JOURNAL OF BACTERIOLOGY, JUlY 1973, p. 330-340Copyright 0 1973 American Society for Microbiology

Vol. 115, No. 1Printed in U.S.A.

Cell Surface Protein of Pseudomonas(Hydrogenomonas) facilis

HARRY G. RITTENHOUSE,1 BRUCE A. McFADDEN, LEWIS K. SHUMWAY,AND JOHN HEPTINSTALL2

The Graduate Programs in Biochemistry and Genetics and Departments of Chemistry and Botany, WashingtonState University, Pullman, Washington 99163

Intact cells of Pseudomonas facilis contain one major molecular weight class ofprotein that is exposed at the cell surface as revealed by lactoperoxidase-cat-alyzed iodination with 1251. All molecular weight classes of protein in derived cellenvelope preparations are apparently saturated by iodination by lactoperoxidaseafter prolonged sonic treatment. The molecular weight of the predominantlyexposed protein in intact cells is approximately 16,000, which is the minimalmolecular weight of a cell envelope protein that precipitates as a complex withphospholipid from extracts of P. facilis. The isolation of labeled phospholipo-protein (PLP) after labeling intact cells with 1251 corroborates previous experi-ments which suggested a surface location for the protein portion of thephospholipoprotein (PPLP)- Solvent extraction of cells and immunologicalevidence, including studies with ferritin-coupled antibodies, indicate that PPLP islocated at the cell surface and may also be within the cell envelope. Theseexperiments suggest that PPLP is the major cell surface protein in P. facilis.

Although proteins are known to be present inconsiderable amounts in the cell envelope ofgram-negative bacteria, little is known of thesecomponents (7, 9, 19). In particular, the proteinin the outer membrane of gram-negative bacte-ria, which amounts to about 60% of the cellenvelope protein in Salmonella typhimurium(20), has been investigated only recently. Polya-crylamide gel electrophoresis of purified outermembrane preparations from gram-negativebacteria has revealed the presence of up to 11proteins including one or two major proteinswhich may account for as much as 70% of thetotal protein in this outermost cell structure (2,20, 28). Selective proteolytic attack of the outermembrane of Escherichia coli has been used asevidence for an asymmetric arrangement ofproteins in the membrane and for the presenceof select molecular weight classes of proteinsexposed at the membrane surface (2). The facilerelease of lipopolysaccharide (LPS)-protein-lipid complexes from some gram-negative bac-teria (5, 10, 15) indicates the lability of surfacecomponents of the outer membrane and thepresence of protein associated with lipid andLPS on the surfaces of these organisms.

Previous work (11, 14, 25) in this laboratory

'Present address: Department of Bacteriology, Universityof Califomia, Los Angeles, Calif. 90024.

2 Present Address: Department of Biological Sciences,Oxford Polytechnic, Oxford, England.

on a hydrophobic phospholipoprotein (PLP),which precipitates when the ionic strength ofthe "soluble" fraction from Pseudomonas facilisis lowered, revealed a cell envelope location forthe protein component (PPLP). PPLP was unusu-ally hydrophobic, apparently pure, and had aminimal molecular weight of 15,000 (25). Anti-PLP, which was directed towards PPLP, ag-glutinated P. facilis, establishing that the pro-tein was located in part on the cell surface (25).In this paper we present immunological andchemical evidence that PPLP is located in theouter membrane portion of the cell envelope ofP. facilis and that a portion of it is very looselybound on the cell surface. By using the enzymelactoperoxidase, shown by Phillips and Morri-son (21) to catalyze radioiodination of proteinsexposed on the membrane surface, we presentdata which indicate that PPLP is the major cellsurface protein in P. facilis.

MATERIALS AND METHODSOrganism and medium. The origin, maintenance,

and culture of P. facilis, previously classified asHydrogenomonas facilis (23), were as described byKuehn and McFadden (13). Cultures were grown in300 ml of fructose medium, transferred to 4.5 liters ofthe same medium, grown at 30 C with slow shaking(160 rpm), and harvested at the late log phase ofgrowth.

