaneously and stained withamido black after electrophoretic transfer (29).

Radioimmunoprecipitation analysis. Radioimmunoprecipi-tation analysis of mycoplasma proteins involved metaboli-cally labeled organisms subjected to TX-114-phase fraction-ation as described above. To prepare immobilized MAbs forprecipitation, hybridoma culture supernatants were incu-bated for 18 h with slow tumbling at 4°C with SepharoseCL-4B beads covalently coupled to affinity-purified goatanti-mouse IgG heavy and light chains (Affibeads, CooperBiomedical) or to protein A (Pharmacia, Uppsala, Sweden).Beads with bound MAbs were washed 4 to 6 times at 4°Cwith Tris-saline and once with Tris-saline containing 0.05%TX-114 and were then added (20-pAl packed volume perprecipitation) to TX-114 detergent fractions containing la-beled proteins, which had been adjusted to a final concen-tration of 0.05% TX-114 in Tris-saline. After incubation for18 h at 40C, beads were rinsed 4 to 6 times with 0.05%TX-114 in Tris-saline and heated at 100°C in SDS-PAGEsample buffer lacking 2-mercaptoethanol to dissociate MAbsand antigens without releasing heavy and light chains fromimmunoglobulin covalently coupled to Affibeads. After theremnoval of beads by centrifugation, the supernatant wastransferred to a new tube, and 2-mercaptoethanol was addedto a final concentration of 5% (vol/vol). Samples were thenheated at 100°C for 1 min and subjected to SDS-PAGE.Autoradiography or fluorography was performed as de-scribed above.Treatment of mycoplasma proteins with trypsin. Aliquots

of a TX-114 fraction from M. hyopneumoniae were supple-mented with graded amounts of acetylated trypsin (Sigma).Samples were incubated for 60 min at 37°C, SDS-PAGEsample buffer was immediately added, and samples wereheated under reducing conditions and subjected to SDS-PAGE as described above. Filter blots of digested productswere prepared and immunologically stained as describedabove.Removal of noncovalently bound lipid from hydrophobic

proteins. To remove noncovalently bound lipid from hydro-phobic proteins isolated by detergent-phase fractionation,0.5 ml of a TX-114-phase-containing [3H]palmitate-labeledM. hyopneumoniae proteins was first precipitated by adding4.5 ml of methanol (high-performance liquid chromatogra-phy [HPLC] grade; Fisher Scientific Co., Pittsburgh, Pa.)and then incubating the preparation for 18 h at -70°C. Aftercentrifugation (10,000 x g for 10 min at 4°C), the supernatantwas removed and the pellet was dried under nitrogen,suspended in 50 ,ul of0.1% SDS (ultrapure grade; BoehringerMannheim Biochemicals, Indianapolis, Ind.), and trans-ferred to a 1.0-ml conical glass Reacti-vial (Pierce). Thesolution was again precipitated with cold methanol andcentrifuged at 1,500 x g for 10 min at 4°C. After removal ofthe supernatant, the pellet was extracted by four additions(each of 0.5 ml) of chloroform-methanol (2:1, vol/vol), whichwere agitated for 5 min and centrifuged at 1,500 x g for 10min after each extraction. A sample (20 p.l) of each sequen-tial extract was assayed for radiolabel by liquid scintillationcounting. Excess solvent was removed after the final extrac-tion under dry nitrogen, and delipidated proteins weresubjected to methanolysis or in some cases prepared insample buffer for SDS-PAGE analysis.

Analysis of fatty acid hydrolysis and methanolysis products.Acid methanolysis of [3H]palmitate-labeled TX-114-phase

proteins of M. hyopneumoniae was performed as describedby Olson et al. (18). Briefly, 0.4 ml of 83% methanolcontaining 2 N HCl was added to delipidated proteins(described above), and the vials were evacuated and incu-bated at 95°C for 16 h. Samples were then extracted fourtimes with 0.3 ml of HPLC-grade petroleum ether (Fisher).The combined petroleum ether extracts received 2 p.l ofmethanol containing 40 p.g of each of the following fatty acidmethyl esters: caprylic (C8:0), capric (C100), lauric (C120),myristic (C140), palmitic (C160), and stearic (C18:0) (Sigma);the extracts also received 4 .1l of methanol containing 40 pLgof palmitic acid. After addition of these internal standards,samples were dried under nitrogen, dissolved in 250 .1l ofHPLC-grade methanol, and applied to isocratic reversed-phase HPLC with a ,uBondapak C18 column (3.9 mm by 30cm) (Waters Associates, Inc., Milford, Mass.), with 80%acetonitrile as the mobile phase at a flow rate of 1.0 ml/min.Fractions were collected at 0.5-min intervals and were addedto vials containing 5 ml of 3a70 scintillation cocktail (Re-search Products, International Corp., Mt. Prospect, Ill.).Radioactivity (3H) was measured in a scintillation counter(Model LS7000; Beckman Instruments, Inc., Fullerton,Calif.) 3H methanolysis products were identified by compar-ison of their profiles with the elution profile (optical densityat 214 nm) of the methyl ester and free fatty acid internalstandards (Sigma).Hydroxylamine treatment of proteins after SDS-PAGE

was performed by the method of Simonis and Cullen (24).Gels were stained with Coomassie blue, destained, andrinsed with water to remove acetic acid. They were theneither left untreated or incubated for 18 h at room tempera-ture with freshly prepared 1.0 M hydroxylamine (Sigma) atpH 10.0 and subsequently processed for fluorography asdescribed above.

