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Cellulose-binding proteins of Fibrobacter succinogenes and the possible role of a 180-kDa cellulose-binding glycoprotein in adhesion to cellulose

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Cellulose-binding proteins of Fibrobacter succinogenes and the possible role of a 180-kDa cellulose-binding glycoprotein in adhesion to cellulose
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  Cellulose-binding proteins of Fibrobactersuccinogenes and the possible role of a 180-kDa cellulose-binding glycoprotein inadhesion to cellulose Jianhua Gong, Emmanuel E. Egbosimba, and Cecil W. Forsberg Abstract: Fibrobacter succinogenes possesses seven cellulose-binding proteins (CBPs) of 40,45,50, 120, 180,220, and240 kDa. The 120-, 180-, 220-, and 240-kDa proteins were present in the outer membrane (OM), while the 40-, 45-, 50-,and 120-kDa proteins were either periplasmic or peripheral membrane proteins. The 120-kDa CBP, which was identified asendoglucanase 2, was a major component in both the OM and periplasm. Zymogram analysis for glucanases showed thatthe major membrane-associated CBPs, with the exception of endoglucanase 2, lacked endoglucanase activity. Affinity-purified antibodies against the 180-kDa CBP cross-reacted strongly with numerous cell envelope proteins of higher and lowermolecular mass, including the previously characterized chloride-stimulated cellobiosidase. Treatment of the 180-kDa CBPand cell envelope proteins with periodate resulted in almost complete loss of antibody binding, suggesting that they possesseda common epitope that was carbohydrate in nature. Immunogold labelling of whole cells using antibodies against the 180-kDaCBP demonstrated that either the 180-kDa CBP or related proteins with a cross-reactive epitope were located at the cellsurface. These epitopes were distributed uniformly on cells not bound to cellulose but congregated on the cell surface at sitesof adhesion of cells to cellulose. Antibodies to the 180-kDa protein caused 62% inhibition of binding of F. succinogenes tocrystalline cellulose, which provides evidence that either the 180-kDa CBP and (or) other related cross-reactive surfaceproteins have a role in adhesion to cellulose. Key words: cellulose, adhesin, adhesion, binding, Fibrobacter, succinogenes, rumen.Rksumk : Fibrobacter succinogenes posskde sept protCines se liant 8 la cellulose (CBPs) de 40,45,50, 120, 180,220 et240 kDa. Les protkines de 120, 180,220 et 240 kDa Ctaient prCsentes dans la paroi exteme alors que les protCines de 40,45,50 et 120 kDa Ctaient pCriplasmiques ou sit6es 8 la pCriphCrie de la membrane. La CBP de 120 kDa, qui a CtCidentifiCecomme l'endoglucanase 2, Ctait une composante majeure de la paroi exteme et du pCriplasme. Un zymogramme desglucanases a dkmontre, qu'8 l'exception de l'endoglucanase 2, les CBPs majeures assocites ? la membrane ne posskdaientpas d'activitt endoglucanase. Des anticorps dirigCs contre la CBP de 180 kDa reagissaient fortement de faqon croisCe avecde nombreuses protkines de l'enveloppe cellulaire de haut et bas masse molCculaire, incluant la cellobiosidase induite par lechlore. Le traitement de la CBP de 180 kDa et des protkines de l'enveloppe cellulaire avec le periodate a rCsult6 en une pertepresque totale de la liaison de l'anticorps, ce qui suggbre que ces protkines possbdent un Cpitope commun constituC d'hydratesde carbone. Le marquage immunologique B 'or colloidal des cellules complbtes en utilisant des anticorps spCcifiques pour laCBP de 180 kDa a dCmontrC que cette CBP ou des protCines relikes qui possbdent un Cpitope responsable d'une rCactioncroisCe Ctaient situCes B a surface bactkrienne. Ces Cpitopes Ctaient distribuks uniformCment sur les bactkries non likes 8 lacellulose mais se localisaient sur la surface des bactkries au site de contact de la bactCrie B a cellulose. Les anticorps dirigCscontre la protCine de 180 kDa induisaient une inhibition de 62% de la liaison de F. succinogenes 8 la cellulose cristalline, cequi dCmontre que la CBP de 180 kDa et (ou) d'autres protCines de surface qui prksentent une rCaction croisCe jouent un r61edans 17adhCsion la cellulose. Mots clks : cellulose, adhCsine, adhCsion, liaison, Fibrobacter, succinogenes, mmen.