Materials. Lysozyme, pepsin, albumin (bovine,fraction V), carbonic anhydrase, lactoperoxidase, Tri-

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ton X-100, agarose, and Coomassie Brilliant Blue Rwere obtained from Sigma Chemical Co., St. Louis,Mo.; carrier-free 125iodine was from Schwarz/MannCo., Orangeburg, N.Y.; ferritin (horse spleen, twicecrystallized), toluene-2, 4-diisocyanate, goat anti-rab-bit immunoglobulin G (IgG) serum, and fluorescein iso-thiocyanate-conjugated goat anti-rabbit (IgG frac-tion) were from Miles Laboratories, Inc., Kankakee,Ill.; and sodium dodecyl sulfate (SDS; sequanalgrade) was from Pierce Chemical Co., Rockford, Ill.All other chemicals were reagent grade or the purestgrade available.

Cell envelope, spheroplast, and spheroplastmembrane preparations. Cell envelope preparationsof P. facilis were obtained from cells in the late logphase essentially by the method of Schnaitman (27).Wet-packed cells were brought to a 50% (wt/vol) sus-pension in 0.05 M tris(hydroxymethyl)-aminomethane(Tris)-chloride buffer (pH 8.0, 25 C) and broken bypassage through a French pressure cell at 2 C and15,000 lb/in2. The treated suspension was centrifugedat 2 C at 3,000 x g for 5 min to remove unbroken cells.The supernatant portion was then centrifuged at100,000 x g for 60 min at 2 C, and the particulatefraction was collected and washed once with the sameTris buffer.

Ethylenediaminetetraacetate (EDTA)-lysozymespheroplasts were prepared by the method of Birdselland Cota-Robles (1). Spheroplast membranes wereobtained by osmotic shock of the EDTA-lysozymespheroplast preparation by a 10-fold dilution of thespheroplast suspensions into deionized water.Iodination procedure. The general procedure of

Poduslo et al. (22) was followed. Samples were sus-pended in 5.0 ml of 0.1 M sodium phosphate (pH 7.4),and lactoperoxidase was added to the reaction mix-ture to give a final concentration of 1 jiM. Either 50 or100 uCi of Na'25I (sp act 1 mCi/mmol) were added,and the reactions were initiated and continued at 30 Cby the addition of 0.02 ml of 8 mM H202 at 15-sintervals. The addition of small amounts of H202 wasdone to minimize undesirable oxidation of intactbacteria or cell envelope preparations by free perox-ide. After either 60 or 90 min, the reactions werequenched by chilling to 2 C, and the samples weredialyzed overnight at 2 C against the phosphatebuffer. Particles were recovered from particulate sam-ples by centrifugation at 100,000 x g (2 C) for 10 minand stored with soluble samples at -20 C untilapplication to SDS polyacrylamide gels and subse-quent electrophoresis. The following samples wereradioiodinated for 90 min (total addition of 7.2 ml ofH202 solution): intact P. facilis which had beenharvested at late log phase (100 ACi of 1251), derivedcell envelope preparations sonically treated for 15 min(100 MCi of 1251), and derived cell envelope prepara-tions sonically treated for 30 s (50 uCi of 1251). Othersamples including intact P. facilis which had beenharvested at late log phase (50 ,uCi of 1251), derived cellenvelope preparations sonically treated for 15 min (50UCi of 1251), PLP, bovine serum albumin (BSA), andlysozyme were incubated in the presence of Na125I for60 min (total addition of 4.8 ml of H202 solution).

Gel electrophoresis. SDS-polyacrylamide gel elec-trophoresis was carried out by the method described

by Osborn et al. (30). The gels were removed, fixed ina 15% acetic acid-25% isopropanol solution for 12 h,and then placed in a 10% acetic acid-25% isopropanolsolution for 6 h before staining for 12 h with a solutionof 0.25% Coomassie Brilliant Blue containing 25%isopropanol and 10% acetic acid. The gels weredestained by soaking in 10% acetic acid. Alterna-tively, 125-mm gels containing radioiodinated mate-rial were sliced laterally in 1.20-mm sections afterelectrophoresis, and the gamma emissions werecounted for each gel section with a gamma-spectrome-ter. Molecular weights of the components in stainedgels or radioactive gels were estimated by comparisonwith migration of standards under identical condi-tions with the assumption that standards and un-knowns were saturated by 2% SDS and that resultantstructures were all in an extended rod-like configura-tion (24).Immunological procedures. The preparation of

antibody to PLP, Ouchterlony double diffusion, im-munoelectrophoresis, and agglutination of bacteria byantibody were as described previously (25). Antiserumto phenol-extracted protein or cell envelope prepara-tions was obtained from rabbits after injecting approx-imately 4 mg subcutaneously twice weekly for 3 weeksand bleeding 1 week after the final injection.