Antisera to M. hyopneumoniae proteins. Antisera raised towhole, disrupted M. hyopneumoniae organisms by inocula-tion of cesarean-derived, colostrum-deprived swine (8) werea gift from M. J. Freeman and C. H. Armstrong, PurdueUniversity, Lafayette, Ind. Preimmune and hyperimmunesera obtained from two animals at 29 weeks postinoculationwere used in this study.Murine hyperimmune antisera to gel-purified proteins iso-

lated from M. hyopneumoniae were raised in BALB/c mice(National Institutes of Health contract colony, Charles RiverBreeding Laboratories, Wilmington, Mass.). To obtain pu-rified proteins, approximately 4 mg of whole organisms wereTX-114-phase fractionated, and the hydrophobic TX-114-phase proteins were methanol precipitated and applied to a31-mm channel of an SDS-PAGE slab gel. Protein bandswere negatively stained for 10 min in 4 M sodium acetate at20°C (12), excised from the gel, rinsed in water, and stored at-70°C until elution. Gel pieces were thawed, rinsed brieflyin water, and eluted with an electroelution unit (InternationalBiotechnologies, Inc., New Haven, Conn.). Gel fragmentswere placed in the apparatus and eluted at 100 V for 2 h inSDS-PAGE electrode buffer (192 mM glycine, 25 mM Tris,0.1% SDS, pH 8.3). Proteins were trapped after anodicmigration in a 0.5-ml salt cushion of 7.5 M ammoniumacetate. Isolated protein was collected and monitored forpurity by silver staining (37) after SDS-PAGE. An elutedsuspension containing purified protein was prepared as a 1:1(vol/vol) emulsion with incomplete Freund adjuvant (DifcoLaboratories, Detroit, Mich.). Two-month-old mice werepre-bled and then injected intraperitoneally on days 0, 12,and 42 with 1 to 5 p.g of protein per inoculation. Sera frommice immunized with p65, p50, and p44 were used for

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A B C 0 E F G1 2 1 2 1 2 1 2 1 2 X X

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FIG. 1. Immunostaining of TX-114-phase-fractionated M. hyo-pneumoniae J proteins with MAbs. Mycoplasmas were phase frac-tionated, and the TX-114-phase (lanes 1) and aqueous-phase (lanes2) proteins were applied in paired channels for SDS-PAGE andimmunoblotted with various MAbs as described in Materials andMethods. Each lane represents fractionated proteins derived fromapproximately 50 p.g of mycoplasmas. MAbs included anti-p82 (A),anti-p70 (B), anti-p66 (C), anti-p65 (D), anti-p50 (E), anti-p44 (F),and anti-p41 (G).

immunoblot analyses at dilutions ranging from 1:500 to1:5,000.

RESULTS

Characterization of integral membrane surface proteins bydetergent-phase partitioning. Murine MAbs generated byimmunization with whole, broth-grown M. hyopneumoniae Jhave been classified by their ability or inability to bind to thesurfaces of intact immobilized mycoplasmas and by the sizeof the cognate protein recognized in immunoblot analysis(32a). Table 1 summarizes the binding properties of sevenMAbs and lists the corresponding antigens bound. Thecommon designation (e.g., anti-p50) will be used in referenceto these MAb reagents.

Analysis of TX-114-phase-fractionated mycoplasmas bySDS-PAGE, followed by immunoblotting with individualMAbs, was used to determine partitioning properties of eachantigen recognized (Fig. 1). Selective partitioning of eachantigen exclusively into the TX-114 (hydrophobic) or aque-ous (hydrophilic) phase demonstrated the high efficiency offractionation by this procedure. Antigens segregating intothe TX-114 phase showed the presence of hydrophobicdomains capable of interacting with detergent micelles andwere classified as integral membrane proteins. These arereferred to for simplicity as hydrophobic proteins, to reflectthis partitioning property. Four antigens partitioned into thehydrophobic phase: p70, p65, p50, and p44. Since MAbs top65, p50, and p44 bound surface epitopes of M. hyopneu-moniae (Table 1), these three proteins were determineddirectly to be integral membrane surface protein antigens(32a). The hydrophobic protein p70 was also shown to be asurface antigen by additional criteria (see below). In con-trast, proteins p82, p66, and p41 partitioned into the aqueoushydrophilic phase, thereby failing by this procedure todemonstrate domains accessible to hydrophobic interactionswith TX-114. However, the recognition of a surface epitopeby anti-p41 (Table 1) also established the location of thisantigen at the mycoplasma periphery (32a). Immuno-fluorescent staining of intact organisms with this MAbshowed an intense signal, possibly suggesting its abundanceat the surface despite relatively weak staining of im-munoblots (Fig. 1). Proteins p82 and p66 were provisionallyclassified as internal cytoplasmic constituents, since theycould not be recognized as surface antigens by MAb binding