[Traduit par la rCdaction]Received September 15, 1995. Revision received December 7,1995. Accepted January 3, 1996. J. Gong; E. Egbosimba, and C.W. Forsberg. Departmentof Microbiology, University of Guelph, Guelph, ONN1G 2W1. Canada.Present address: Department of Medical and MolecularGenetics, School of Medicine, Indiana University,975 West Walnut St., Indianapolis, IN 46202, U.S.A.Author to whom all correspondence should be addressed(e-mail: cforsber@micro.uoguelph.ca). Introduction In the rumen, plant materials are colonized by large numbersof microorganisms. For many, this involves adhesion to thesurfaces, a process considered essential for efficient digestionof cellulosic materials (Cheng et al. 1991; Weimer 1992).Attachment of cellulolytic ruminal microbes, particularly thebacteria, to cellulose has received considerable attention (Akinand Barton 1983; Bhat et al. 1990; Cheng et al. 1984; Gong andForsberg 1989; Kudo et al. 1987; Minato and Suto 1978;Morris 1988; Pel1 and Schofield 1993; Roger et al. 1990). Can. J. Microbiol. 42: 453-460 (1996). Printed in Canada / ImprimC au Canada  Can. J. Microbiol. Vol. 42, 1996 However, the mechanism of attachment is still not fully under-stood. In Clostridium thermocellum, (a nonruminal cellulolyticbacterium), a noncatalytic scaffolding protein (CipA) of thecellulosome was found to play a central role in cellulosedigestion (BCguin and Aubert 1994; Kruus et al. 1995). Thisprotein comprises a cellulose-binding domain (CBD) and ninehighly similar internal repeats of 166 amino acid residues each.The repeats are receptors for endoglucanases. A terminal repeatmay serve either as a site for binding to the cell surface or maybe a site for concatenation of the CipA subunits (BCguin andAubert 1994). A noncatalytic scaffolding protein required forhydrolysis of crystalline cellulose has also been reported inClostridium cellulovorans (Doi et al. 1994). When the cellu-losomes are cell associated, the scaffolding proteins have a rolein the adhesion of cells to cellulose (Beguin and Aubert 1994).Cellulases may also have a direct role in adhesion of cells tocellulose. Expression of an exoglucanase containing the CBDfrom Cellulomonasfimi on the cell surface of ~scherichiaoliconferred on cells the ability to bind cellulose with high affinityand specificity (Francisco et al. 1993). In cellulolytic ruminalbacteria, it is not known whether adhesion of the bacteria tocellulose is mediated by surface-associated cellulolyticenzymes or specific noncatalytic adhesins. This question wasaddressed by Mitsumori and Minato (1993a, 1993b) with theirreported isolation and purification of 120- and 225-kDa non-catalytic CBPs from Fibrobacter succinogenes. However, theydid not localize the proteins and had limited evidence that theproteins had a role in adhesion of cells to cellulose.To further advance our knowledge of adhesion of F. succino-genes to cellulose, we have surveyed the bacterium for thepresence of surface-associated CBPs. After an initial screeningof CBPs, we focused on the role of a 180-kDa CBP in theadhesion process. Materials and methods Bacterium and growth conditions Fibrobacter succinogenes subsp. succinogenes S85 (Montgomeryet al. 1988) was grown at 37OC in a chemically defined medium(CDM) with ammonium sulfate as the sole source of nitrogen andeither 0.5% w/v glucose or 0.3% w/v microcrystalline cellulose(Avicel PH105, FMC Corp. Marcus Hook, Pa.) as a carbon source(Groleau and Forsberg 198 1). Labelling cells with [14C]isobutyrate [14C]Isobutyrate labelling of cells was performed as described byWegner and Foster (1963). Fibrobacter succinogenes was grown for10 h from a 10% v/v inoculum in 9 mL of CDM with glucose as thecarbon source but lacking fatty acids, except for 0.46 nmol/mL ofvalerate and 0.38 nmol/mL of isobutyrate, and containing 0.2 yCi/mLof [l-14C]isobutyrate (56 mCi/mmol, 1 Ci = 37 GBq; ICN Radio-chemicals, Irvine, Calif.).To determine the concentration of 14C n samples, 0.05-mLvolumeswere