Ferritin-labeled antibody studies employed either(i) antibody obtained from fractionation of antiserumto PLP with ammonium sulfate (25) or (ii) goat anti-rabbit IgG serum conjugated to ferritin by using thebifunctional reagent, toluene-2,4-diisocyanate (29).

For best results it was found that conjugated anti-body-ferritin should be prepared and used for electronmicroscope studies within 1 day. For the directferritin-coupled antibody studies, the following prep-arations derived from late log P. facilis were in-cubated with ferritin-labeled anti-PLP in 0.01 MTris-chloride buffer (pH 8.0, 25 C) at 37 C for 4 hand then at 2 C for 24 h: lysozyme-EDTA spheroplastsfrom plasmolyzed cells, phenol-extracted intact cells,and spheroplast membranes derived from lysozyme-EDTA spheroplasts. The Tris buffer solution for thelysozyme-EDTA spheroplasts contained 0.5 M su-crose to prevent lysis of the spheroplasts. The abovesamples were centrifuged at 2,000 x g for 10 min at 2C, washed in the same cold Tris buffer, and pelletedat 5,000 x g at 2 C. The pelleted samples were thenprepared for electron microscope examination. Nor-mal rabbit serum conjugated to ferritin was used forthe control samples.

For the indirect ferritin-coupled antibody method,P. facilis cells were incubated with antibody to PLP in0.01 M Tris-chloride (pH 8.0, 25 C) for 4 h at 37C and then for 24 h at 2 C. The cells were washedtwice with the same cold Tris buffer at 2 C andincubated with ferritin-coupled goat anti-rabbit IgGserum in the Tris-chloride buffer for 4 h at 37 Cand 24 h at 2 C. After centrifugation at 2,000 x g for10 min (2 C), the cells were washed twice with the coldTris buffer, and the 5,000 x g pellet (2 C) wasprepared for electron microscopy. In the controlsample, P. facilis cells were incubated initially withnormal rabbit serum instead of antibody to PLP.The indirect method was used for the immunofluo-

rescence study of P. facilis. Cells which had been

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grown heterotrophically on fructose to early log phaseor late log phase or autotrophically with 90% H2, 5%02, and 5% CO2 by the procedure of Kuehn and Mc-Fadden (13) were mixed with antibody to PLP in 0.01M Tris-chloride (pH 8.0, 25 C) and incubated for 2 h at40 C. The cells were then washed twice with the Trisbuffer at 2 C and resuspended in the same buffercontaining fluorescein isothiocyanate-conjugated goatanti-rabbit (IgG fraction). After incubation at 40 C for2 h, the stained cells were mounted on a microscopeslide by using a mounting agent composed of 9 vol ofglycerol per 1 vol of 0.05 M sodium phosphate buffer(pH 7.3) containing 0.85% NaCl. For control samples,normal rabbit serum was used instead of anti-PLP inthe first incubation step. The samples were examinedunder oil immersion by fluorescence microscopy with aZeiss Fluorite 100x objective. The light source was amercury vapor lamp and dark-field illumination wasused.

Electron microscopy. Samples were prepared forthe electron microscope as previously described (11).

Solvent extraction procedures. Various solventextractions were used to aid in locating PPLP withinthe complex layers of the cell envelope and finding thecondition required to dissociate PPLP from cells enve-lope preparations and intact cells.

Phenol extraction of intact P. facilis was conductedby the general procedure of Weidel et al. (31). A 5-gamount of P. facilis was suspended in 20 ml ofphenol-water (1:1, vol/vol) at 65 C for 10 min. Themixture was then cooled in an ice bath and cen-trifuged at 20,000 x g. After separation from theaqueous phase, the phenol phase was dialyzed at 2 Cfirst against 50 vol of 0.01 M Tris-chloride (pH 8.0,25 C) containing 1% Triton X-100 and 1 M urea andfinally against 0.01 M Tris-chloride (pH 8.0, 25 C)to remove most of the residual phenol. The resultantprecipitate is referred to as phenol-extracted protein.Residual phenol was also removed from phenol-ex-tracted P. facilis cells by the above dialysis procedure.