(Table 1). Treatment of samples with trypsin before SDS-PAGE or trypsin treatment of blots after transfer as previ-ously described (19, 35) abrogated antibody binding. Thisindicated that all epitopes recognized by this MAb serieswere associated with proteins.Although MAb binding was observed in the presence of

excess medium components and the MAbs had been initiallyscreened for inability to bind serum constituents (32a), directdemonstration of MAb binding to metabolically labeledintegral membrane proteins was confirmed by radioimmuno-precipitation. All MAbs to hydrophobic antigens (p70, p65,p50, and p44) selectively precipitated corresponding proteinsfrom the TX-114 phase of [35S]methionine-labeled organisms(Fig. 2), thereby establishing that (i) all of these antigenswere in fact metabolically labeled mycoplasma gene prod-ucts bearing distinct epitopes, (ii) they did not physicallyinteract under the mild detergent conditions used for immu-noprecipitation, and (iii) they were not involved in disulfidecross-linking to other components.

Radioimmunoprecipitation was also used to show that thehydrophobic p70 protein was localized at the mycoplasmasurface. TX-114-phase fractionation of intact mycoplasmasvectorially surface labeled with 125I revealed selective label-ing of TX-114-phase proteins p44, p60, p65, p68, p70, andlarger components of 90 to 95 kilodaltons (kDa) (data not

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FIG. 2. Radioimmunoprecipitation of [35S]methionine-labeledhydrophobic proteins of M. hyopneumoniae J with MAbs. TX-114-phase proteins (lane 1) were prepared by phase fractionation of[35S]methionine-labeled mycoplasmas as described in Materials andMethods. Immunoprecipitates obtained with MAbs anti-p70 (lane2), anti-p65 (lane 3), anti-p50 (lane 4), or anti-p44 (lane 5) wereprepared and analyzed by SDS-PAGE and autoradiography asdescribed in Materials and Methods. Arrows indicate positions ofselected proteins in the TX-114 phase used for immunoprecipitation.

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A2 3 4

B2 3 4

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FIG. 3. TX-114-phase fractionation of M. hyopneumoniae J pro-teins metabolically labeled with "'C-amino acids and [3H]palmitate.Mycoplasmas were labeled with 14C-amino acids (A) or [3H]palmit-ate (B), as described in Materials and Methods, and phase fraction-ated with TX-114. Samples of whole organisms (lanes 1), TX-114-phase proteins (lanes 2), aqueous-phase proteins (lanes 3), orTX-114-insoluble material (lanes 4) were subjected to SDS-PAGE,and gels were fluorographed as described in Materials and Methods.Each channel represents proteins derived from 50 ,ug of rpyco-plasma. Arrows indicate the positions of TX-114-phase proteinslabeled with [3H]palmitate. The inset represents TX-114-phase pro-teins from [31H]palmitate-labeled mycoplasmas (lane 1) immunopre-cipitated as in Fig. 2 with MAbs to p65 (lane 2), p5O (lane 3), or p44(lane 4).

shown) but not other metabolically labeled components ofthis phase (Fig. 2). Anti-p70 specifically precipitated 125I-surface-labeled p70 from the TX-114 phase (data not shown),indicating that whereas the epitope recognized by this MAbwas not expressed at the mycoplasma surface (as measuredby indirect immunofluorescence [Table 1]), the protein wasaccessible to surface iodination. A contrasting situation wasfound for protein p50; although anti-p50 directly recognizeda surface epitope, p50 was not significantly labeled with 125Iby this method.

Covalent lipid modification of integral membrane surfaceprotein'avitigens. Recent reports of lipid-associated proteinsin a number of mycoplasmas and other Mollicutes (5-7, 17,37a) and observations from our own laboratory demonstrat-ing numerous hydrophobic lipid-associated proteins in M.hyorhinis (3) prompted the investigation of possible lipidassociation with specific membrane proteins of M. hyo-pneumoniae. The overall TX-114-phase fractionation patternof "'C-amino-acid-labeled M. hyopneumoniae proteins isdepicted in Fig. 3A. Approximately 15 proteins efficientlypartitioned into the TX-114 phase (see also Fig. 2), whereasmost mycoplasma proteins were found exclusively in theaqueous hydrophilic phase. In contrast to the complexity ofthis pattern, TX-114-phase partitioning of mycoplasmas met-

abolically labeled with [3H]palmitate revealed that only fourproteins of the organism were associated with lipid (Fig. 3B).