mixed with 10 mL of one-step scintillation cocktail, Fluorosol(National Diagnostics) and incubated at 42OC for 4 h prior to countingwith a model 2000 TRI-CARB liquid scintillation analyzer (PackardInstrument Company, Downers Grove, Ill.). Greater than 70% of theradioactive isobutyrate was incorporated into cells and 85% was inlipids. Assay of adhesion to cellulose Adhesion to cellulose was determined using a radioactive adhesionassay. Anaerobic conditions were maintained by performing allmanipulations in an anaerobic chamber with an atmosphere of90% V/V f C02and the balance H2. Carbon dioxide was the gas phasefor the CDM, while nitrogen was the gas phase for all other suspendingmedia, unless indicated otherwise. Cells grown for 10h with [14C]iso-butyrate reached an optical density at 675 nm of 3.0 (1.2 mg dryweight/mL). They were harvested by centrifugation, washed oncewith CDM lacking carbohydrates, and diluted in the same medium. A0.5-mL sample of the appropriately diluted cell suspension was thenincubated at 22OC for 30 min with 40 mg of autoclaved micro-crystalline cellulose (Avicel PH102) in 0.2 mL of CDM lacking carbo-hydrates. The samples were briefly mixed on a vortex mixer at 10-minintervals during the incubation. The cellulose was then washed twicein 1-mL volumes of CDM lacking carbohydrates by centrifugation(377 x g, 20°C, 1 min), the supernatant fluid containing unbound cellswas removed, and the cellulose was resuspended in new medium. A0.05-mL sample of the cellulose with bound cells was mixed with10 mL of scintillation fluid and the radioactivity was determined usinga liquid scintillation counter.To examine adhesion to cellulose of cells lacking outer membrane(OM), labelled cells treated to remove the OM by sequential washesin 0.5 M NaCl and 25% w/v sucrose (Gong and Forsberg 1993) weremixed with cellulose. To examine inhibition of adhesion of I? succino-genes S85 to cellulose by different antibodies, each of the appropri-ately diluted antibodies or preimmune serum was incubated withwashed 14C-labelled cells (0.05 mg/mL, dry weight) at 37OC for30 min with shaking (100 rpm) prior to the adhesion assay. Themixtures of antibodies and cells were also tested for agglutination byincubation (without cellulose) in a microtiter plate at 37OC for 2 h andthen at 4OC for 16 h to allow settling of the cells. Isolation of cell envelopes, outer membranes, periplasm,and inner membranes Cell envelopes were prepared by washing cells once in 50 mM sodiumphosphate buffered saline (pH 6.7) and disrupting them by 8- to 30-streatments using an ultrasonicator with I-min cooling intervals on ice,followed by centrifugation (30 000 x g, 4"C, 30 min). After one washof the envelopes in buffered saline, the envelopes were used. Themethod for isolation of OMS, periplasm, and inner membranes (IMs)was that described previously (Gong and Forsberg 1993) and is brieflysummarized here. Glucose-grown cells were sequentially washed(centrifugation, 10 000 x g, 4OC, 10 min) with a salts solution corre-sponding to that of the growth medium, twice in 0.5 M NaCl in 10 mM piperazine-N,N1-bis(2-ethanesulfonic cid (PIPES) buffer (pH 6.8),once in 25% w/v sucrose - 1 mM EDTA, once in water, and once in25% sucrose. After recentrifugation of all supernatant samples toremove residual cells, the OM was recovered from the NaCI, sucrose,and water washes by centrifugation (100 000 x g, 4OC, 2 h) of thecell-free supemates. The nonsedimentable materials contained theperiplasmic contents and any peripheral membrane proteins that mayhave been released. The IM was recovered from the remaining treatedcells by ultrasonication and centrifugation as described previously(Gong and Forsberg 1993). Solubilization of membrane proteins and binding tocellulose Membrane samples, including cell envelopes, OMS or cytoplasmicmembranes (CMs) prepared from glucose-grown cells, in 50 mMsodium phosphate buffered saline (pH 6.5) were extracted with0.5% w/v of 3-((3-cholamidopropyI)dimethylammonio)-l-propane- sulfonate (CHAPS) at 22OC for 2 h. Insoluble materials were removedby centrifugation (100 000 x g, 4"C, 2 h). A 