P. facilis cells were also extracted by either (i)suspension of 1 g of late log-phase cells in 1 ml of 0.9%saline at 25 C for 5 min and collection of thesupematant fraction from centrifugation at 100,000 xg at 2 C for 10 min or (ii) incubation of 1 g of late logcells in 10 ml of 0.1 M Tris-hydrochloride (pH 8.0, 25C) containing 0.005 M EDTA at 100 C for 15 min withsubsequent collection of cells by centrifugation. Cellenvelope preparation (1 g) of P. facilis was extractedwith 50 ml of chloroethanol-water (1: 1, vol/vol) at 25 Cfor 4 h. After centrifugation at 10,000 x g, the pelletwas washed with water five times (P-1). The chloro-ethanol-water extract was dialyzed against water, andthe white precipitate which appeared in the superna-tant portion during dialysis (P-2) was pelleted at10,000 x g.

RESULTS

lodination experiments. When intact P. fa-cilis is labeled with 125I and derived cell en-velopes are examined by gel electrophoresis, aselect size of protein (mol wt 16,000) is predomi-

nantly labeled (Fig. 1). This experiment wasperformed with both 100 ACi and 50 ,uCi of125iodide. The similar gel profile for both experi-ments indicates lactoperoxidase-catalyzedequilibration of 125I with all proteins exposed tothe surface of intact cells.The gel profile of cell envelope protein labeled

after isolation of the cell envelope fraction fromintact cells (Fig. 2) also reveals select molecularweight classes of protein exposed to the enzymelactoperoxidase. The incorporation of relativelylittle label into this preparation is similar tothat in experiments with intact cells, as is thepredominant labeling of protein of a molecularweight of about 16,000. More proteins, however,are labeled in this fraction than in intact cells.Presumably this results from iodination of pro-teins at the cytoplasmic face of the cell enve-lope.

Extensive incorporation of 125I into proteinwas found (Fig. 3) when the cell envelopefraction was sonically treated prior to reactionwith radioiodine and lactoperoxidase. A briefsonic treatment (30 s) apparently exposes moreof the cell envelope protein since considerableiodine is incorporated into all molecular weightclasses of protein, although a major peak corre-sponding to a molecular weight of about 16,000is still evident. More extensive sonic treatment(15 min) exposes still more protein as evidencedby the higher incorporation of label in more cellenvelope molecular weight classes (Fig. 3). Thevery similar gel profile of cell envelope proteinlabeled with either 50 ,uCi or 100 IsCi of 125I afterthe 15-min sonic treatment indicates equilibra-tion of label with exposed proteins. As a conse-quence of longer sonic treatment, protein of16,000 daltons becomes a relatively minor radio-active peak in marked contrast to the experi-ments with intact cells.

Cell and cell envelope samples incubatedwith 100 ,Ci of Na125I in the absence of lac-toperoxidase incorporated very little label(1-2% of lactoperoxidase-treated samples). Thegel profiles (not shown) of these samples re-vealed only one small peak which was locatedslightly ahead of the tracking dye. This mayrepresent non-enzymatic iodination of lipid.When intact P. facilis was labeled with 100

jsCi of 1251I by using lactoperoxidase, the PLPsubsequently isolated by the method of Kuehnet al. (14) contained gamma emissions of 2,050counts per min per mg of protein (16). Extrac-tion of lipid from the labeled PLP preparation asdescribed by Kuehn et al. (14) yielded materialhaving 1,565 counts per min per mg of protein,demonstrating that the protein component ofPLP in intact P. facilis contained most, if not

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0?!0l

Relative MobilityFIG. 1. Iodination of intact P. facilis. After iodination of 1 g of wet-packed intact cells, the cell envelope

fraction was prepared, and the 126I distribution in counts per minute was determined after SDS-polyacrylamidegel electrophoresis. Symbols: 100 .uCi of 1251- initially added, 0; 50,uCi of 1251- initially added, A. Positions ofstandards of specified molecular weights are shown across the top.