Moreover, all four partitioned exclusively into the hydro-phobic TX-114 phase and aligned with '4C-labeled proteinsp65, p60, p50, and p44. No other proteins were labeled withpalmitate over this extended period (18 h), indicating thatlabel was not dispersed into general metabolic pools and thatassociation of label with these four protein's was highlyspecific. Radioimmunoprecipitation of 3H-labeled proteinsfrom TX-114-phase preparations with the use of MAbs top65, p50, and p44 directly established that each of thesesurface proteins was associated with lipid (Fig. 3, inset).The nature of lipid association was further investigated by

attempting to remove 3H label by extraction with organicsplvents. Methanol 'precipitation of TX-114-phase proteinsfollowed by five cycles of extr,action with chloroform-methanol (2:1, vol/vol) failed to remove bound 3H label asmonitored by fluorography after SDS-PAGE, nor did it affectthe antigenicity of hydrophobic proteins in immunoblotanalysis (data not shown). The stability of label in samplessubjected to SDS-PAGE and the resistance of label toextraction by organic solvent suggested possible covalentlipid modification of these proteins.

Confirmation of covalent lipid attachment was obtained byanalysis of products derived from acid methanolysis of[3H]palmitate-labeled TX-114-phase proteins (18). After pre-cipitation and extraction to remove noncovalently boundlipids, proteins were treated by acid methanolysis, whichtypically released over 80% of the nonextractable label.Reversed-phase HPLC analysis of released products re-vealed a major peak coelpting (41.3 min) with an internalstandard of methyl palmitate (Fig. 4B). Surprisingly, asecond major peak (representing a nearly identical amount oflabel) was eluted later (52 min). This component did notcorrespond to any of the standards used, including free fattyacid, as well as methyl ester forms of C8.0, C10:0, C12:0, C14:0,C16:0, C18:0, or oleic acids. Two additional m,inor peaks wereobserved, but their presence varied among individual exper-iments: one corresponded to methyl myristate (23 min), andthe other was eluted at a position (30 min) not correspondingto any standard used. No other peaks were eluted within 120min, and the peaks observed represented 67% recovery oflabel applied to the column. These results indicated that amajor portion of label was present as covalently boundpalmitic acid, consistent with findings reported for othermycoplasma species (3, 5-7, 17, 37a). However, the pres-ence of an additional major product was unexpected andcontrasted markedly with the single methyl palmitate prod-uct previously obtained by the same procedure to analyzelipid-modified proteins of M. hyorhinis (3). This raised thepossibility that palmitic acid might be converted to otherlipid intermediates in M. hyopneumoniae before or aftercovalent linkage to protein.Although covalent lipid modification was limited to four

proteins in this organism, it was possible that each proteinmight contain only one of the two major lipid speciesidentified. To determine whether an individual protein mightgenerate the same or more restricted pattern of methanolysisproducts, the p44 protein was excised from an SDS-PAGEgel loaded with methanol-precipitated and extracted TX-114-phase proteins from [3H]palmitate-labeled cells. Acidmethanolysis of this gel-isolated protein resulted in an HPLCelution pattern (Fig. 4C) very similar to that derived from thecombined TX-114-phase proteins (Fig. 4B), including boththe prominent methyl palmitate and the unidentified productat 52 min, which occurred in the same ratio as that found inanalysis of total TX-114-phase proteins. The lack of methylmyristate and the component at 30 min among the products

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LIPID-MODIFIED SURFACE ANTIGENS OF M. HYOPNEUMONIAE

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10 20 30 40 50 60

Time (minlFIG. 4. HPLC analysis of acid methanolysis products derived

from [3H]palmitate-labeled hydrophobic proteins of M. hyopneu-moniae J. TX-114-phase proteins prepared from [3H]palmitate-labeled mycoplasmas were precipitated with methanol and exten-sively extracted with chloroform-methanol as described in Materialsand Methods. Extracted proteins were subjected to acidmethanolysis, and the resulting ether-extractable, 3H-labeled prod-ucts released were analyzed by reversed-phase HPLC as describedin Materials and Methods. (A) Chromatogram of standards indicat-ing the elution position of selected fatty acid methyl esters includingpalmitate (mC16:0), myristate (mC14:0), laurate (mC12:0), caprate(mC1o:o), or free palmitic acid (C16:0). (B and C) Profiles of 3H-labeled products derived from TX-114 proteins (B) or from isolatedprotein p44 (C), purified by SDS-PAGE as described in Materialsand Methods. Internal standards were included in 3H-labeled prep-arations but are not shown in panels B or C.

generated from p44 (Fig. 4C) was not considered significantin light of their minor and variable presence in analyses oftotal proteins. These findings indicate that at least one of thefour lipid-modified proteins contains both lipid species. The

other three modified proteins may individually contain theselipids in similar ratios, or (perhaps less likely) they may beselectively modified yet generate the same overall ratio ofthese lipids when analyzed together.The nature of covalent lipid modification was further

investigated by attempting to remove lipid by hydrolysiswith alkaline hydroxylamine. This procedure has beenwidely used to differentiate more labile thioester- and 0-ester-linked fatty acids from more stable amide bonds (18,24). Hydroxylamine treatment of [3H]palmitate-labeled,SDS-PAGE-separated TX-114-phase proteins had no effecton the labeling pattern or fluorographic intensity of any ofthe four modified proteins (data not shown), indicating thatthe labeled lipids associated with these proteins were boundby amide or highly resistant O-ester bonds.Immunogenicity and antigenic uniqueness of lipid-modified