0.5-mL sample of thesolubilized membrane proteins (about 0.5 mg) in 0.05 M sodiumphosphate buffer was incubated with 40 mg of autoclaved micro-crystalline cellulose (Avicel PH-105) using continuous mixing byinversion at 8 rpm for 45 min at 22OC. The samples were transferredto 0.5-mL microcentrifuge tubes with a pin hole in the bottom of each  Gong et al. and supported in 1.5-mL microcentrifuge tubes and centrifuged(3000 rpm, 22OC, 5 min, in a microfuge). The cellulose was resus-pended in 0.5 mL of the same buffer lacking CHAPS and recentri-fuged. This was repeated three times. Proteins bound to cellulose wereeluted with 0.1 mL of the sodium dodecyl sulfate - polyacrylamidegel electrophoresis (SDS-PAGE) sample buffer at 100°C for 5 min,and 30 pL of the eluate was used for SDS-PAGE or Western blotanalysis. The unbound proteins from the incubation mixture weresubjected to lipidldetergent extraction as noted below and thenmixed with SDS-PAGE sample buffer and heated as above prior toelectrophoresis. Preparation of antibodies Antigens including the OMS, cell envelopes (CEs), or intact cells wereprepared from F. succinogenes S85 grown on glucose. To prepare OMantigen, the OM released from ,385 cells by washes in 0.5 M NaClwere pooled and dialyzed against saline. Then 1 mL of each antigencontaining 150 pg protein was emulsified in Freund's incompleteadjuvant and administered subcutaneously in the back of the neck ofa New Zealand white rabbit at day 1, and intramuscularly into thehindquarters at days 8 and 15. An intravenous injection of the sameantigen containing 75 pg protein in sterile saline was given at day 29.Rabbits were bled from an ear vein 4 days after the final injection. Theblood was clotted by incubation without agitation for 1 h at 37OC and2 h at 4OC, followed by centrifugation at 10 400 x g for 10 min. Theupper serum phase free from red blood cells was saved. Preimmuneserum was prepared prior to immunization.To prepare antibodies against the 180-kDa CBP, CHAPS-solubilized cell envelope proteins were bound to cellulose, washedwith 0.05 M phosphate buffer (pH 6.5), released with electrophoresissample buffer, and separated by preparative SDS-PAGE. The180-kDa CBP was localized by briefly staining the gel with 0.1%Coomassie brilliant blue R-250.The portion of the gel containing the180-kDa CBP was excised and rinsed with water. The gel slice wasemulsified in Freund's incomplete adjuvant and two rabbits wereimmunized with approximately 50 pg of the protein per injection asdescribed previously (McGavin and Forsberg 1989).The immunoglobulin G (IgG) fraction was purified on a protein A - Sepharose Fast Flow column (Sigma) equilibrated in 10 mM sodiumphosphate buffered saline (PBS, pH 7.3). Protein-A-bound IgG waseluted with 0.1 M glycine hydrochloride buffer (pH 2.5) containing0.5 M NaCl and neutralized immediately with 1.5 M Tris-HC1(pH 8.8).The method for preparation of monospecific polyclonal antibodieswas described previously (Huang and Forsberg 1988), except that theprotein band was localized in the SDS-polyacrylamide gel byCoomassie staining of a vertical slice of the gel, and the band wasexcised and transblotted to nitrocellulose.Titers of antibodies against intact cells of F. succinogenes S85 andvarious cell membrane preparations were determined by an enzyme-linked immunosorbent assay (ELISA) described by Lam et al. (1987),except that wells were coated with F. succinogenes cells. Titers ofantibodies against the 180-kDa CBP and the chloride-stimulated cel-lobiosidase were determined by preparative separation of the proteinsby SDS-PAGE, followed by Western blotting using a Bio-Rad~ini-~rotein~11ulti Screen apparatus. A doubling dilution series ofantibodies were added to the slots. The subsequent staining steps wereperformed as described for the standard Western blotting procedure. SDS-PAGE and Western blot analysis SDS-PAGE was performed by using the method of Laemmli (1 970).Proteins in SDS-polyacrylamide gels were visualized by stainingwith Coomassie. OM, and IM, and protein samples containing deter-gent exhibited poor resolution of protein bands without a treatment toremove the lipid or detergent prior to electrophoresis. The resolutionwas substantially improved by a rapid solvent extraction that removesboth lipids and detergents (Wessel and Fliigge 1984). Zymogramanalysis for endoglucanases and Western blotting were performed asdescribed previously (Gong and Forsberg 1993). The first antibodieswere the appropriately diluted polyclonal antiserum or monospecificpolyclonal antibody as indicated in the figure legends. Endoglucanase2 (EG2) and polyclonal antisera against it were prepared as previouslydescribed by McGavin and Forsberg (1988,1989) and McGavin et al.