300

E 240

180

120-

0'1 02 03 04 05 06 07 08 09

Relative Mobility

FIG. 2. Iodination of the cell envelope preparationfrom P. facilis derived from 1 g of wet-packed cells.The cell envelope fraction was iodinated by using 1001sCi of 1251-, and the 125I distribution was determinedafter SDS-polyacrylamide gel electrophoresis. Forother specifications see Fig. 1.

all, of the label. Intact cells exposed to 100 ,uCiof 126I without lactoperoxidase yielded PLPwhich contained no significant label (86 countsper min per mg of protein).

Figure 4 shows the gbl profile of PLP labeledafter isolation from intact cells compared with126I-labeled BSA and lysozyme. The peak ofradioactive material for the PLP sample corre-sponds to a molecular weight of 16,000 whencompared to the relative mobilities of the la-beled standards BSA and lysozyme (Fig. 4) and

of BSA, aldolase, pepsin, carbonic anhydrase,and lysozyme in gels stained with CoomassieBrilliant Blue.

Ferritin-coupled antibody and immuno-fluorescence studies. Fig. 5A shows the speci-fic labeling of the cell surface of intact P. facilis(incubated initially with antibody to PLP) withferritin-coupled goat anti-rabbit IgG serum.The control sample (Fig. 5B), which had beenincubated initially with normal rabbit serum,contained small amounts of ferritin with nolocalized concentration on the cell surface.The direct method, using ferritin-coupled

antibody to PLP, resulted (not shown) in asmall amount of label specifically attached tothe cell surface with moderate amounts ofscattered ferritin, whereas the control samplecontained essentially no ferritin label. Thisindicated that the antigenic determinants ofPPLP on the cell surface were sterically lessavailable to the very large ferritin-coupled anti-PLP molecules used in the direct method thanto the relatively smaller unconjugated anti-PLPused in the initial step of the indirect method.Alternatively, the antibody-antigen complexmay be more easily detached from the cellsurface when the direct method is employed;this would explain the presence of scatteredferritin in the samples in which ferritin-labeledantibody to PLP had been used.

In contrast, phenol-water-extracted P. faciliscontained practically no ferritin attached to the

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16,000

0.1 0.2 0.3 0.4 0.5 0.6 07 0.8 0.9 1.0 1.1

Relotive Mobility

FIG. 3. Iodination of sonically treated cell envelope fractions each derived from 1 g of wet-packed P. facilis.The cell envelope fractions were sonically treated for the indicated time intervals at 20 kc before reaction with1251-. The 125I distribution was determined after SDS-polyacrylamide gel electrophoresis. Symbols: cellenvelope fraction sonically treated for 30 s and labeled by using 50 ACi of 125L-, 0; cell envelope fractionsonically treated for 15 min and labeled by using 50 ,uCi of 125k-, A; cell envelope fraction sonically treated for 15min and labeled by using 100 ,uCi of 125P-, 0. For other specifications see Fig. 1.

684

40 35 294 4 4

14.3

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

Relative MobilityFIG. 4. Iodination of PLP, BSA, and lysozyme. PLP, BSA, and lysozyme were labeled by using 100 MCi of

125iodide, and the distribution of radioactivity was determined after SDS-polyacrylamide gel electrophoresis.Symbols: PLP, 0; BSA, A; lysozyme, 0. For other specifications see Fig. 1.

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FIG. 5. Electron micrographs of P. facilis with ferritin-coupled goat anti-rabbit IgG serum. A, Cells werepreincubated with antibody to PLP; no post-stain. B, Cells were preincubated with control serum. Bar markersin this and all electron micrographs represent 0.5 gm.

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extracted surface (not shown). Large masses ofscattered ferritin were visible, however. SincePPLP can be partially eluted from the intact cellswith various solvent techniques, including phe-nol-water extraction, the scattered ferritin mayhave been due to attachment to fragments ofthe cell wall containing PPLP. The presence ofalmost no scattered ferritin in the control sam-ple was consistent with this possibility.