membrane surface proteins of M. hyopneumoniae. A set ofhydrophobic, lipid-modified integral surface membrane pro-teins of M. hyopneumoniae was defined by the experimentsdiscussed above by using murine MAbs recognizing discreteepitope structures. Because surface antigens are importantas potential targets of humoral responses in the natural swinehost, it was of interest to determine whether these proteinswere immunogenic in swine (particularly as measured inresponse to immunization with whole-organism prepara-tions). In addition, since antiserum from swine immunizedwith M. hyopneumoniae has been shown to react withcomponents of other mycoplasma species found in swine (8)and because surface antigens bearing common modificationscould in general contribute to the well-documented serologiccross-reactivity among swine mycoplasmas (8, 32), an at-tempt was made to identify additional epitopes sharedamong the lipid-modified surface proteins of M. hyopneu-moniae by using polyclonal antibodies (PAbs) to specific,gel-purified proteins.

Titration of swine preimmune serum and antiserum to M.hyopneumoniae on immunoblots of SDS-PAGE-separatedTX-114-phase proteins revealed (data not shown) that p70,p65, p50, and p44 were all selectively recognized by hyper-immune serum, even at high serum dilution (1:1,000 to1:20,000). Swine immunized with whole organisms thereforeproduced antibodies reacting with epitopes present on thesespecific surface membrane antigens.To assess possible antigenic relatedness among lipid-

modified hydrophobic membrane proteins p65, p50, and p44,the individual proteins were eluted from preparative SDS-PAGE of TX-114-phase proteins and used to generate hy-perimmune PAbs in mice. The resulting antisera were testedon immunoblots of TX-114-phase proteins to determinewhether antibodies raised to one antigen could recognizeepitopes on other proteins in this preparation. The hyperim-mune sera were highly specific (Fig. 5); despite the sensitiv-ity of this assay system, they bound only the correspondingimmunizing protein and showed no reaction with otherproteins, including all of those modified with lipids. Epitopemapping studies (Fig. SB) were performed to further analyzepossible structural relationships among these antigens. Im-munostaining of blots representing a series of graded trypsindigestion reactions of TX-114-phase proteins showed thatPAbs to p65, pS0, and p44 each stained distinct sets ofepitope-bearing tryptic peptides. MAbs to these respectiveproteins stained only a few of the peptides recognized by thecorresponding PAbs (data not shown), confirming that PAbsrecognized additional epitopes on these proteins distinctfrom those detected by MAbs. Collectively, these datastrongly suggest that lipid-modified proteins p65, p50, and

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p44 are antigenically and structurally unrelated, yet are allimmunogenic in swine.

Evidence for species specificity and intraspecies structuralvariation among hydrophobic surface membrane protein an-tigens of M. hyopneumoniae. Antigenic cross-reactivityamong species of swine mycoplasmas has been widelyobserved (e.g., see references 8 and 32) and has raisedpractical problems in the use of serologic techniques toclearly distinguish these organisms. Similar problems arisefrom reported antigenic variation within mycoplasma spe-cies (11, 19, 25, 34). Determination of individual antigens or

A

4

B

1 2 3 4 5 6

32

5()

44

A

1 2 3 4 5 6 7 a

p650

FIG. 6. Immunoblotting of mycoplasma species with MAbs toM. hyopneumoniae J. Whole organisms representing a panel of sixmycoplasma species were subjected to SDS-PAGE and im-munoblotted with combinations of MAbs to either TX-114-phaseproteins p70, p65, p5O, and p44 (A) or aqueous-phase proteins p82,p66, and p41 (B). Species included M. hyopneumoniae J (lane 1), M.flocculare (lane 2), M. hyorhinis (lane 3), M. pulmonis (lane 4), M.fermentans (lane 5), and M. bovis (lane 6). Approximately 50 ,ug ofmycoplasma was added to each well.

p65 p50

1 2 3 4 5 1 2 3 4 5

FIG. 5. Immunoblot and epitope mappingbic lipid-modified M. hyopneumoniae J prolmonospecific polyclonal antisera. TX-114-phajected to SDS-PAGE and immunoblotted as dto Fig. 1. (A) Replicate filter blots stained witiantibodies: a combination of the four MAbsp44 (lane 1); preimmune (lane 2) or hyperimmto purified p65; preimmune (lane 4) or h3antibody to purified p5O; preimmune (lane 6) (7) antibody to purified p44; and a combinatiorantibodies to these three proteins (lane 8). Aiand used as described in Materials and Mcreactions representing TX-114 proteins subjecting (from lanes 1 to 5) trypsin concentrationimmunoblotted with hyperimmune mouse aproteins p65 (left panel), p50 (middle panel), (

p44 epitopes having properties of species specificity, relatednessamong species, and variation within a species is therefore of