(1989). The chloride-stimulated cellobiosidase and polyclonal anti-serum against this enzyme were prepared as described by Huang andForsberg (1988) and Huang et al. (1990). The second antibody wasgoat anti-rabbit antibodies conjugated to alkaline phosphatase.Oxidative cleavage of carbohydrate epitopes of antigens immobi-lized on nitrocellulose was carried out by using the method ofWoodward et al. (1985). Proteins from SDS-polyacrylamide gelsblotted to nitrocellulose were oxidized with 10 mM periodic acid in50 mM sodium acetate buffer (pH 4.5) at 22OC for 1 h in the dark priorto probing with antibodies. In the investigation of the cross-reactivityamong the 180-kDa CBP, chloride-stimulated cellobiosidase, andEG2, the proteins were subjected to SDS-PAGE, excised, and thenre-electrophoresed in a second SDS-polyacrylamide gel as describedby Cleveland et al. (1977), except the protease digestion step wasomitted. Other analytical methods Protein was determined by the method of Bradford (1976), withbovine serum albumin as a standard. Dry weight of the cells wasdetermined as described by Forsberg and Lam (1977). Treatment ofthe cell envelopes with proteases was performed in two steps. A1.2-mg sample of protein was treated with trypsin (1100 U, frombovine pancreas type 111, Sigma) in 20 mM Tris-HC1 buffer (pH 7.5)containing 0.02% wlv sodium azide, and then with 13units of protein-ase K in the same solution containing 5 mM EDTA at 37OC for 22 h. Immunogold labelling Immunogold labelling of cells grown on glucose (10 h) or Avicelcellulose (40 h) was conducted as described by Huang and Forsberg(1990) for prefixation labelling, except that the pH of N-2-hydroxy- ethylpiperazine-N'-2-ethanesulfonic cid (HEPES) buffer was 7.0instead of 6.7. The control samples were treated in an identical fashionexcept that the first antibodies were replaced by preimmune sera. Goldparticles (10 nm) conjugated with a goat anti-rabbit IgG antibody(Sigma) used in the staining procedure were first examined for clusterson carbon-formvar coated 200-mesh copper grids and none werefound in the preparations used. The methods for preparation of nega-tively stained samples were essentially those described by McGavinet al. (1990). Results CBPs of Z? succinogenes Fihrohacter succinogenes cells grown on glucose were pre-viously shown to have high binding capacity for cellulosefibres, which suggests that they have OM associated CBPs. Toidentify these OM CBPs, it was essential to solubilize themembrane for cellulose-binding assays. CHAPS is one of themost efficient nondenaturing detergents available for solubiliz-ing membranes (Hjelmeland 1980). When cell envelopes of thebacterium were solubilized with the 0.5% wlv CHAPS andthen mixed with microcrystalline cellulose, 10% of the totalsolublized protein bound to the cellulose, which indicated thepresence of CBPs. The samples tested for CBPs includednonsedimentable extracellular culture fluid (ECCF), perip-lasm, and OM and IM fractions. Figure 1 shows SDS-PAGEprotein profiles of cellulose-bound and -unbound proteins. TheECCF contained CBPs of 180 and 220 kDa. The periplasmic  456 Can, J. Microbial. Vol. 42, 1996 Fig. 1. SDS-PAGE proteln profiles of nonsed~mentable roteins Fig.2. SDS-PAGE zymogram analysls for endoglucanasefrom the concentrated ECCF, the penplasmic fraction (PenP), activity in of cell envelope CBPs samples. Lane 1, Coomassieand CHAPS-solubilized OM and IM proteins either bound toblue stained CBPs; lane 2, Congo red stained CBPs. In lane 