Spheroplast membranes prepared fromEDTA-lysozyme spheroplasts (1) by osmoticshock show very heavy label with ferritin-cou-pled antibody to PLP (Fig. 6A), whereas the con-trol sample (Fig. 6B) contains practically no fer-ritin. The fragmentation of the EDTA-sphero-plasts exposes considerably more antigenic de-terminants of PPLP compared with spheroplasts(not shown) and affords heavy labeling by thebulky ferritin-coupled anti-PLP molecules.

Late log-phase P. facilis cells were labeledindirectly with fluorescein-conjugated goatanti-rabbit IgG (not shown) in a uniform man-ner. This again indicates the presence of uni-formly distributed PPLP on the cell surface. Inthe control sample (preincubation with normalrabbit serum) the cells were not stained. P.facilis which had been grown heterotrophicallyon fructose to early log phase or autotrophicallywith H2, 02, and CO2 were also stained withfluorescein-conjugated goat anti-rabbit IgG.Solvent extraction experiments. Immuno-

electrophoresis with anti-PLP and the proteinextracted from intact P. facilis with phenolresulted in a single, long precipitin arc locatedslightly toward the cathode (Fig. 7A). Moresignificantly, Fig. 7B shows the fusion of pre-cipitin lines resulting from double diffusion ofphenol-extracted protein and PLP against anti-body to PLP. The aqueous layer was found tocontain large amounts of saccharide measuredas hexose (8), presumably as LPS, and smallamounts of protein (16) in a ratio of 9:1 byweight, in accord with the data of Weidel et al.(31) for E. coli. Antiserum prepared againstphenol-extracted protein also yielded a precipi-tin line of identity between PLP and phenol-extracted protein after double diffusion (notshown).Both P-1 and P-2 fractions from chloro-

ethanol extraction of cell envelopes yielded pre-cipitin lines of identity with PLP after doublediffusion against antibody to PLP (Fig. 7B).The precipitin reaction with P-1 indicates thatsome PPLP is associated tightly with the cellenvelope structure since P-1 represents mate-rial which can not be extracted with chloro-ethanol-water. Interestingly, hexose (0.20 mg permg of protein), pentose (0.22 mg per mg of pro-

tein), and phosphate (0.02 mg per mg of protein)were found in the P-1 fraction, indicating thepossibility of covalent attachment of carbohy-drate and lipid to protein.The presence of some PPLP which is very

tightly bound to intact cells is indicated by theagglutination of heat-treated P. facilis (Table1). Considerable amounts of this protein may bedenatured or extracted, or both, by the heattreatment, however, since the titer is reducedfrom 2,048 for untreated cells to 256 for heat-treated cells. Antiserum either to phenol-extracted protein or cell envelopes agglutinatesintact P. facilis (Table 1). P. facilis extracted bythe phenol-water procedure is not agglutinatedby antibody to PLP, indicating that the re-moval of the outer membrane removes all of theexposed antigenic sites.A portion of the PPLP on the cell surface is

probably very loosely bound as indicated by themild 0.9% saline extraction of intact cells at 25C which removes material yielding a precipitinline of identity with PLP after double diffusionagainst anti-PLP (Fig. 7B, well no. 5).

Cell envelope protein composition. The pro-tein composition of cell envelope preparationsfrom P. facilis (Fig. 3 and 8) is quite similar tothe cell envelope protein compositions reportedfor E. coli (2, 27) and Salmonella typhimurium(20). A very heterogeneous pattem of proteins isobtained with major protein peaks located atapproximate molecular weights of 34,000 and42,000. Protein of a molecular weight of 16,000 isabout 8% of the total cell. envelope protein onthe basis of 1251 incorporation into cell envelopepreparations that had been sonically treated for15 min (Fig. 3). This calculation assumes equalfractions of tyrosine and histidine residues in allthe cell envelope proteins since the enzymelactoperoxidase specifically iodinates only theseamino acid residues (21). From a scan of aCoomassie Brilliant Blue-stained gel, protein ofthis size (arrow, Fig. 8) was estimated to com-pose about 12% of the total cell envelope pro-tein. This calculation is also approximate be-cause the absorbance of Coomassie BrilliantBlue-stained protein deviates from Beer's law athigher protein concentrations (3). Both thesecalculations indicate, however, that protein of amolecular weight of 16,000 is present in P.facilis cell envelopes in substantial amountsalthough it is not the major protein species.