1 2 3 4 5 general importance for improving serologic classification.More broadly, however, identification of these properties isalso essential in defining the structural basis of variabilityamong mycoplasma antigens that might affect the nature ofthe mycoplasma-host interaction. Distribution of epitopesdefined by MAbs to M. hyopneumoniae was therefore inves-tigated with a panel of mycoplasma species and strains.Immunoblot analysis of SDS-PAGE-separated proteins

was first performed by using combinations of MAbs recog-nizing TX-114-phase proteins (p70, p65, p5O, and p44) oraqueous-phase proteins (p82, p66, and p41) to stain a seriesof blots of whole organisms, representing M. hyopneu-moniae J, M. flocculare, M. hyorhinis, M. pulmonis, M.fermentans, and M. bovis. All four MAbs to hydrophobicmembrane proteins (Fig. 6A) were highly specific for M.hyopneumoniae. In contrast, epitopes recognized by thethree MAbs to aqueous-phase proteins of M. hyopneu-moniae showed marked differences in their distribution (Fig.6B). Anti-p82 reacted exclusively with M. hyopneumoniae,

analysis of hydropho- anti-p41 (binding a surface epitope of M. hyopneumoniae)teins with the use of reacted with a slightly smaller protein from only one otherse proteins were sub- species (M. bovis), and anti-p66 specifically recognizediescribed in the legend proteins of similar but distinct sizes (64 to 68 kDa) in allh the following mouse species except M. bovis. Staining with individual MAbsto p7O, p65, p5O, and confirmed this interpretation (data not shown).iune (lane 3) antibody This same group of MAbs also defined two types ofyperimmune (lane 5) antigenic differences within the species M. hyopneumoniae.)r hyperimmune (lane When strains J and VPP11 were compared by immunoblot

ntof the hyperimmune analysis (Fig. 7), all MAbs were shown to react with both

nthodse (B) Sepres of organisms except anti-p82, which failed to identify anyted to fivefold increas- epitope in strain VPP11 (Fig. 7A). Although the exact naturee

(0 to 1 mg/ml) and and location of the p82 protein are not established, itntibodies to purified represents a highly specific and identifiable antigenic differ-or p44 (right panel). ence between these two strains of M. hyopneumoniae.

p50 no

p44 51

B

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1 2 3

-wm*i

.:::.- 90

_NMONW-4s__ 6S

FIG. 7. Intraspecies variation of M. hyopneumoniae antigensrecognized by MAbs. (A) Whole mycoplasmas of M. hyopneu-moniae strains J (left lanes) and VPP11 (right lanes) subjected toSDS-PAGE in paired channels and immunoblotted. Pairs were

stained with MAbs to p82 (lane 1), p66 (lane 2), p65 (lane 3), p50(lane 4), p44 (lane 5), or p41 (lane 6). (B) Immunoblot of M.hyopneumoniae strain J (lane 1), strain VPP11 (lane 3), or a

combination of both strains (lane 2) prepared as for panel A aboveand stained with MAb to p70. The position of p70 in strain J ismarked. (C) TX-114-phase proteins prepared from [35S]methionine-labeled M. hyopneumoniae strains J and VPP11 and separated bySDS-PAGE as described in the legend to Fig. 2. Autoradiographshows the pattern obtained with strain J (lane 1), strain VPP11 (lane2), or a combination of proteins from both strains (lane 3). Numbersindicate the position of selected hydrophobic proteins (see also Fig.2 and 3). The two components bracketed in lane 3 correspond to theproteins recognized by anti-p70 MAb.

A particularly interesting, additional form of variationamong strains was detected with MAb to the hydrophobicsurface protein p70 (Fig. 7B). Immunoblotting with this MAbidentified antigens differing in size between the two strains,both forms of which partitioned into the TX-114 phase. Thisdifference was also confirmed by comparing autoradiographsof TX-114-phase proteins from organisms labeled with[35S]methionine. Strain VPP11 expressed an alternative pro-tein slightly larger than p7O found in strain J (Fig. 7C). Theseresults established that two ATCC type strains of M. hyo-pneumoniae expressed different forms of an integral mem-

brane surface protein. The nature of this difference and the

structural relationship of these proteins have not yet beenestablished. Mixed preparations of labeled TX-114-phaseproteins also revealed slight differences in doublet forms ofp65 between these strains (Fig. 7C).

DISCUSSION

These studies provide direct evidence that major proteinsexposed at the surface of M. hyopneumoniae are covalentlymodified by lipid. Recently, we have also demonstrated fattyacylated proteins on the surface of another member of thegenus Mycoplasma, M. hyorhinis (3; M. J. Boyer and K. S.Wise, manuscript in preparation). These observations sup-port the concept that many of the fatty acylated membraneproteins now demonstrated to be prevalent in other speciesof Mollicutes (5-7, 17, 37a) may be located at the externalface of the single limiting membrane of these procaryotes.The function of these modified proteins has yet to bedetermined, although it is clear from the current study andfrom others (3, 19, 37a) that they can represent importantantigenic structures mediating immune responses affectinggrowth and survival of the organism.Two features of protein fatty acylation in M. hyopneu-