1, cellulose or in the unbound eluate. (A) Membrane samples werethe 180-kDa CBP is indicated by an arrowhead, while in lane 2solubilized wlth 0.5% w/v CHAPS in 50 mM sodium phosphatearrowheads point to bands of endoglucanase activity. (pH 6.5) as descnbed in the Materials and methods. (B) Twicethe amount of eluted sample was electrophoresed tounequ~vicolly emonstrate the presence of the 220- and 1 2 240-kDa CBPs. The samples bound to cellulose were releasedby using SDS -PAGE sample buffer. A ECCF PerlP me- "A ILI d *Y ""d .,, fraction obtained by washing cells in NaC1, which includedboth peripheral membrane proteins and periplasmic proteins,contained CBPs of 40,45,50, and 120 kDa. The OM containedCBPs of 120, 180, 220, and 240 kDa. Similar bands wereobserved whether the binding was performed in sodium phos-phate buffer in the presence of CHAPS or after extensivedialysis to remove CHAPS, and in the presence of 250 mMNaC1. The 120- and 180-kDa CBPs from the OM bound tocellulose more tenaciously and were not detected in theunbound fraction, but the 220- and 240-kDa bands wereroutinely detected in the unbound fraction. The IM preparationsusually contained very low concentrations of CBPs, except fora weakly binding 140-kDa protein. Zymogram analysis ofSDS-PAGE gels of CBPs from cell envelopes (including bothIM and OM) using carboxymethylcellulose (CMC) as thesubstrate revealed endoglucanase activity for CBPs of approxi-mately 60, 80-90, and 120 kDa but no band of activity for the180-kDa CBP (Fig. 2). The 60- and 120-kDa activity bandswere weak, but the diffuse 80- to 90-kDa band was moreintense. Neither the 60- nor the 80- to 90-kDa enzyme wasdetected as a major band among the CBPs by Coomassiestaining. The lack of glucanase activity amongst the largerCBPs, except for the 120-kDa CBP, does not preclude themfrom being endoglucanases, because their catalytic domainsmay have been irreversibly denatured during SDS -PAGE.Of the CBPs, only the 120-kDa CBP was found to be apreviously studied protein. It was identified as EG2 (McGavinet al. 1989) by Western blotting with the corresponding anti-body (data not shown). Characteristics of the 180-kDa CBP Mitsumuri and Minato (19936) had identified 120- and220-kDa CBPs of F. succinogenes. Therefore, we have focusedon the role of the 180-kDaCBPin adhesion of cells to cellulose.Polyclonal antiserum prepared against the 180-kDa CBP andmonospecific antibodies prepared by affinity adsorptionreacted with the 180-kDa protein in Western blotting, as wellas with numerous proteins of both higher and lower molecularmass in cell envelope preparations (Fig. 3). The banding patternwas similar for both monospecific antibodies and the anti-serum. Preparations of OM and IM were probed by Westernblotting and both exhibited similar banding patterns to cellenvelopes (data not shown). This finding was expected for thecytoplasmic membrane fraction owing to contamination byremnants of OM and the high sensitivity of Western blotting.Cross-reactivity with a broad range of cellular proteins waspreviously observed when using monospecific antibodiesagainst the chloride-stimulated cellobiosidase (Huang et al.1990). Therefore, we were interested in determining whetherthe cellobiosidase shared common antigenic epitopes with the180-kDa CBP. Antibodies against the 180-kDa CBP reactedstrongly with the chloride-stimulated cellobiosidase (Fig. 4).Similarly those against the chloride-stimulated cellobiosidasereacted with the 180-kDa CBP. Both antibodies also reactedweakly with EG2. However, antibodies against EG2 showedno detectable reactivity with either the 180-kDa CBP or thechloride-stimulated cellobiosidase. Weak cross-reactivity waspreviously observed between antibodies against EG2 andthe chloride-stimulated cellobiosidase (Huang et al. 1990);however, in that study four times the amount of protein waselectrophoresed.To determine whether the shared antigenic epitope of the180-kDa CBP and cross-reactive epitopes of cell envelopeproteins fromF. succinogenes S85 were carbohydrate in nature,samples of the 180-kDa CBP and cell envelope proteins wereseparated by