DISCUSSIONThe radioiodination studies of intact cells,

envelopes, and sonically treated envelopes of P.facilis provide evidence for a very asymmetricdistribution of protein at the cell surface. In

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FIG. 6. Electron micrographs (no post-stain) of spheroplast membranes derived from EDTA-lysozymespheroplasts of P. facilis. A, Ferritin-coupled antibody to PLP was used; B, ferritin-coupled gamma-globulinfrom control serum was used.

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Phenol extract

+

A BFIG. 7. Immunoelectrophoresis and immunodiffu-

sion experiments of extracted materials from P.facilis. The 1.5% agarose slides were made with 0.05MTris-chloride buffer (pH 8.0) containing 1% TritonX-100 and 1 M urea. A, Immunoelectrophoresis ofphenol-extracted protein from P. facilis (left andright wells) reacted with antibody to PLP in centertrough. Electrophoresis was conducted for 30 min in0.05 M Tris-chloride buffer (pH 8.0) at 5 mA perslide. B, Ouchterlony double-diffusion experiment ofmaterials extracted from intact cells and cell en-velopes of P. facilis. The center well contained anti-body to PLP, and the sample wells contained thefollowing: (1) PLP, (2) phenol-extracted protein fromP. facilis; (3) chloroethanol-insoluble material (P-1);(4) precipitate (P-2) obtained during dialysis of chlo-roethanol extracts of cell envelopes; (5) saline extractof intact P. facilis; and (6) aqueous layer fromphenol-water extraction of intact P. facilis.

particular, protein of a molecular weight of16,000 contains the majority of label in studiesof whole cells and is also a major labeledfraction resulting from lactoperoxidase-cat-alyzed iodination of cell envelopes. In contrast,with sonically disrupted cell envelopes, 125I isincorporated into a wide molecular weight rangeof proteins, and much more label is incorpo-rated (100- to 1,000-fold). Lactoperoxidase pre-sumably catalyzes iodination of all cell envelopeproteins once they have been exposed. Phillipsand Morrison (21) have previously demon-strated the selectivity of this process in labelingsurface proteins of intact erythrocytes by com-paring species labeled before and after erythro-cyte membrane disruption.The incorporation of 125I into PPLP upon

labeling intact P. facilis is further evidence of acell surface location for this protein. The rela-

tive mobility of 125I-labeled PLP in 2% SDSpolyacrylamide gels after electrophoresis corre-sponds to the major radioactive peak (mol wt16,000) obtained with cell envelopes from la-beled intact cells. The presence of more thanone protein species in the peak, of course,remains a possibility. The dense ferritin label-ing of intact cells with ferritin-coupled goatanti-rabbit IgG serum (indirect method) sug-gests the presence of high levels of the protein atthe cell surface. The small amount of attachedferritin on intact cells with the direct method

TABLE 1. Agglutination of P. facilisa

Pretreatment of P. Antibody directed Titerbfacilis against

None PLP 2,048At 100 C for 15 min PLP 256

in 0.1 MTris-chloridebuffer (pH 8.0)containing 0.005M EDTA

Phenol-water-ex- PLP No aggluti-tracted cells nation

None Phenol-extracted 256protein

None Cell envelope 4,096fraction

a In all cases, P. facilis was harvested at the late logphase of growth for use in the agglutination studies.

Titer is the reciprocal of the highest dilution ofantibody that still gives an observable agglutination.

08

06-

0

010 I

2 3 4 5 6 7 8 9 10loDistance (cm)

FIG. 8. SDS-polyacrylamide gel profile of cell en-velope preparation of P. facilis. The gel was stainedwith Coomassie Brilliant Blue and scanned at anoptical density of 620 nm. The arrow indicates proteinwith molecular weight about 16,000.