moniae distinguish this organism from other Mollicutesstudied to date. First, the highly restricted labeling of onlyfour proteins with palmitate contrasts with the numerous(typically more than 20) proteins similarly labeled in otherspecies (5-7, 17, 37a), including M. hyorhinis (3) grown inmedium and conditions identical to those used in the currentstudy. Second, the apparent conversion of palmitate toanother form of lipid covalently linked to proteins has notbeen reported for other Mollicutes and represents a markeddifference from one other genomically unrelated myco-plasma (M. hyorhinis [26a]) studied under identical condi-tions, in which only labeled palmitic acid could be demon-strated on fatty acylated proteins of palmitate-labeledorganisms (3; M. J. Boyer and K. S. Wise, manuscript inpreparation). Neither the nature of lipid linkage nor theoverall lipid composition of modified proteins has been fullyestablished for M. hyopneumoniae or other Mollicutes.Evidence reported by Dahl and co-workers (5, 6) suggestingO-ester linkage of oleate to proteins of M. capricolum, andlabeling of these proteins by [2-3H]glycerol are consistentwith one established model of bacterial lipoprotein structureand biogenesis (for a review, see reference 38). Whetherlipid modification of M. hyopneumoniae will resemble thismodel or entail additional modifications such as those pro-posed by Dahl et al. (6) remains to be determined. Interest-ingly, all four M. hyopneumoniae proteins (p65, p60, p50,and p44) labeled with [3H]palmitate can also be labeled with[3H]oleate, although the nature of this linkage has not beendetermined (K. Wise and M. Kim, unpublished observa-tion). Given the extreme evolutionary divergence repre-sented within the Mollicutes (20, 36), the presence of multi-ple mechanisms of protein fatty acylation may not besurprising. Analysis of the Mollicutes may indeed reveal anumber of procaryotic lipoprotein modification systems asvaried as those currently emerging from studies of eucary-otes (4, 15, 18, 22, 23).

Detergent-phase fractionation of M. hyopneumoniae re-vealed fewer intrinsic membrane proteins partitioning intothe TX-114 phase than were previously demonstrated in asimilar analysis of M. hyorhinis (19). This provided both anexcellent means of initially purifying membrane proteins ofM. hyopneumoniae and, because of the efficiency of parti-tioning, an unambiguous property useful in defining specific

A

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surface proteins. In contrast to partitioning of the intrinsicmembrane proteins into the TX-114 phase by virtue ofhydrophobic domains accessible to detergent micelle inter-action, exclusive partitioning of the surface protein p41 intothe aqueous phase could reflect either the absence of majorhydrophobic domains or masking of these regions by stronghydrophobic protein-protein interactions. Although the na-ture of the interaction between p41 and the membrane is notfully established, its possible extrinsic properties and itsprominence at the cell surface (32a) prompt speculation thatthis protein may be part of the extracellular "coat" structureexpressed on cultured M. hyopneumoniae cells and impli-cated in mediating interactions between the organism andhost cells in vivo (26, 31).The host response to M. hyopneumoniae membrane pro-

teins measured in this study by immunoblots of TX-114-phase proteins showed that p70, p65, p50, and p44 are majorimmunogenic surface structures recognized in their nativeconfiguration by antibodies of swine immunized with wholeorganisms (8). Furthermore, PAbs to gel-purified p65, p50,or p44 were monospecific and identified quite distinctive setsof tryptic fragments from each protein. Identification ofthese antigenically distinct and immunogenic proteins maytherefore provide a set of defined antigenic targets to moni-tor specific responses to M. hyopneumoniae infection, with-out the interference of antibodies to other components ofimmunologically related microbial flora, including mycoplas-mas of swine (8).

Construction of antibodies to individual surface proteinsof M. hyopneumoniae has also provided a means to identifygenes encoding specific surface antigens of this organism.PAb to p65 has recently been used to isolate a clonedgenomic DNA fragment of M. hyopneumoniae expressingthis antigen in Escherichia coli from recombinant bacterio-phage (S. Hopkins, M. Kim, M. McIntosh, and K. Wise,Abstr. 6th Int. Congr. Int. Organ. Mycoplasmol., p. 188,1986). Such DNA fragments may be useful for (i) generatinginformation from DNA sequencing on the primary structureand processing of lipid-modified proteins in this organismand (ii) overproducing antigenic recombinant gene productspossibly useful for diagnosing disease (as described above)or for prophylactic immunization against enzootic pneumo-nia caused by this agent (21, 32). In analogous studies withanother strain of M. hyopneumoniae, Klinkert et al. (13)have reported identification of surface antigens by usingantisera to recombinant fusion proteins produced in E. colifrom cloned M. hyopneumoniae DNA fragments. Mycoplas-mal antigens recognized by these reagents included proteinsof 90, 68, and 50 kDa, which were characterized by trypsinsensitivity, by comigration in one-dimensional SDS-PAGEwith components of whole organisms 1251 surface labeledwith lactoperoxidase, and in one case (p90), by binding ofantibody to the surface of the organism. It is not clearwhether these antigens correspond to any of the specificTX-114-phase surface proteins reported here, since a num-ber of proteins in this size range are present in the wholeorganism. Antibody to the fusion protein related to p68 wasreported also to bind a 26-kDa protein (13). Since hyperim-mune serum to purified p65 described in the current studyfailed to bind a protein of this size even when tested onimmunoblots of whole organisms, the p65 we report isprobably distinct from p68 reported by these investigators.Two proteins were identified by MAbs representing pre-