SDS-PAGE, transferred to nitrocellulose, oxi-dized with periodate, and subjected to immunoblottingwith antiserum against the 180-kDa CBP. As shown in Fig. 5,  Gong et al. Fig. 3. SDS-PAGE and Western blot analysis of CE and the180-kDa CBP prepared from glucose-grown F: succinogenes S85. SDS-PAGE-separated CE proteins and CBPs were stainedwith Coomassie (arrowhead shows the Coomassie stainedpurified 180-kDa CBP). The lanes subjected to Western blottingwere either probed with monospecific polyclonal antibodies(MSAb) or untreated polyclonal antiserum (antiserum) againstthe 180-kDa CBP. The second antibody was goat anti-rabbitantibody conjugated to alkaline phosphatase (CE, 10 pgproteinDane; CBP, 0.3 pg proteinDane). SDS-PAGE Western blot MSAb Antiserum &I & ww wm ma UVU UUV periodate oxidation greatly reduced the immunoreactivity ofboth the 180-kDa CBP and all cell envelope protein bands.However, it did not affect the staining of either the 180-kDaCBP or the cell envelope proteins by the protein stain arnidoblack, thereby showing that the proteins were not removed bythe treatment. From these results it appeared that one or moreglycosyl substitutions of the 180-kDa CBP are present on awide variety of other cell envelope proteins of both higher andlower molecular mass. When cell envelopes were digested withtrypsin and proteinase K, immunoreactive bands weredestroyed and reaction with the antiserum was no longerdetected (data not shown). This indicated that the antigens wereglycoproteins rather than lipopolysaccharide or other polysac-charides that are not degraded by protease action. Immunogold labelling of cells To determine whether there were sites on the cell surface of F. succinogenes that react with antibodies against the 180-kDaCBP, cells grown on cellulose were incubated with a 10-folddilution of polyclonal antiserum against the 180-kDa CBP, andthen, after washes in buffer, were incubated in a 10-fold dilutionof anti-rabbit IgG - gold conjugate. Gold particles were uni-formly distributed over the surface of cells not attached tocellulose and instead were free in the medium (Fig. 6A). Incontrast, all cells examined (at least 20 cells) that were boundto cellulose were intensely labelled at the junctions where theywere attached to cellulose, with very little label on the rest ofthe cell surface (Fig. 6B). Alarge number of gold particles were Fig. 4. Western blot analysis showing cross-reactivity ofantibodies against the 180-kDa CBP, the chloride-stimulatedcellobiosidase (Cbsase), and EG2. The first antibodies for theWestern blots were the respective 1000-fold diluted polyclonalantiserum against the 180-kDa CBP and the monospecificpolyclonal antibodies against each of the chloride-stimulatedcellobiosidase and EG2. The second antibody was a goatanti-rabbit antibody conjugated to alkaline phosphatase asin Fig. 3; the 180-kDa CBP, 0.2 pg/lane; chloride-stimulatedcellobiosidase, 0.2 pgllane; EG2,0.3 pg/lane. Arrowheadsshow the locations of a weak reaction of EG2 with antibodies. SDS-PAGE Western blot (1st Ab) CBP Cbsase EG2 Fig. 5. Effect of periodate oxidation of the 180-kDa CBP andcell envelopes on their reactions with antibodies against the180-kDa CBP. OX, periodate oxidation, Ab, immunostainingwith a 1000-fold dilution of polyclonal antiserum against180-kDa CBP as the first antibody. ABS, amido black staining.The 180-kDa CBP was 0.4 pghane, while cell envelopes,prepared from glucose-grown cells of F: succinogenes S85, were10 pg proteinhane. The plus sign indicates that the treatmentwas performed, while a minus sign indicates that the treatmentwas omitted. Samples subjected to antibody treatments show theimmunoblot profile, while those of samples stained with arnidoblack stain show the SDS-PAGE Coomassie staining profile. 180 kDa CBP Cell enveIopes OX ++- +-f- Ab ++-- +f- ABS - 180, also found on cellulose particles with no attached bacteria,indicating that the glycoproteins were also released from cellsduring growth and became bound on cellulose. No nonspecificbackground staining of either cells or cellulose was observed
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