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CELL SURFACE PROTEIN OF P. FACILIS

(ferritin-coupled anti-PLP) may be a conse-quence of a high degree of steric hindrance tothe approach ofvery bulky molecules to PPLP. Inthis situation, the initial binding of anti-PLP(indirect method) may then be favored over thedirect binding of the more bulky ferritin-cou-pled anti-PLP molecules. The specific heavylabeling of spheroplast membranes directlywith ferritin-coupled anti-PLP is consistentwith this interpretation since the fragmentationof spheroplasts would be expected to exposemore protein. The heavy ferritin labeling ofspheroplast membranes also indicates that PPLPis present in substantial amounts in the cellenvelope of P. facilis. It should be noted thatdouble diffusion of spheroplast membranes andPLP against antibody to PLP previously re-vealed a single precipitin line of identity (25).

Phenol-extracted cells showed a lack of ag-glutination with anti-PLP and absence of at-tached ferritin-coupled anti-PLP, indicatingthe removal of the exposed protein from intactcells by phenol-water extraction. Apparently,phenol-water extraction of intact cells removesthe outer membrane of P. facilis as is the casefor Veillonella sp. (181). The presence of PPLP inthe phenol phase after phenol-water extractionof intact cells revealed by immunoelectrophore-sis and immunodiffusion also indicates an outermembrane location for this protein. The aque-ous phase of the phenol-water extraction con-sists mostly of saccharide (probably lipopoly-saccharide) but does not contain this protein,reflecting the known extremely hydrophobicnature of the protein (25).

Partial dissociation of PPLP from intact cellsand cell envelope preparations by extractionmethods (ranging from very mild to ratherharsh) provides evidence for great variation inthe degree of integration of this protein withinthe cell envelope. The presence of the protein inthe insoluble fraction of chloroethanol-extractedcell envelopes (P-1) demonstrates that some ofthis protein is extremely tightly bound. Inaddition, the agglutination of cells of P. facilisafter extraction at 100 C for 15 min in thepresence of 5 mM EDTA demonstrates thatsome PPLP cannot be removed from intact cellseven with this harsh treatment and thereforemust be quite strongly bound to, or integratedinto, the cell envelope. On the other hand, somePPLP can be eluted from intact cells by extrac-tion with 0.9% saline at 25 C.

It is likely that, during cell growth and celldivision, the components of the cell wall are in adynamic state. At various stages of cell elonga-tion and subsequent cell division, some cell wallcomponents may be initially integrated within

the cell wall but become increasingly labile ascell wall synthesis continues. This would resultin release of surface components from the cellduring cell growth. LPS which is located in theouter membrane and in part on the cell surfacehas been found associated with protein andlipid as a LPS-protein-lipid complex releasedfrom bacterial cells (4, 5, 10, 12, 26, 30, 32). Ofspecial relevance is the finding by Weckesser etal. (30) that extraction of Rhodopseudomonascapsulata with 0.9% NaCl at 37 C for 2 h resultsin elution of a LPS-protein-lipid complex. Sac-charide is also present in the mild saline extractof P. facilis which contains PPLP. The LPS-protein-lipid complexes found in other laborato-ries may represent a structure which exists inthe outer membrane in vivo. It will be of interestto see whether PPLP is associated with LPS as aLPS-protein-lipid complex after mild elutionfrom intact P. facilis. Whether related proteinsoccur in the complete somatic 0 antigens ofvarious gram-negative bacteria will also be ofdeep interest since it has long been known thatprotein is a component of 0 antigens (17). Thewidespread occurrence of proteins similar tothat of PPLP on bacterial cell surfaces has beeninferred recently on the basis that anti-PLPagglutinates a wide variety of bacteria (H. G.Rittenhouse, J. B. Rodda, and B. A. McFadden,J. Bacteriol., in press).

ACKNOWLEDGMENTSWe thank the Veterinary Pathology Department at Wash-

ington State University for use of the equipment for fluores-cence microscopy and the Animal Science Department atWashington State University for use of a gamma spectrome-ter (1185 series, automatic gamma counting system, Nuclear-Chicago).We gratefully acknowledge the expert technical assistance

of Russ Jones.These studies were supported by Public Health Service

grant AM-14400 (to B.A.M.) from the National Institute ofArthritis and Metabolic Diseases and predoctoral fellowshipno. GM-43746 (to H.G.R.) from the National Institute ofGeneral Medical Sciences. This manuscript was completedwhile B.A.M. was a Guggenheim Fellow at University ofLeicester. He thanks H. L. Komberg for many courtesiesextended during that period.

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