cise differences between American Type Culture Collectiontype strains of the organism. The presence of protein p82 instrain J and the failure to detect it in strain VPP11 may reflect

the absence of a gene, lack of gene expression, or a subtleantigenic alteration in a gene product in the latter strain. Thisreagent is a useful marker for studying intraspecies variationof M. hyopneumoniae. Alternative forms of a surface protein(p70 and p73, respectively) were also identified on strains Jand VPP11. The molecular basis of this difference is unclear,but investigation of these variants may be useful in under-standing membrane protein expression, as well as mecha-nisms of diversification involving mycoplasma antigenicstructure.A final observation from these studies revealed a specific

component that may contribute to the wide-spread phenom-enon of antigenic cross-reactivity among mycoplasmas, in-cluding those of swine origin (8). One MAb identified antigenp66 or similar-sized components in swine mycoplasma spe-cies M. hyopneumoniae, M. flocculare, and M. hyorhinis, aswell as species from other hosts, including M. pulmonis, M.fermentans, and M. bovis. Analysis of detergent-fractiona-ted aqueous-phase protein staining patterns showed thisprotein to be the most abundant in each organism. As acomponent expressing conserved structure and possiblyfunction, it may be useful in analyzing diversification of aprotein possibly conserved among mycoplasma species, aswell as antigenic relatedness within this group of procary-otes.

ACKNOWLEDGMENTS

We thank M. J. Freeman and C. Armstrong for providing swineantisera to M. hyopneumoniae and Karen Ehlert for preparation ofthe manuscript.

This work was supported in part by grant 85-CRCR-1-1805 fromthe U.S. Department of Agriculture. K. Wise is a recipient of PublicHealth Service Research Career Development Award AR00848from the National Institute of Arthritis and Musculoskeletal andSkin Diseases.

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method for tritium-labeled proteins and nucleic acids in poly-acrylamide gels. Eur. J. Biochem. 46:83-88.

2. Bordier, C. 1981. Phase separation of integral membrane pro-teins in Triton X-114 solution. J. Biol. Chem. 256:1604-1607.

3. Boyer, M. J., and K. S. Wise. 1987. Fatty acylated integralsurface membrane proteins of mycoplasmas are abundant andexpress marked intraspecies variation. J. Cell. Biochem. Suppl.llB:1111.

4. Cross, G. 1987. Eukaryotic protein modification and membraneattachment via phosphatidylinositol. Cell 48:179-181.

5. Dahl, C. E., and J. S. Dahl. 1984. Phospholipids as acyl donorsto membrane proteins of Mycoplasma capricolum. J. Biol.Chem. 258:10771-10776.

6. Dahl, C. E., J. S. Dahl, and K. Bloch. 1983. Proteolipidformation in Mycoplasma capricolum. J. Biol. Chem. 258:11814-11818.

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8. Freeman, M. J., C. H. Armstrong, L. L. Sands-Freeman, and M.Lopez-Osuna. 1984. Serological cross-reactivity of porcine ref-erence antisera to Mycoplasma hyopneumoniae, M. flocculare,M. hyorhinis, and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirecthemagglutination test. Can. J. Comp. Med. 48:202-207.

9. Freundt, E. A. 1983. Culture media for classic mycoplasmas.Methods Mycoplasmol. 1:127-135.

10. Freundt, E. A., and D. G. Edward. 1979. Classification andtaxonomy, p. 1-42. In M. F. Barile and S. Razin (ed.), Themycoplasmas, vol. 1. Academic Press, Inc., New York.

11. Gois, M., F. Kuksa, J. Franz, and D. Taylor-Robinson. 1974.The antigenic differentiation of seven strains of Mycoplasma

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12. Hunkapillar, M. W., E. Lujan, F. Ostrander, and L. E. Hood.1983. Isolation of microgram quantities of proteins from poly-acrylamide gels for amino acid sequence analysis. MethodsEnzymol. 91:227-236.

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14. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

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16. Markwell, M. A. K. 1982. A new solid-state reagent to iodinateproteins. Anal. Biochem. 125:427-432.

17. Nystrom, S., K.-E. Johansson, and A. Wieslander. 1986. Selec-tive acylation of membrane proteins in Acholeplasma laidlawii.Eur. J. Biochem. 156:85-94.

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19. Riethman, H. C., M. J. Boyer, and K. S. Wise. 1987. TritonX-114 phase fractionation of an integral membrane surfaceprotein mediating monoclonal antibody killing of Mycoplasmahyorhinis. Infect. Immun. 55:1094-1100.

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22. Schlesinger, M. J. 1981. Proteolipids. Annu. Rev. Biochem.50:193-206.

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